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THE IN TERNATIONAL JOURNAL OF F UNGAL TAXONOM Yr & NOMENCLATURE
- Volume 94° Het ase $ October-December 2005 (2006)
CONTENTS
: Tuber umbilicatum, a new species from China, with a key to the spinose-reticulate ‘ 4 spored Tuber species _ Chen, Juan, Pei-Gui Liu & Yun Wang Blumenavia toribiotalpaensis: a new species of Clathraceae from Jalisco, Mexico
! oes Luisa eee Reggae? & J. Antonio Vazquez-Garcia
ris Giege Tufan, Hiiseym Siimbiil & Aysen Ozdemur Tiirk
ul gic lan ealecuiee characterization of the yi eieeposs of —
eae hs ae . . Rosario Medel a new w record of Lepiota o occurring in ‘the Gulf of Mexico area Jota ha anes yom: Letiaa Montoya & Victor M. Bandala rection of] Dothidotthia aspera “ane adenageaucla toa a byphomycetous
phic fungus, Thyrostoma negundinis _ Annette W. Ramaley
“Alan w. enrages & John A. Elix
sof Pertusaria nerore Western Australia
. iii Huang, Chunru Li, Smear A. Hiiaben Kathie T. Hodge: Meizhen Fan & Zengzhi Li
et SP nov, a bambusicolous, synnematous fungus from tad Gawas & Darbhe Jayarama Bhat
De ies es of / Anthracoidea ( ae from China . = Guo
. SPyle tte VWs ache esa a Y Jiang & ¥-J. Yao
ber paid and I. Suen poe ste Mirco ett, Mauro Marchetti,
‘Sl
55
ate 1
85
89 , 103 111
127 133
137
149
[Content continued from front cover] Two new species of Hypogymnia (Lecanorales, Ascomycota) with pruinose lobe tips from China Xinli Wei & Jiangchun Wei Discovery and description of a teleomorph for Leptographium koreanum H. Masuya, J.-J. Kim, M. J. Wingfield, Y. Yamaoka, S. Kaneko, C. Breuil & G.-H. Kim Two parasitic fungi on a new host, Syringa (Oleaceae) Ovidiu Constantinescu, Vadim A. Melnik & Gerard J.M. Verkley Further notes on the molecular taxonomy of Metarhizium Bo Huang, Richard A. Humber, Shigui Li, Zengzhi Li & Kathie T. Hodge The genus Hymenochaete (Basidiomycota, Hymenomycetes) in the Hawaiian Islands Erast Parmasto & Robert L. Gilbertson A new predatory fungus from China Dongshen Yang, Weimin Chen, Ying Huang, Minghe Mo & Keqin Zhang New species of sterile crustose lichens from Australasia John A. Elix Some interesting pyrenomycetous fungi on bark of Quercus spp. from Spain Julia Checa & M.N. Blanco Biogeography and hosts of poroid wood decay fungi in North Carolina: species of Fomes, Fomitopsis, Fomitella and Ganoderma L.F. Grand & C.S. Vernia Two new species of Ramaria from southwestern China Ping Zhang, Zhu-Liang Yang & Zai-Wei Ge The world’s second record of Neoheteroceras flageoletii reported from Turkey Elsad Hiiseyin, Faruk Selcuk & Ahmet Sahin Lewia chlamidosporiformans sp. nov. from Euphorbia heterophylla Bruno S. Vieira & Robert W. Barreto Weddellomyces turcicus, a new species on a grey Acarospora from Turkey M. Gokhan Halici, Alan Orange & Ahmet Aksoy Cordyceps spegazzinii sp. nov., a new species of the C. militaris group Monica S. Torres, James F. White, Jr. & Joseph F. Bischoff A phylogeny of Ramariopsis and allied taxa Ricardo Garcia-Sandoval, Joagin Cifuentes,Efrain De Luna, Arturo Estrada-Torres & Margarita Villegas A dichotomous key to Scutellospora species (Gigasporaceae, Glomeromycota) using morphological characters Gladstone A. Silva, Leonor C. Maia & Sidney L. Stiirmer New species and phylogenetic relationships of Hypoxylon species found in Thailand inferred from the internal transcribed spacer regions of ribosomal DNA sequences _N. Suwannasai, S. Rodtong, S. Thienhirun & A.J.S. Whalley Tricholoma equestre, the correct name for T: flavovirens (Agaricales) H. Deng & Y. -J. Yao A new species of Lecanicillium isolated from the white pine weevil, Pissodes strobi Harry H. Kope & Isabel Leal Two new species of Hymenochaetaceae from eastern China Yu-Cheng Dai & Bao-Kai Cui Some entomogenous fungi from Wuyishan and Zhangjiajie nature reserves 2. Three new species of the genus Hirsutella Zongqi Liang, Yanfeng Han, Aiying Liu & Jianzhong Huang
[Content continued on inside back cover]
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MYCOTAXON
THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE
Volume 94, 2005 (2006)
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MYCOTAXON
VOLUME NINETY-FOUR TABLE OF CONTENTS
Tuber umbilicatum, a new species from China, with a key to the spinose- reticulate spored Tuber species Chen, Juan, Pei-Gui Liu & Yun Wang Blumenavia toribiotalpaensis: a new species of Clathraceae from Jalisco, Mexico Yalma Luisa Vargas-Rodriguez & J. Antonio Vazquez-Garcia Studies on Basidiomycetes in Greece 1: The genus Crepidotus . Z. Gonou-Zagou & P. Delivorias The lichen flora of the Termessos National Park in Southwestern Turkey Ozge Tufan, Hiiseym Siimbiil & Aysen Ozdemir Tiirk Two new species of Anthracoidea (Ustilaginales) from China Lin Guo A new species of Lepiota (Agaricaceae, Basidiomycetes) from China Han-Chen Wang & Zhu-Liang Yang ITS sequence analysis and ascomatal development of Pseudogymnoascus roseus Y. Jiang & Y.-J. Yao Morphological and molecular characterization of the mycorrhizas of Inocybe rufuloides and I. splendens Mirco Iotti, Mauro Marchetti, Enrico Bonuso & Alessandra Zambonelli Russula in Himalaya 1: A new species of subgenus Amoenula K. Das, S.L. Miller, J.R. Sharma, P. Sharma, & R.P. Bhatt Streptopodium passiflorae comb. nov. on Passiflora rubra J.R. Liberato & R.W. Barreto Type revision of three Termitomyces species from India B.-H. Tang, T.-Z. Wei & Y.-J. Yao A review of the genus Gyromitra (Ascomycota, Pezizales, Discinaceae) in Mexico Rosario Medel A new species and a new record of Lepiota occurring in the Gulf of Mexico area Leticia Montoya & Victor M. Bandala The connection of Dothidotthia aspera (Botryosphaeriaceae) to a hypho- mycetous anamorphic fungus, Thyrostoma negundinis Annette W. Ramaley A new species of Pertusaria from Western Australia Alan W. Archer & John A. Elix Molecular evidence for the taxonomic status of Metarhizium taii and its teleomorph, Cordyceps taii (Hypocreales, Clavicipitaceae) Bo Huang, Chunru Li, Richard A. Humber, Kathie T. Hodge, Meizhen Fan & Zengzhi Li Vamsaprija indica gen. et sp. nov., a bambusicolous, synnematous fungus from India Puja Gawas & Darbhe Jayarama Bhat Two new species of Hypogymnia (Lecanorales, Ascomycota) with pruinose lobe tips from China Xinli Wei & Jiangchun Wei Discovery and description of a teleomorph for Leptographium koreanum H. Masuya, J.-J. Kim, M. J. Wingfield, Y. Yamaoka, S. Kaneko, C. Breuil & G.-H. Kim
ili
103
War
135
137
149
I e}s)
159
IV
Two parasitic fungi on a new host, Syringa (Oleaceae) Ovidiu Constantinescu, Vadim A. Melnik & Gerard J.M. Verkley Further notes on the molecular taxonomy of Metarhizium Bo Huang, Richard A. Humber, Shigui Li, Zengzhi Li & Kathie T. Hodge The genus Hymenochaete (Basidiomycota, Hymenomycetes) in the Hawaiian Islands Erast Parmasto & Robert L. Gilbertson A new predatory fungus from China Dongshen Yang, Weimin Chen, Ying Huang, Minghe Mo & Keqin Zhang New species of sterile crustose lichens from Australasia John A. Elix
Some interesting pyrenomycetous fungi on bark of Quercus spp. from Spain Julia Checa & M.N. Blanco Biogeography and hosts of poroid wood decay fungi in North Carolina: species of Fomes, Fomitopsis, Fomitella and Ganoderma L.E Grand & C.S. Vernia Two new species of Ramaria from southwestern China Ping Zhang, Zhu-Liang Yang & Zai-Wei Ge The world’s second record of Neoheteroceras flageoletii reported from Turkey Elsad Hiiseyin, Faruk Selcuk & Ahmet Sahin Lewia chlamidosporiformans sp. nov. from Euphorbia heterophylla Bruno S. Vieira & Robert W. Barreto Weddellomyces turcicus, a new species on a grey Acarospora from Turkey M. Gokhan Halici, Alan Orange & Ahmet Aksoy Cordyceps spegazzinii sp. nov., a new species of the C. militaris group Monica S. Torres, James F. White, Jr. & Joseph F. Bischoff A phylogeny of Ramariopsis and allied taxa Ricardo Garcia-Sandoval, Joaqin Cifuentes, Efrain De Luna, Arturo Estrada-Torres & Margarita Villegas A dichotomous key to Scutellospora species (Gigasporaceae, Glomeromycota) using morphological characters Gladstone A. Silva, Leonor C. Maia & Sidney L. Stiirmer New species and phylogenetic relationships of Hypoxylon species found in Thailand inferred from the internal transcribed spacer regions of ribosomal DNA sequences N. Suwannasai, S. Rodtong, S. Thienhirun & A.J.S. Whalley Tricholoma equestre, the correct name for T: flavovirens (Agaricales) H. Deng & Y. -J. Yao A new species of Lecanicillium isolated from the white pine weevil, Pissodes strobi Harry H. Kope & Isabel Leal Two new species of Hymenochaetaceae from eastern China Yu-Cheng Dai & Bao-Kai Cui Some entomogenous fungi from Wuyishan and Zhangjiajie nature reserves 2. Three new species of the genus Hirsutella Zongqi Liang, Yanfeng Han, Aiying Liu & Jianzhong Huang
|W a7 181 189
215 Z19
MapAs,
Jel 20 241 245 249
Pape,
265
293
303
220
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Paecilomyces parvosporus, a new species with its relatives from Yunnan
Province, China Yanfeng Han, Zongqi Liang, Huali Chu & Jichuan Kang Notes on Otidea from Xinjiang, China Wen-Ying Zhuang Book reviews and notices David L. Hawksworth
Indices & Information
Nomenclatural novelties proposed in volume 94
Author index for volume 94
Reviewers for volume 94
MycoTaxon online resources—Index to Fungous and Lichen Taxa, cumulative indices, distributional checklists, past volumes online, information for authors e reviewers, search features
Errata
MycoTaxon Instructions to authors
Recent books from MycoTAxon
Vi
PUBLICATION DATE FOR VOLUME NINETY-THREE
MYCOTAXON for JuLY-SEPTEMBER, VOLUME 93 (1-426 + I-VI)
was issued on November 11, 2005
MYCOTAXON
Volume 94, pp. 1-6 October-December 2005
Tuber umbilicatum, a new species from China, with a key to the spinose-reticulate spored Tuber species
JUAN CHEN *”, PEI-Gu1 Liu * & YUN WANG?
pgliu@mail.kib.ac.cn 1 Kunming Institute of Botany, Chinese Academy of Sciences Kunming 650204, P. R. China
2 Graduate School of Chinese Academy of Sciences Beijing 100039, P. R. China
3 Institute of Applied Ecology, Chinese Academy of Sciences Shenyang 110015, P. R. China
Abstract—Tuber umbilicatum, a truffle associated with Pinus yunnanensis in southwestern China, is described and illustrated as a new species. It is characterized by inconspicuously papillate ascomata with an umbilicate basal cavity and ellipsoid ascospores ornamented with spines connected by low ridges to form an alveolate reticulum 6-8 meshes across the spore width. A key to seven Tuber species bearing spinose-reticulate spores is presented.
Key words—Tuberaceae, truffle, spore ornamentation, hypogeous fungus
Introduction
Since Liu (1985) reported the genus Tuber from Shanxi province, China, several additional papers on Chinese truffles have been published. The export of Chinese Tuber spp. to Europe has stimulated further interest in members of this genus in China. Currently, up to 20 species, including 10 new species, have been reported in this country (Liu 1985, Tao & Liu 1989, Wang & Li 1991, Hu 1992, Wang et al. 1998, Zhuang 1998, Xu 1999, Wang & He 2002, He et al. 2004, Zhang et al. 2005, Song et al. 2005). During study of truffles associated with Pinus yunnanensis, a dominant tree in southwestern China, we found a new truffle, characterized by inconspicuously papillate ascomata with an umbilicate basal cavity and ellipsoid, spinose-reticulate ascospores. To distinguish this new species from others with spinose-reticulate spores, a world-wide key to the seven Tuber species with such spores is provided.
Materials and Methods
Macroscopic characters are described from fresh specimen. Microscopic methods of Yang & Zhang (2003) were followed. For scanning electron microscopy (SEM), spores were scraped from the dried gleba onto doubled-sided tape, which mounted directly
*corresponding author
2
on an SEM stub, coated with gold-palladium, and examined and photographed with a JEOL JMS-5600LV SEM. Herbaria that provided specimens are abbreviated and cited according to Holmgren et al. (1990), except HKAS (Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences) and IFS (Herbarium of Institute of Applied Ecology, Chinese Academy of Sciences), two not yet listed in the Index Herbariorum.
Taxonomy
Tuber umbilicatum Juan Chen & P. G. Liu, sp. nov. Figs. 1-5 Ascomata ochracea, glabra vel subglabrabasi umbilicata. Peridium bistratum, 320-500 um crassum: stratum exterius pseudoparenchymaticum, stratum interius hyphi intertextis. Gleba solida, purpureobruneola vel griseobruneola, venis albis. Asci sporis 1-4 (6). Ascosporae ellipsoideae, ochraceae, 21-40 x 14-32 um, spinulis 3-5 um altis, reticulato alveolato < 1um alto connexis ornatae. Holotypus hic designatus: HKAS 44316.
Etymology: Latin, umbilicatum, in reference to the umbilicate depression of the ascomata.
Ascomata (Fig. 1) 1.2-1.9 cm broad, globose, with an umbilicate depression at the base, surface smooth or with minute papillae up to 50 um high, pale yellow, becoming yellow-brown or brown when dried. Peridium (Fig. 2) brittle, peeling easily from the gleba, mostly 320-500 um thick, composed of two layers: outer layer 90-250 um thick, pseudoparenchymatous, composed of subglobose to subangular, yellow-brown cells 7-16 x 5-11 um, the walls 1-3 um thick; inner layer 150-400 um thick, of intricately interwoven, hyaline hyphae 2-5 um in diam, the walls thin to somewhat thickened. Gleba purple-brown or grey-brown with pink tint at maturity, marbled with numerous, narrow, branching, white veins radiating from the basal cavity.
Asci (Fig. 3) 50-83 x 37-67 um excluding the stalk, globose to subglobose, ellipsoid or irregular, sessile or sometimes with a short stalk, 1-4 (-6) spored. Ascospores (Fig. 4, 5) ellipsoid, yellow-brown at maturity, the walls up to 2 um thick, in 1-spored asci (28-) 33-40 x 20-32 um excluding ornamentation, 2-spored asci (23-) 25-37 x 17-26 um, 3- spored asci (21-) 23-33 (-36) x 17-23 um, 4-spored asci 21-30 (-32) x 15-22 (-25) um, 5-spored asci 21-26 x 14-20 um; Q = (1-) 1.2-1.6 (-1.8), Q = 1.4 + 0.13; ornamentation of . spines 3-5 (-6) um tall connected by an alveolate reticulum < 1 um tall, the alveolae 3-6 (-7) x 2.5-5 um, 6-8 across the spore width and (6-) 7-10 along the spore length.
Habitat: Hypogeous under Pinus yunnanensis.
Specimen examined—CHINA: Yunnan province, Chengjiang county, Tigu Village, elev. 1900-2000 m, 31 Oct. 2003, Juan Chen 145 (HKAS 44316 — holotype).
Discussion— The surface of the ascomata of T: umbilicatum is glabrous to subglabrous to the naked eye, but very fine papillae can be seen with a stereomicroscope. These papillae give the peridium a wavy outer edge under the compound microscope. The combination of umbilicate, nearly smooth ascomata and spinose-reticulate spores is distinctive.
Species of Tuber can be divided on the basis of spore ornamentation into three groups: 1) spiny (representative species: T. melanosporum Vittad.), 2) reticulate (representative species: T. borchii Vittad.) and 3) spinose-reticulate (representative species:
4
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SS K | Wee fi NS Woe in ANKLES KX RANI? Nie LS ai
Figs. 1-5 Tuber umbilicatum (HKAS 44316, Holotype) 1. Fresh ascomata; 2. Vertical section of the peridium; 3. Asci; 4. SEM of spore, showing the detail of ornamentation; 5. Ascospores.
T. spinoreticulatum Uecker & Burds.). T: umbilicatum belongs to the T: spinoreticulatum group, which includes T. taiyuanense B. Liu, T. pseudoexcavatum Y. Wang et al. and T. huidongense Y. Wang, known only from China; T. lyonii Butters (=T. texense Heimsch) and T. spinoreticulatum from North America; and T. malacodermum E. Fisch. from
4
Germany and Switzerland (Fischer 1923, Heimsch 1958, Uecker & Burdsall 1977, Liu 1985, Wang et al. 1998, Wang & He 2002, Trappe et al. 1996). These species can be difficult to distinguish because of their morphological similarity. Accordingly, to clarify differences among these species we re-examined the holotypes of T. huidongense (IFS 89923) and T. texense (OSC 42353), isotype of T’ spinoreticulatum (OSC 38860), a neotype of T: taiyuanense (HMAS 75888) and two specimens of T. pseudoexcavatum (HKAS 47617, 41313). Ascomata and SEM photomicrographs of spores are illustrated in Figs. 6-11. Although we did not obtain specimens of T: malacodermum, Fischer's (1923) description as confirmed by study of the type collection by J. M. Trappe (personal communication) showed it to have a pseudoparenchymatous peridium composed of cells inflated up to 20-70 um broad. A key to the spinose-reticulate species is presented here, based on these studies.
Key to species with spinose-reticulate spores
1. Peridium with rounded cells inflated to 20-70 um. . . . . . . I: malacodermum 1. Peridium of interwoven hyphae or with rounded cells mostly < 20 um broad . . 2 2. Ascomata With'a. basal cavity ea tet ee a © ee rane 2. Ascomata without a basal cavity ..-.. ....... OP ES os 6 ee) 3. Peridium smooth or with Waa oes’ ieee 10-50 um high; spores ellipsoid,
Oslin : . . . To umbilicatum 3. Peridium with conspicuous warts 100- 150 um. Bent spores ae: broadly ellipsoid,
QOS Sue 25 Aare Gg ee UR ee AL ee ed ee eee 4 4. Cavity conspicuous; asci 1-8 spored, sessile. . . . . . . . . IT. pseudoexcavatum 4. Cavity inconspicuous; asci 1-4 spored, stipitate. . . . . . . T. spinoreticulatum
5. Ascomata smooth; ascospores Pra dial O.<i73; apa 1-1.25. T. taiyuanense Ds Recon emis minute veanvTiy: up to ag! lum. REDE ascospores ellipsoid, Q > 1.3, MOStly L371 65.) Mea oman. so cette Tere CMe 5 o> i ee 6
6. Spores with longer spines mostly > 3 um long, reticulum complete and regular, meshes ° size bigger 4-8 x 4-6um .. . . ... . . DT huidongense
6. Spores with shorter spines 2-3 um fone Feelin Aa to complete, meshes size smialleri326002-4 (irate ae LT oni(— i texense)
Acknowledgments
Prof. James Trappe and Dr. Michael Castellano reviewed the manuscript and offered invaluable comments and suggestions. Specimens were generously lent to us by the herbaria of the Department of Botany & Plant Pathology, Oregon State University, Royal Botanic Gardens, Kew, and Institute of Microbiology, Chinese Academy of Sciences. Sincere gratitude is offered to Prof. M. Zang, Dr. Zhu L. Yang and Mrs. X. H. Wang, Kunming Institute of Botany, for their valuable suggestions on earlier version of the manuscript. Many thanks to my colleagues, F Q. Yu, H. D. Zheng and J. Y. Chen for providing morphological photos. The study was supported in part by National Science Foundation of China (No. 30470011), the Knowledge Innovation Program of Chinese Academy of Sciences (No. KSCXZ-1-09-06 & KSCXZ-SW-101C) and the Nature Science Foundation of Yunnan province (No. 2004C0050M).
Figs. 6-11. T. taiyuanense (HMAS 75888, neotype): 6. Ascomata; 7. SEM micrograph of ascospore. T. pseudoexcavatum (HKAS 47617): 8. Ascomata. T: huidongense (IFS 89923, holotype): 9. Ascospore.
T. spinoreticulatum (OSC 38860, isotype): 10. Ascospore. T. texense (OSC 42353, holotype): 11. Ascospore.
Literature cited
Fischer E. 1923. Zur Systematik der Schweizerischen Triiffeln aus den Gruppen von Tuber excavatum und rufum. Verhandlungen Naturforschenden Gesellschaft Basel 35: 34-50.
He XY, Li HM, Wang Y. 2004. Tuber zhongdianense sp. nov. from China. Mycotaxon 90: 213-216.
Heimsch C. 1958. The first recorded truffle from Texas. Mycologia 50: 657-660.
Holmgren PK, Holmgren NH, Barnett LC. 1990. Index Herbariorum: part I: herbaria of the world. 8th edition, New York Botanical Garden: New York. 693 pp.
Hu HT. 1992. Tuber formosanum sp. nov. and its mycorrhizal associations. Quarterly Journal of the Experimental Forest, National Taiwan University 6: 79-86.
Liu B.1985. New species and new records of hypogeous fungi from China (I). Acta Mycologica Sinica 4; 84-89.
Song MS, Cao JZ, Yao YJ. 2005. Occurrence of Tuber aestivum in China. Mycotaxon 91: 75-80.
Tao K, Liu B. 1989. A new species of the genus Tuber from China. Journal of Shanxi University (Natural Science Edition) 12: 215-218.
Trappe JM, Jumpponen AM, Cazares E. 1996. NATS truffle and truffle-like fungi 5: Tuber lyonii (=T. texense), with a key to the spiny-spored Tuber species groups. Mycotaxon 60: 365-372. Uecker FA, Burdsall HH. 1977. Tuber spinoreticulatum, a new truffle from Maryland. Mycologia
69: 626-630.
Wang Y, He XY. 2002. Tuber huidongense sp. nov. from China. Mycotaxon 83: 191-194.
Wang Y, Li ZP. 1991. A new species of the genus Tuber from China. Acta Mycologica Sinica 10: 263-265. (in Chinese).
Wang Y, Moreno G, Riousset LJ, Manjon JL, Riousset G, Fourré G, Massimo GD, Garcia Montero LG, Diez J. 1998. Tuber pseudoexcavatum sp. nov., a new species from China commercialized in Spain, France and Italy with additional comments on Chinese truffles. Cryptogamie Mycologie O71 ES-120!
Xu AS. 1999. Notes on the genus of Tuber from Tibet. Mycosystema 18: 361-365. (in Chinese).
Yang ZL, Zhang LF. 2003. Type studies on Clitocybe macrospora and Xerula furfuracea var. bispora. Mycotaxon 88: 447-454.
Zhang LF, Yang ZL. Song DS. 2005. A phylogenetic study of commercial Chinese truffle and their allies: Taxonomic implications. FEMS Microbiology Letters 245: 85-92.
Zhuang WY. 1998. A list of discomycetes in China. Mycotaxon 67: 365-390.
MYCOTAXON
Volume 94, pp. 7-14 October-December 2005
Blumenavia toribiotalpaensis: a new species of Clathraceae from Jalisco, Mexico
"YALMA L. VARGAS-RODRIGUEZ & 7J. ANTONIO VAZQUEZ-GARCIA
yvargal@lsu.edu '107 Life Sciences Building, Dept. of Biological Sciences, Louisiana State University Baton Rouge 70803, Louisiana, U.S.
*Depto. de Botanica y Zoologia, CUCBA, Universidad de Guadalajara km 15 carr. Guadalajara-Nogales, Las Agujas Nextipac, 45110, Zapopan, Jalisco, Mexico
Abstract—A new stink horn species, Blumenavia toribiotalpaensis sp. nov. (Clathraceae) from Mexico, is described and illustrated. Blumenavia toribiotalpaensis differs from previously described species of the genus by its larger receptacle and basidiospores, distribution of the gleba over the upper half of the columns and presence of no more than four columns.
Key words—Basidiomycota, phalloid, systematics, Talpa de Allende
Introduction
During the course of studies of the distribution and demography ofa recently discovered Acer saccharum subsp. skutchii (cloud forest sugar maple) population in Jalisco, Mexico (Vargas-Rodriguez 2005), we collected fungal specimens with novel features. A survey of the literature revealed that the specimens are an undescribed species of Blumenavia (Clathraceae).
The genus Blumenavia was established to accommodate B. rhacodes Moller (Méller 1895). Currently, two species, B. rhacodes and B. angolensis (Welw. & Curr.) Dring, are included in the genus (Dring 1980). The genus is characterized primarily by the small number of columns that lack transverse arms. In addition, the columns are free at the base and united at their apices with glebifers consisting of membranes attached by one side to each of the two inner angles of the column (Dring 1980, Saenz 1980).
The new Blumenavia species occurs in a pine-cloud forest transition considered a Tertiary refuge (Vargas-Rodriguez 2005). The subtropical montane cloud forest is unique in having high plant species richness and includes a number of endangered and relict plants, comparable only to certain Asian forests (Graham 1999, Vargas-Rodriguez 2005). The forest contains temperate disjunct tree genera with East Asia and East North America, such as Acer, Magnolia, Carpinus, Cornus, Fraxinus, Juglans, Tilia, and Ostrya (Graham 1999, Vazquez-Garcia et al. 2000). This exceptional community is proposed for protection as a biosphere reserve with 3,000 inhabitants supporting the movement
8
(Vargas-Rodriguez 2005). The discovery of the novel Blumenavia species increases our knowledge and relevance of the biota of this unique region.
Materials and Methods
We collected fruiting bodies at different stages of development in a pine-montane cloud forest transition (1,800 m a.s.l.), near Talpa de Allende, Jalisco, Mexico, among fallen leaves under the canopy of adult Pinus spp. and Carpinus caroliniana trees. A second collection was made a year later in the same area and a third one in 2005. Five fruiting bodies were fixed in FAA solution (five parts 40% formaldehyde: five parts glacial acetic acid: 90 parts 95% ethyl alcohol) and seven were dried. A free hand cross section was made to one egg. Spores were mounted on slides in lactophenol and in 3% KOH and examined with a NIKON Microphot compound microscope using differential interference contrast and bright field optical systems. Hand-cut sections of the columns were made from a sample that had been fixed in FAA for nine months. Sections were dehydrated with ethanol, critical point dried, mounted, and coated with gold:palladium 60:40 in an Edwards S-150 sputter coater. We used a Cambridge S-260 scanning electron microscopy (SEM) for observation.
Taxonomic Description
Blumenavia toribiotalpaensis Vargas-Rodriguez sp. nov. FIGS. 1-14
Ovum fulvum, superficies interdum findens aquamis fulvis angularibus, elipsoidale 2.2- 3.8 cm longum x 2.1-3.9 cm altum; adhaesae albae rhizomorphae 1-1.5 cm diametro. Receptaculum expansum 12.1-15.3 cm altum, 3.8-5.6 cm longum, colore vario ab albulo ad dilutum bombax, cylindrale (or cylindratum) ex 3 vel 4 columnis robustis constans; columnis 0.9-1.1 cm diametro ex parte gracillima, 1.9-2.1 cm ex parte latissima, conjunctis superne, inferne liberis, cum sulco in superfacie; semicirculares sectione, constantes ex 9 tubis compositis in 3 ordinibus ab canali abaxiali; proximi canali sunt 5 tubuli circulares sectione in material nova; medius ordo constat ex 3 latioribus tubis, polygonalibus sectione; atque ex singulari magna polygonali tuba constat ordo extremus ab canali abaxiali. Columnae tela glebifera incrassata in facie interna informante cristam per anterior- laterales angulos columnae, glebifera incrassata marginibus unita, crista blebam ferente. Gleba coercita intra laceratam glebiferam cristam, sita in superior parte columnarum, atro-brunnescenti-olivacea, aroma simili piscibus mortuis et nauseosa. Basidiosporae 3.8- 4,2 x 1.7-1.9 um. Associati basidiocarpis siti Scarabaei (Staphilinidae et Leiodidae) intra brachia et muscae (Tephritidae) in gleba maturorum basidiocarporum.
Egg pale brown, outer surface sometimes cracking into angular brown scales, ellipsoidal, 2.2-3.8 cm wide, 2.1-3.9 cm high, gelatinous layer 5-7 mm thick, traversed by peridial sutures corresponding to each of the four columns, immature glebal mass about 4 mm diameter among columns, glebiferous tissue separated by a cavity opened in one extreme and joined to the column only by the opposite extreme, medullar zone rectangular-like in shape; attached white rhizomorphs 1-1.5 mm in diameter. Expanded receptacle 12.1- 15.3 cm high and 3.8-5.6 cm wide, whitish to pale beige, cylindrical, with 3 to 4 robust columns; columns 0.9-1.1 cm diameter at the thinnest part to 1.9-2.1 cm at the widest part, united above, free below with a groove in the outer surface, semicircular in section,
Figure 1-11. Blumenavia toribiotalpaensis, IBUG 422a. 1, Habitat photograph showing receptacle and volva. 2, Receptacle, volva, and rhizomorphs from specimen preserved in FAA. IBUG 456, 3, Receptacle and volva. IBUG 422a. 4, 5, 9, 10, Detail of gleba, showing its distribution over the upper half of the receptacle. IBUG 456, 6 & 11, Detail of gleba. Yalma L. Vargas-Rodriguez 455, 7 & 8. Freehand cross section of an egg. IBUG 422a, 9, Detail of the volva from specimen preserved in FAA.
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Figure 12-14. Blumenavia toribiotalpaensis, LSUM 422b. 12, Basidiospores. 13, Sections of the arm showing number and arrangement of tubes and associated staphylinid beetle. 14, Inner (upper side) and outer (bottom) arm surfaces.
1]
comprised of 9 tubes arranged in three ranks from the abaxial groove; nearest to the groove are five small tubes, circular in section in fresh material; the middle rank consists of three wider tubes, polygonal in section; and a single, large polygonal tube comprises the outermost rank from the abaxial groove (Fig. 13). Columns with a thickened glebiferous tissue on the inner surface forming a crest along the anterior-lateral angles of the column, glebiferous tissue united to the arms by the edges, crest bearing the gleba. Gleba restricted to the lacerate glebiferous crest, situated on the upper half of the columns, dark olive-brownish, odor of dead fish, nauseous. Basidiospores 3.8-4.2 x 1.7-1.9 um, cylindrical, hyaline in KOH. Beetles [Staphilinidae (Fig. 13), Leiodidae] and flies (Tephritidae) associated with basidiocarps, beetles located inside the arms reach the interior through holes in basidiocarps, holes appear in decaying basidiocarps; flies located in gleba of mature basidiocarps.
Specimen examined — HOLOTYPE here designated. MEXICO, JALISCO: Talpa de Allende municipality, pine-cloud forest (Acer-Podocarpus-Abies) transition, “Ojo de Agua del Cuervo” (“Crow spring”) locality, west of Cumbre de Los Arrastrados (20°11”N; 105°16"W), 1800 m a.s.l., on Pinus spp. fallen trunk and debris, 10 Sep 2002, Yalma L. Vargas-Rodriguez 240, with Javier Curiel, J. Antonio Vazquez-Garcia and Toribio Quintero Moro, dry specimen (BPI); 10 Sep 2002, Yalma L. Vargas-Rodriguez 240a, with Javier Curiel, J. Antonio Vazquez-Garcia and Toribio Quintero Moro, dry specimen (IBUG) (Holmgren et al. 1990).
PARATYPE here designated. MEXICO, JALISCO: Talpa de Allende municipality, pine-cloud forest (Acer-Podocarpus-Abies) transition, “Ojo de Agua del Cuervo” (“Crow spring”) locality, west of Cumbre de Los Arrastrados (20°11”N; 105°16”W), 1800 m a.s.l., on fallen leaves of Pinus spp. and Carpinus caroliniana, 14 Sep 2003, Yalma L. Vargas-Rodriguez 422b, 423a, 423b, 424a and J. Antonio Vazquez-Garcia, FAA preserved material (LSUM); 14 Sep 2003, Yalma L. Vargas-Rodriguez 422a and J. Antonio Vazquez- Garcia, FAA preserved material (IBUG); 13 Sep 2005, Yalma L. Vargas-Rodriguez 454, 456, 462 and J. Antonio Vazquez-Garcia, dry specimen (IBUG); 13 Sep 2005, Yalma L. Vargas-Rodriguez 459, 463 and J. Antonio Vazquez-Garcia, dry specimen (LSUM).
Etymology—From the Latin talpaensis, referring to the municipality where the fungus was collected and toribio, referring to Toribio Quintero Moro, a remarkable forest conservationist. He has promoted the protection of the Talpa de Allende forests by collecting 3,000 signatures from Talpa de Allende habitants and petitioning state and federal Mexican authorities for the creation of a new biosphere reserve in the area.
Known distribution—Jalisco: Only known from the type locality. The species is not common in the area; individuals are patchy distributed along 100 m. The species was not previously known by local people. Only three other species (Clathrus crispus, C. cancellatus, and C. ruber) of the Clathraceae family are known for the Jalisco state and these do not co-occur with Blumenavia toribiotalpaensis. This is the first record of the genus for western Mexico and the second one for the country.
Habit and habitat—Occasionally gregarious. Among plant leaves and debris, under Pinus spp., Carpinus caroliniana and Acer saccharum subsp. skutchii canopy, in transitional pine forest to montane cloud forest.
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Discussion
Blumenavia differs from similar stink horn genera in the glebifer form. Especially notable is the unique gleba borne on lateral flaps of tissue that is lacking in Clathrus, Laternea, and Ligiella.
The differences between Blumenavia toribiotalpaensis and the two previously described species include the distribution of the gleba and the size of the receptacle and basidiospores (Table 1). The gleba is distributed along half the length of the column in B. toribiotalpaensis; in B. rhacodes the gleba is present over the entire length of the column and in B. angolensis restricted to the upper quarter or one third of the receptacle. The receptacle and basidiospores are larger in B. toribiotalpaensis than in the other two species (Table 1).
Table 1. Differences among species of Blumenavia
' Blumenavia Blumenavia rhacodes Blumenavia angolensis Lia toribiotalpaensis Expanded 8.5-13x8 cm 10x3 cm 12.1-15.3x3.8-5.6 cm receptacle Receptacle Clear orange to yellow White Whitish to pale beige color Sections of Triangular and trapezoidal; Subtriangular or Semicircular; 9 tubes columns; about 10 tubes quadrangular; about 6 number of tubes tubes Number of 3-6 3-5 3-4 columns Groove along Present Lacking Present outer side of column Basidiospores 3-4x1-1.5 um 3-3.5x1.5 um 3.8-4.2 X 1.7-1.9 pm Gleba Entire length of the column Upper quarter or one Upper half distribution third of the receptacle Habitat and In coffee plantations in Habitat not noted. Africa: Pine-montane cloud distribution cloud forest in Mexico. Angola, Tanzania, South forest transition. Mexico: Veracruz; The Africa; US: Texas; The Mexico: Jalisco.
Caribbean: Trinidad; South Caribbean: Puerto Rico; America: Brazil. South America: Brazil.
tS
Mature specimens of B. toribiotalpaensis usually have four columns but one immature specimen has only three. The white columns in fresh specimens separate B. toribiotalpaensis from B. rhacodes with orange-yellow columns; B. angolensis however, also has white columns. The spongy texture of the columns of B. toribiotalpaensis distinguishes this species from the more rigid, less spongy texture of B. angolensis. Number, shape and arrangement of tubes in transverse sections of columns differ, being more numerous in B. toribiotalpaensis than in B. angolensis, with a semicircular shape, and arranged differently from B. rhacodes (Table 1).
Habitat and known geographical distribution differ among the three Blumenavia species. Although both B. toribiotalpaensis and B. rhacodes are known from Mexico, B. rhacodes has been found in coffee plantations established under the canopy of cloud forest trees at 1300 m a.s.l. in Teocelo, Veracruz, Mexico (Lopez et al. 1981), while B. toribiotalpaensis occurs at higher elevations (1800 m a.s.l.) in the transition of pine and cloud forest. In Mexico, B. rhacodes is also known from Xalapa, Veracruz (Calonge et al. 2004). This is the first record of the genus for western Mexico and is the second for the country, which was previously found in eastern Mexico (Veracruz state). Individuals of Blumenavia species have low density in Mexico. Blumenavia angolensis is known from Angola, Tanzania, South Africa and Brazil, although the habitats were not reported (Dring 1980) (Table 1).
Acknowledgments
The authors are grateful to Meredith Blackwell for her helpful comments to the manuscript and for her technical support. Margaret C. Henk kindly helped with the photomicrographs. Diane Ferguson and Vesna Karaman helped with the photographs. Richard Warga wrote the Latin description. We acknowledge the assistance of David Farr (USDA National Fungus Collection, BPI), who made material of B. rhacodes available for comparison. Erick Soto-Cantu, Heather Passmore and Angeles Vargas acquired literature needed in the study. We especially appreciate the helpful suggestions of Laura Guzman-Davalos, Silvia Cappello Garcia and Joaquin Cifuentes throughout the study and the peer reviews of Peter Roberts and Kentaro Hosaka.
Literature Cited
Calonge FD, Guzman G, Ramirez-Guillén F. 2004. Observaciones sobre los Gasteromycetes de México depositados en los herbarios XAL y XALU. Boletin de la Sociedad Micoldgica de Madrid 28: 337-371.
Dring DM. 1980. Contributions towards a rational arrangement of the Clathraceae. Kew Bulletin eiep Ie Ieh
Graham A. 1999. Late Cretaceous and Cenozoic history of North American vegetation. Oxford University Press, New York, New York, USA.
Holmgren PK, Holmgren NH, Barnett LC. 1990. Index herbariorum. Part I. Ed. 8. Regnum Vegetation 120: 1-693.
Lopez A, Martinez D, Garcia J. 1981. Adiciones al conocimiento de los Phallales del estado de Veracruz. Boletin de la Sociedad Mexicana de Micologia 16: 109-116.
Moller A. 1895. Botanische Mittheilungen aus den Tropen. Heft 7. Jena: G. Fisher, 152 pp. Saenz JA. 1980. Ligiella, a new genus for the Clathraceae. Mycologia 72: 338-349.
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Vargas-Rodriguez YL. 2005. Ecology of disjunct cloud forest sugar maple populations (Acer saccharum subsp. skutchii) in North and Central America. Master of Science Thesis, Louisiana State University, Baton Rouge, Louisiana, USA.
Vazquez-Garcia JA, Vargas-Rodriguez YL, Aragon F. 2000. Descubrimiento de un bosque de Acer- Podocarpus-Abies en el municipio de Talpa de Allende, Jalisco, Mexico. Boletin del Instituto de Botanica, Universidad de Guadalajara 7: 159-183.
MY COTAXON
Volume 94, pp. 15-42 October-December 2005
Studies on Basidiomycetes in Greece 1: The genus Crepidotus
Z. GONOU-ZAGOU & P. DELIVORIAS
zgonou@biol.uoa.gr panadeli@biol.uoa.gr University of Athens, Faculty of Biology, Department of Ecology & Systematics Panepistimioupoli, GR-157 84 Athens, Greece
Abstract—The diversity of Crepidotus in the Eastern Mediterranean region is poorly known, and data from Greece are scarce. The present work aims at the record and study of the diversity of the genus in Greece and at the contribution to the knowledge of the distribution of the genus in Europe. Forty-four collections have been examined and ten taxa have been identified. Crepidotus autochthonus, C. lundellii, C luteolus, C. subverrucisporus and C. applanatus var. subglobiger are newly recorded from Greece and most taxa are recorded on new substrates for both Greece and Europe. Detailed descriptions, ecological notes and taxonomical comments on all studied taxa are given.
Key words—lignicolous fungi, mycodiversity, biodiversity, taxonomy, Crepidotaceae
Introduction
Crepidotus is a distinct and well-defined genus, and although most species remain not yet completely documented and clarified, several monographic works, as well as critical revisions and regional studies based mainly on morphological studies, provide comprehensive systematic treatment of many species (Singer 1947; Pilat 1948; Hesler & Smith 1965; Singer 1973; Watling & Gregory 1989; Nordstein 1990; Stangl et al. 1991; Senn-Irlet 1995; Senn-Irlet & De Meijer 1998; Aime 2001; Krisai-Greilhuber et al. 2002; Bandala & Montoya 2002a, 2002b, 2004). The systematic position of Crepidotus was until very recently debatable, as it was placed either in family Crepidotaceae (Moser 1978; Jiilich 1981; Singer 1986; Hawksworth et al. 1995), Strophariaceae (Kihner 1980) or Cortinariaceae (Bas 1988; Kirk et al. 2001). Recent phylogenetic analyses (Aime 1999; Moncalvo et al. 2002; Aime et al. 2005) allow to redefine the Crepidotaceae within a broader phylogenetic framework of the agarics, and the genera Crepidotus and Simocybe are better supported in family Crepidotaceae s.s., which represents a separate lineage of dark-spored euagarics.
In the Mediterranean region some studies on Crepidotus exist, mainly concerning N. Africa (Malencon & Bertault 1975), Spain (Ortega & Buendia 1989) and Italy (Lonati 2000). However, the knowledge of the diversity of Crepidotus in the Eastern Mediterranean region is poor, as few papers have been published and most of these are not easily accessible. The data from Greece are scarce. To date, only seven taxa were
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recorded from Greece, usually without descriptions, and most collected very few times (Diapoulis 1939; Maire & Politis 1940; Avtzis & Diamandis 1988; Minter 1988; Diamandis & Perlerou 1990; Pantidou 1991; Diamandis 1992; Zervakis et al. 1998; Dimou et al. 2002a; Dimou et al. 2002b; Konstantinidis 2002; Polemis et al. 2002). Few papers from neighboring countries have been recently published, such as Croatia (Tkaléec & Meéi¢ 2003) and Turkey (Oztiirk et al. 2003; Ersel & Solak 2004; Sesli & Denchev 2005). Most of the above mentioned papers are floristic studies, lacking detailed descriptions or exsiccata.
The aim of the present study is to provide a better understanding of the biodiversity of the genus in Greece, concerning its morphology (including the range and importance of its variability), ecology and chorology. Collections have been made from various regions of central and southern continental Greece, mainly from coniferous and deciduous forests, as well as riparian and maquis vegetation.
The following ten taxa are identified and described in this work: C. applanatus var. subglobiger, C. autochthonus, C. calolepis, C. cesatii var. cesatii, C. epibryus, C. lundellii, C. luteolus, C. mollis, C. subverrucisporus and C. variabilis.
Materials and methods
Microscopical observations were made in bright field or phase contrast using a standard light transmission microscope. Sections of dried material were mounted in 3% KOH, with or without the addition of Phloxine. All measurements were made under 1000x magnification. At least 20 spores and 10 basidia and cheilocystidia were measured per specimen. The spores were measured from the surface of the pilei or from a spore deposit (when available). The spore sizes are given in approximation to 0.5 um, with extreme values given in parentheses, followed by the length-width ratio of the spores (Q). Habitat references in the descriptions refer exclusively to the collected material. Greek localities are transcribed into latin according to ISO 843: 1997 (E). Authorities’ abbreviations are in accordance to Authors of Fungal Names by Kirk & Ansell (1992). We have adopted the infrageneric classification proposed by Senn-Irlet (1995).
Material from other collectors or researchers (published or unpublished), was examined when available. The specimens collected from the authors are deposited in’ the Mycological Herbarium of the University of Athens (ATHU-M).
Taxonomic descriptions
Crepidotus (Fr.: Fr.) Staude 1857 Crepidotus subgenus Crepidotus
Crepidotus calolepis (Fr.) P. Karst. Figs la—b; 9b, d; 11g Crepidotus calolepis (Fr.) P. Karst., Bidr. Kann. Finl. Nat. Folk 32: 414 (1879); Crepidotus mollis var. calolepis (Fr.) Pilat, Ann. Hist. -Nat. Mus. Natl. Hung., n.s. 2B: 74 (1940); Crepidotus mollis ssp. calolepis (Fr.) Nordstein, Synopsis Fungorum (Oslo) 2: 67 (1990).
Pileus 10-70 mm, semicircular to flabelliform, convex to plano-convex, laterally or almost laterally attached to the substrate, with incurved and later even margin, surface viscid-sticky to dry, densely minutely tomentose-scaly with yellowish brown to brown
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fibrillose scales on a dirty whitish, cream to ochre-yellowish background, consistency tough, elastic. Lamellae whitish in young specimens, then spotted brownish and finally uniformly ochre-brown to cinnamon, moderately crowded, emarginately adnate, margin minutely fimbriate, remaining whitish. Stipe absent or rudimentary. Spore print yellowish brown.
Basidiospores 7.5-11.0 x 5.5-7.0 um, Q = 1.21-1.69, ellipsoid, amygdaliform in side view, smooth, yellowish brown in KOH, thick-walled, apex obtuse, depressed or occasionally mucronate; in some spores inner wall curving inwards at apex, resembling a callus or an indistinct germ pore when accompanied by a small apical depression (Figs la, 9b). Basidia 25-32 x 7-9 um, cylindrical-clavate, 4-spored. Cheilocystidia 25-65 x 4-14 um, clavate, cylindrical, irregularly cylindrical, somewhat fusiform to narrowly lageniform, sometimes branched, apex obtuse, often subcapitate (Fig. 1b), frequently embedded in gelatinous material entirely covering the lamellar edge. Pleurocystidia absent but pleurocystidioid-like bodies present in some specimens, clavate, with a short, apical or rarely lateral, finger-like protuberance (Fig. 11g). Lamellar trama often gelatinized. Pileipellis with an underlying layer of parallel hyphae, 3-5 um wide, hyaline, not encrusted, and an upper layer of parallel to somewhat ascending hyphae, 4-12 um wide, hyaline to pale ochraceous, with encrusting zebra-like pigment, amongst which ascending scale-forming hyphae, 4-15 um wide, brown to dark brown, thin- to somewhat thick-walled, short-celled, with strongly encrusting, zebra-like pigment (Fig. 9d), in some specimens with markedly large, plate-like encrustations. Pileal trama partly gelatinized, gelatinous layer underlying the pileipellis usually distinct, up to 200 um thick, but occasionally thin, rudimentary, and hence difficultly observed. Secretory hyphae occasionally present in pileipellis, pileal trama and lamellar trama, hyaline to golden yellow in KOH. Clamp connections absent in all tissues.
Habitat: Solitary to gregarious on standing or fallen trunks and branches of coniferous or deciduous trees.
Specimens examined — Mt. Taygetos, Messinia, on wood of Platanus orientalis, 29 Nov. 1968, Pantidou, ATHU-M 1071 (as C. mollis); Mt. Kandilio, Pagontas-Prokopi, Evvoia, forest of Pinus sp., on fallen branches of Pinus sp., 13 Dec. 1986, Gonou, ATHU- M 3781; Mt. Taygetos, Messinia, forest of Pinus nigra & Abies cephalonica, on fallen branches, 17 Nov. 1997, Gonou, ATHU-M 3782; Mt. Aroania, Zarouchla, Achaia, forest of A. cephalonica, on stump of A. cephalonica, 27 Nov. 1997, Delivorias, ATHU-M 3970; Mt. Parnitha, Attiki, forest of A. cephalonica, on fallen branches of A. cephalonica, 4 Dec. 1997, Gonou, ATHU-M 3783; Mt. Vardousia, Artotina, Fokida, forest of P. nigra & Abies sp., on fallen branches, 26 Sep. 1999, Gonou, ATHU-M 5107; Mt. Parnonas, Agios Petros, Arkadia, forest of Castanea sativa & Quercus sp., on fallen branches, 21 Nov. 1999, Gonou, ATHU-M 5105; river Agrafiotis, Epiniana, Evrytania, riparian vegetation, on living trunk and branches of Pl. orientalis, 15 Oct. 2000, Delivorias, ATHU-M 5122; Gardiki, Fthiotida, forest of Alnus glutinosa, 15 Oct. 2000, Dimou, 731; Ano Chora, Nafpaktia, Aitoloakarnania, dead trunk of Pl. orientalis, 22 Nov. 2001, Dimou, 988; Mt. Tymfristos, Agios Nikolaos, Evrytania, forest of Pl. orientalis, Quercus sp. & C. sativa, on trunk base of Pl. orientalis, 8 Nov. 2003, Gonou, ATHU-M 5108; Aetos, Messinia, on branches of PI. orientalis, 6 Dec. 2003, Kapsanaki, ATHU-M 5111; Mt. Liakoura, Granitsa, Evrytania, forest of Abies borisii-regis, on fallen twigs of A. borisii-regis, 27 Sep. 2004, Delivorias, ATHU-M 5161; Mt. Liakoura, Limeri, Evrytania, forest of P. nigra, on
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fallen branches of P. nigra, 13 Oct. 2004, Delivorias, ATHU-M 5174; Mt. Tymfristos, Agios Nikolaos, Evrytania, mixed forest of Pl. orientalis, Quercus frainetto, C. sativa and A. borisii-regis, on branches of PI. orientalis, 23 Oct. 2004, Gonou, ATHU-M 5169; Mt. Liakoura, Granitsa, Evrytania, forest road, on living branches of Pl. orientalis, 11 Noy. 2004, Delivorias, ATHU-M 5175; Mt. Liakoura, Granitsa, Evrytania, forest of A. borisii-regis and PI. orientalis, on fallen trunk of PI. orientalis, 11 Nov. 2004, Delivorias, ATHU-M 5178.
Remarks: C. calolepis is very closely related to C. mollis and is considered by some authors as a variety or subspecies of the latter (Pilat 1948; Nordstein 1990). Others (Singer 1973; Watling & Gregory 1989; Senn-Irlet 1995, Bandala & Montoya 2004) consider the two taxa distinct at a specific level. Both species are characterized by the presence of a gelatinous layer in the pileal trama, considered to be more developed in C. mollis and less developed or absent in C. calolepis. This, however, has not been considered a reliable distinguishing feature (Nordstein 1990, Senn-Irlet 1995). We have accepted the species-concept of C. calolepis as portrayed by Senn-Irlet (1995), who has performed the most detailed work on the European species of the genus. According to this concept, C. calolepis is distinguished from C. mollis by the somewhat broader basidiospores and the presence of yellowish brown, fibrillose scales on the pileal surface formed by brownish hyphae with encrusting pigment. The pileal surface of C. mollis is glabrous or with scattered innate fibrils that may form indistinct pale scales, but the hyphae of the pileipellis are not pigmented and never heavily encrusted.
Our collections include specimens with strongly fibrillose-scaly pilei and specimens with almost or completely glabrous pilei, as well as many transitional forms. We cross-examined the morphology of the pileal surface, the structure of the pileipellis and the spore dimensions and concluded that two distinct forms exist amongst our collections. The first form is characterized by whitish pilei, glabrous throughout or with few fibrillose scales at the centre, in which the pileipellis consists of hyaline to pale yellowish hyphae with minute encrustations and the spores are consistently narrower (only exceptionally exceeding 6 tm in width and never more than 6.5 um). The second form is characterized by yellowish to yellowish-brown, minutely to strongly fibrillose- scaly pilei, often throughout, in which the scale-forming hyphae of the pileipellis are consistently more or less strongly pigmented (yellowish brown to dark reddish brown — in KOH) and in all cases heavily encrusted, and the spores are broader (most exceeding 6 um and frequently reaching 7 um in width). We identified the former as C. mollis and the latter as C. calolepis.
The Mediterranean variety C. calolepis var. squamulosus (Cout.) Senn-Irlet is not clear to us. It is reported by Senn-Irlet (1995) to have slightly larger basidiospores than var. calolepis (8.5-12 x 6-7.5 um versus 7.5-10 x 5-7 um) and broader scale-forming hyphae (up to 22 um wide, instead of 14 um wide). The spore-size in our specimens holds an intermediate position between the two varieties, as, in most collections, a significant portion of the spores exceed 10 um in length, but we have not measured any spores larger than 11 um in length or 7 um in width. Also, we have not encountered any scale-forming hyphae broader than 15 um. Although the spores in our specimens are slightly larger than those reported by Senn-Irlet, this has been reported by other authors as well (Bandala & Montoya 2004) and cannot be considered a significant enough deviance to justify a distinction at a variety level. We have therefore concluded
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that all of our specimens must belong to a single taxon, i.e. var. calolepis. Lonati (1993) reports C. mollis var. squamulosus Cout. with a spore size of 7-10 x 5-6 (-6.5) um and scale-forming hyphae 3-8 tum wide. Judging on his description, Lonati’s C. mollis var. squamulosus must in fact be C. calolepis var. calolepis. Malencon & Bertault (1975) also report having found C. mollis var. squamulosus, but they distinguish it from C. calolepis on the grounds that the latter represents a small-sized species with a non-gelatinized or little-gelatinized pileipellis, features that are not considered diagnostic (Nordstein 1990). They make no reference to the spore-size or the structure of the pileipellis. It is unclear to us whether their C. mollis var. squamulosus represents C. calolepis var. calolepis or var. squamulosus.
An interesting deviant feature in our examined collections is that many basidiospores present a curving of the inner wall at the spore apex, with the outer wall either remaining obtuse and thus giving the impression of a callus-like formation, or having a small depression, resembling an indistinct germ pore (Figs la, c; 9a—b). This was constantly observed in all examined specimens of both C. mollis and C. calolepis and should be considered characteristic for the two species. Singer (1973) also reports similar characteristics. Senn-Irlet (1995) refers that in SEM analyses, spores of C. calolepis and C. mollis reveal a small apical depression, which may be interpreted as an apical thinning of the wall, yet neither a truncate spore apex or an apical thinning was visible under the light microscope. When describing in general the spores of the genus, Singer (1986) reports that the spores may occasionally have an indistinct callus or rarely an indistinct germ pore.
In some specimens of C. calolepis, as well as C. mollis, the lamellar trama and margin are distinctly gelatinous, the gelatinous material often covering the lamellar edge entirely. The presence of this material is possibly related to environmental humidity.
Differentiatied pleurocystidioid-like bodies were observed in the hymenium of some C. calolepis specimens, being more frequent near the margin. Singer (1986) reports that cystidioles may often be present on the sides of the lamellae in Crepidotus species and Hesler & Smith (1965) go as far as to acknowledge these elements as pleurocystidia. Senn- Irlet (1995) observed such bodies in C. cesatii and interpreted them as abnormalities induced by drought and therefore of no taxonomic significance. Apart from C. calolepis, we have also encountered such bodies in specimens of C. subverrucisporus. It is doubtful that these elements are of taxonomical significance in either case, as their presence is not constant and could not be correlated with any other deviant feature.
C. calolepis seems to be by far the most common representative of the genus in Greece, as we have collected it on several substrates from various locations. However, it was formerly recorded only by Maire & Politis (1940) on a stump of Pinus halepensis and dead trunks of Platanus sp., collections dating back to 1904 and 1906. On the other hand, the closely related C. mollis is recorded a number of times in the literature (Diapoulis 1939; Maire & Politis 1940; Pantidou 1991; Zervakis et al. 1998; Dimou et al. 2002a). We examined a collection of Pantidou (ATHU-M 1071), identified as C. mollis. It consists of a single, well-preserved specimen. The pileus is covered almost throughout with minute fibrillose scales and the scale-forming hyphae are yellowish brown to brown with strongly encrusting pigment. The spore dimensions fit accurately to our measurements of other specimens of C. calolepis. We have therefore concluded that this specimen formerly attributed to C. mollis in fact belongs to C. calolepis as here
20
satan naPeEree reo :
Fig. 1. a-b. C. calolepis: a. basidiospores, b. cheilocystidia, c-d. C. mollis: c. basidiospores, d. cheilocystidia. Scale bars = 10 um.
interpreted. We also examined three specimens collected by Dimou (pers. com.), two of which he had identified as C. mollis var. calolepis (731, 988, unpubl.), and one as C, mollis var. mollis (961, unpubl.), and we concur with his judgement. Typical forms of C. mollis and C. calolepis are easily distinguished from one another in the field, but we have encountered many non-typical specimens, transitional in appearance, that could easily be misidentified if not carefully examined under the microscope. The presence of fibrillose scales on the pileal surface can be variable in abundance, and specimens collected in wet weather often have apparently glabrous pilei, to the naked eye, as stressed by Bandala & Montoya (2004). It is our conviction that C. calolepis may have occasionally been mistaken for C. mollis in the past and is in fact much more common in Greece than the latter. Pilat (1948) states that C. calolepis is more common in dry areas or drier inland climates. This may explain the frequent occurrence of C. calolepis in Greece, a country with a drier climate in comparison to most European countries. Most authors (Malencon & Bertault 1975, Ortega & Buendia 1989, Watling & Gregory 1989, Nordstein 1990, Senn-Irlet 1995, Breitenbach & Kranzlin 2000, Krisai- Greilhuber et al. 2002) report either or both C. mollis and C. calolepis solely on wood of broad-leaved trees. Scarce reports exist from wood of coniferous trees (Maire & Politis
21
1940, Bandala & Montoya 2004). In Greece, C. calolepis has been reported on a stump of Pinus halepensis (Maire & Politis 1940), now it is newly recorded on branches of Abies cephalonica, Abies borisii-regis and Pinus nigra. Of the material collected in this work, seven collections of C. calolepis, as well as two collections of C. mollis, were found on wood of coniferous trees (Abies and Pinus), which have not been recorded as substrates of either species in Europe. It has also been recently collected on branches of Alnus glutinosa (Dimou, pers. com.). Furthermore, eight collections of C. calolepis were found on wood of Platanus orientalis, a host also not included in the substrates of this species for Europe (Senn-Irlet 1995).
Crepidotus mollis (Schaeff.: Fr.) Staude Figs 1c-d; 9a, c; 11b, d, k Crepidotus mollis (Schaeff.: Fr.) Staude, Schwamme Mitteldeutschl. 25: 71 (1857)
Pileus 10-50 mm, semicircular to flabelliform, convex to plano-convex, laterally or almost laterally attached to the substrate, with incurved and later even margin, surface viscid-sticky to dry, white to cream, glabrous to minutely fibrillose, forming scattered, indistinct fibrillose scales, more evident in dried specimens, consistency tough, elastic. Lamellae whitish in young specimens, then spotted brownish and finally uniformly ochre-brown to cinnamon, moderately crowded, emarginately adnate, margin minutely fimbriate, remaining whitish. Stipe absent or rudimentary.
Basidiospores 7.0-10.0 x 5.0-6.0 (-6.5) um, Q = 1.42-1.82, ellipsoid, amygdaliform in side view, smooth, yellowish brown in KOH, thick-walled, usually with one and less often two large oil drops as well as few small ones, apex often mucronate and thin-walled, in some spores inner wall folding inwards resembling a callus or an indistinct germ pore (Figs 1c, 9a). Basidia 12-30 x 6-9 um, cylindrical-clavate, 4-spored. Cheilocystidia 32-55 x 6-10 um, irregularly cylindrical, lageniform to slightly fusiform, apex obtuse, sometimes subcapitate, rarely branched or septate, frequently embedded in gelatinous material entirely covering the lamellar edge (Fig. 1d, 11b). Basidioles, basidia and cheilocystidia rarely with golden-yellow, smooth content (Fig. 11d). Lamellar trama seldom gelatinized. Pileipellis with an underlying layer of parallel hyphae, 3-5 um wide, hyaline, not encrusted, and an upper layer of hyaline to pale yellowish hyphae, 4-12 um wide, with granular or minutely encrusting pigment but never heavily encrusted or strongly pigmented (Fig. 9c). Pileal trama partly gelatinized, gelatinous layer underlying the pileipellis, usually well-developed. Secretory hyphae occasionally present in pileipellis, pileal trama and lamellar trama (Fig. 11k), scarce to abundant, hyaline to golden yellow in KOH. Clamp connections absent in all tissues.
Habitat: Gregarious on living or dead trunks and branches of Abies borisii-regis and Platanus orientalis.
Specimens examined — Mt. Zygourolivado, Pefkofyto, Karditsa, forest of Abies borisii- regis, on a fallen trunk of A. borisii-regis, 19 Sep. 1999, Delivorias, ATHU-M 5117; Mt. Zygourolivado, Pefkofyto, Karditsa, forest of A. borisii-regis, on a fallen trunk of A. borisii-regis, 17 Nov. 2001, Delivorias, ATHU-M 5123; Tatoi, Attiki, on fallen twigs of a deciduous tree, Dimou, 961; Mt. Tymfristos, Agios Nikolaos, Evrytania, mixed forest of Platanus orientalis, Quercus frainetto, Castanea sativa and A. borisii-regis, on branches of P. orientalis, 23 Oct. 2004, Gonou, ATHU-M 5170; Mt. Liakoura, Granitsa, Evrytania, forest of A. borisii-regis and P. orientalis, on living trunk and branches of P. orientalis, 11 Nov. 2004, Delivorias, ATHU-M 5177; Mt. Liakoura, Granitsa, Evrytania, forest of A.
22
borisii-regis and P. orientalis, on dead branches of P. orientalis, 11 Nov. 2004, Delivorias, ATHU-M 5180.
Remarks: The aforementioned collections are those we have encountered to fit C. mollis. For further notes on C. mollis and comparison with C. calolepis see remarks under C. calolepis.
Secretory hyphae were observed in abundance in the lamellar trama of basidiocarps from collection ATHU-M 5123. However, in basidiocarps of ATHU-M 5117, collected two years earlier from the same trunk, these hyphae were scarce, and in specimen 961 collected by Dimou, no secretory hyphae were found. The presence of these hyphae is most likely due to environmental conditions or the stage of maturity of the basidiocarps, and is of little or no taxonomical merit. Similar secretory hyphae were also observed in some specimens of C. calolepis.
C. mollis is newly recorded for Greece on Abies borisii-regis. According to the substrate’s list of the species in Europe (Senn-Irlet 1995), there is only one reference of the species from Platanus and none from conifers.
Crepidotus subgenus Dochmiopus (Pat.) Pilat, 1948 Section Dochmiopus
Crepidotus applanatus var. subglobiger Singer Figs 2a-d; 10h; 11h-j Crepidotus applanatus var. subglobiger Singer, Nova Hedwigia Beih. 44: 478 (1973)
Pileus 5-30 mm, semicircular, flabelliform to petaloid, convex to plano-convex, laterally attached to the substrate, margin incurved to even, hygrophanous, translucently striate at margin when wet, surface smooth, somewhat felty at point of attachment, pure white to pale cream, becoming yellowish brown in dried specimens. Lamellae whitish in young specimens, then buff to snuff brown, moderately distant, adnate to decurrent, margin minutely fimbriate, remaining whitish (observed under lens). Stipe rudimentary, with white tomentum on the substrate.
Basidiospores (5.0-) 5.5-6.5 (-7.0) x (5.0—) 5.5-6.0 (7.0) um, Q = 1.00-1.20, globose to subglobose, yellowish brown in KOH, with pinkish content, finely verrucose, with a perispore (Figs 2a, 10h). Basidia 18-26 x 6-8 tm, cylindrical-clavate, 4-spored, with. basal clamp, content with numerous small oil drops. Cheilocystidia 45-110 x 5-12 tum, variable, typically cylindrical to narrowly lageniform, rather often subcapitate or curved at apex, some branched, some septate, in some cases with two septa on the same cystidium, hyaline, thin-walled but rather often thick-walled at the medial part, in some cystidia markedly so, up to 3 um thick (Fig. 2b, d; 11h-j). Pleurocystidia absent but abundant pleurocystidioid-like bodies present, 14-22 x 5-6 um, irregularly cylindrical to clavate, often curved, twisted or constricted, frequently with an apical to lateral, finger-like protuberance (Fig. 2c). Pileipellis a cutis, hyphae 4-8 um wide, hyaline or with pale yellowish, diffuse to somewhat granular intracellular pigment, occasional hyphal ends exerting as pileocystidia, 30-70 x 6-9 um, narrowly lageniform, cylindrical to subcapitate, hyaline. Secretory hyphae present in pileipellis, pileal trama and lamellar trama. Clamp connections present in all tissues.
Habitat: Scattered on a rotten fallen trunk of Picea abies.
23
OOO OO ©
a
js28
- oy re Ce ee
ee Sey,
Fig. 2. C. applanatus var. subglobiger: a. basidiopores, b, d. cheilocystidia, c. pleurocystidoid bodies. Scale bars = 10 um. Specimens examined — W. Rodopi Mts., Elatia, Drama, alt. 1550 m, on a fallen trunk of Picea abies, 5 Oct. 2005, Gonou & Floudas, ATHU-M 5332.
Remarks: C. applanatus var. subglobiger is distinguished from the typical variety by the shape of the cheilocystidia which are longer, narrowly lageniform to cylindrical instead of clavate to capitate. Furthermore, var. applanatus prefers hardwoods, whereas var. subglobiger seems to be restricted to coniferous wood.
We have collected this taxon only once, from a Picea forest in Northern Greece. Microscopic examination revealed a few deviant features, such as the presence of septa on many cystidia, and even, in many occasions, two septa on the same cystidium. This was not a constant feature however, as showed by examination of different lamellar margins from the same basidiocarp. In one lamella the cheilocystidia were almost predominately septate, whereas in a nearby lamella the septate cystidia were scarce to almost absent. Also, many cystidia with a markedly thick-walled medial part were encountered, with the remaining cystidium being thin- to slightly thick-walled. Finally, we observed many branched cystidia at the apices, either forked or laterally branched, in most cases with
24
two, rarely three, branches. It was suprisingly difficult to find basidia, although most specimens were fully mature, with abundant basidiospores in all preparations. We encountered instead many basidioles and pleurocystidioid bodies, these most probably being abnormally developed basidia. All the above mentioned abnormalities, if they be such, might be induced by environmental conditions.
Hesler & Smith (1965) described two varieties of C. applanatus based on the morphology of the cheilocystidia: var. phragmocystidiosus with septate cystidia and var. diversus with branched or knobbed cystidia. These varieties are considered conspecific by Aime (2001) with C. applanatus s. Joss. She concludes, after detailed examination, that cheilocystidia in this taxon are, under the influence of environmental conditions, capable of secondary growth that can alter their shape and size as well as the number of septa per cystidium. This, however, does not seem to be the case in our specimens, as the septate and non-septate cystidia are morphologically similar. We agree, nevertheless, with Aime’s deduction that taxonomic delineation in Crepidotus cannot be based on cystidial morphology alone, as the form of the cheilocystidia may vary greatly within a single taxon and, as observed in this case, even within individual collections.
C. applanatus has been reported twice from Greece, from a fallen trunk of Abies borisii-regis (Diamandis & Perlerou 1990) and from dead branches of Fagus (Diamandis 1992). The latter collection is accompanied by a description, but with no reference to the morphology of the cheilocystidia. Judging by the habitat, the first collection might be var. subglobiger and the second var. applanatus but the authors make no such distinction.
C. applanatus var. subglobiger is newly recorded for Greece.
Crepidotus cesatii var. cesatii (Rabenh.) Sacc. Figs 3a—-b; 9e Crepidotus cesatii (Rabenh.) Sacc., Michelia 1: 2 (1877); Dochmiopus sphaerosporus (Pat.) Pat., Hyménomyc. Eur.: 113 (1887); Crepidotus sphaerosporus (Pat.) J.E. Lange, Dansk Bot. Ark. 9 (6): 52 (1938); Crepidotus cesatii var. sphaerosporus (Pat.) A. Ortega & Buendia, Int. J. Myc. Lich. 4 (1-2): 96 (1989)
Pileus 3-22 mm, circular to semicircular or roundedly flabelliform, rarely somewhat lobed, convex to plano-convex, centrally to eccentrically or almost laterally attached to the substrate, margin incurved, becoming even only in fully mature specimens, surface dry, felty, pure white, remaining so in dried specimens or becoming pale cream. Lamellae whitish in young specimens, often with a pinkish tint, later cream, pinkish’ buff to pale cinnamon, never significantly darker, distant to subdistant, adnate, margin minutely fimbriate, remaining whitish. Stipe absent or rudimentary.
Basidiospores (5.5-) 6.5-8.0 (-9.0) x 4.5-7.0 um, Q = (1.00-) 1.10-1.33 (-1.46), globose, subglobose to broadly ellipsoid, pale yellowish in KOH, finely echinulate (Figs 3a, 9e). Basidia 20-37 x 6-9 um, cylindrical-clavate, 4-spored. Cheilocystidia 28-80 x 4~11 um, diverticulate, clavate, cylindrical, irregularly cylindrical, fusiform, lageniform, usually branched, frequently multiply so, apices obtuse, hyaline, thin-walled (Fig. 3b). Pileipellis a trichodermium of loosely interwoven hyphae with transitions to a loose cutis, hyphae often coiled, 3-6 um wide, hyaline, thin-walled. Clamp connections present in all tissues.
Habitat: Solitary or in small groups on dead or living branches of Platanus orientalis and, in one case, Pinus nigra.
2D
Fig. 3. C. cesatii var. cesatii: a. basidiopores, b. cheilocystidia. Scale bars = 10 um.
Specimens examined — Mt. Zygourolivado, Anthochori, Karditsa, riparian vegetation, on a living branch of Platanus orientalis, 19 Sep. 1999, Delivorias, ATHU-M 5116; Mt. Katachloro, Kedros, Karditsa, riparian vegetation, on branches of Pl. orientalis, 13 Nov. 1999, Delivorias, ATHU-M 5118; Mt. Tymfristos, Raches Tymfristou, Evrytania, forest of Pinus nigra and Abies borisii-regis, on branches of P. nigra, 25 Oct. 2003, Delivorias, ATHU-M 5125; Mt. Tymfristos, Agios Nikolaos, Evrytania, forest of Pl. orientalis, Quercus sp. & Castanea sativa, on fallen twigs and branches of Pl. orientalis, 8 Nov. 2003, Gonou, ATHU-M 5109; Mt. Liakoura, Granitsa, Evrytania, forest of A. borisii- regis and Pl. orientalis, on dead twigs and branches of PI. orientalis, 27 Sep. 2004, Delivorias, ATHU-M 5162; Mt. Tymfristos, Agios Nikolaos, Evrytania, mixed forest of Pl. orientalis, Quercus frainetto, C. sativa and A. borisii-regis, on branches of PI. orientalis, 23 Oct. 2004, Gonou, ATHU-M 5173; Mt. Liakoura, Granitsa, Evrytania, forest road, on living branches of PI. orientalis, 11 Nov. 2004, Delivorias, ATHU-M 5176; Mt. Liakoura, Granitsa, Evrytania, forest of A. borisii-regis and Pl. orientalis, on dead branches of PI. orientalis, 11 Nov. 2004, Delivorias, ATHU-M 5179.
Remarks: The typical variety of C. cesatii is characterized by the distant lamellae, the globose to subglobose, finely echinulate basidiospores, the diverticulate cheilocystidia and the coiled hyphae of the pileipellis. The only other variety recognized by Senn- Irlet (1995), C. cesatii var. subsphaerosporus (J.E. Lange) Senn-Irlet, is reported to have broadly ellipsoid basidiospores, mostly straight hyphae on the pileipellis and to grow on branches of coniferous trees. Also, Watling & Gregory (1989), report that the latter variety lacks the characteristic pink tinge on the lamellae and has a darker spore print, although Senn-Irlet does not make such a reference. In all specimens examined in this work, at least a portion of the basidiospores were found to be broadly ellipsoid, and, in some specimens, these spores predominate. However, in all specimens the hyphae of the pileipellis were clearly coiled and the only collection made from coniferous trees does not seem to deviate microscopically from the remaining collections. As we have been unable to determine a correlation between the variation in shape of the basidiospores and either the structure of the pileipellis, the colour of the lamellae or the habitat, we have concluded that our findings consist of a single taxon, C. cesatii var. cesatii, in which
26
the basidiospores may range from perfectly globose to broadly ellipsoid in their extreme variation, with a Q ratio reaching up to 1.46.
C. cesatii var. cesatii is newly recorded for Greece on twigs and branches of Pinus nigra and Platanus orientalis, the latter seeming a rather common substrate for the species in Greece, in contrast to the references for the distribution of the species in Europe (Senn-Irlet 1995).
Crepidotus variabilis (Pers.: Fr.) P. Kumm. Figs 4a-b, 10g Crepidotus variabilis (Pers.: Fr.) P. Kumm., Fuhr. Pilzk.: 74 (1871)
Pileus 5-15 mm, circular to semicircular or roundedly flabelliform, often lobed, convex to plano-convex, centrally to eccentrically or almost laterally attached to the substrate, with incurved and later even margin, surface dry, felty to smooth, pure white to dirty white, remaining so in dried specimens. Lamellae whitish in young specimens, often with a pale pinkish tint, later clay buff and finally cinnamon brown, moderately crowded to crowded, emarginately adnate, margin minutely fimbriate, remaining whitish. Stipe absent or rudimentary.
Basidiospores (5.0-) 5.5-7.0 (-7.5) x (2.5-) 3.0-3.5 (-4.0) um, Q = 1.57-2.17, short cylindric to elongate, oblong, pale yellowish in KOH, minutely but distinctly punctate-warty (Figs 4a, 10g). Basidia 20-25 x 5-7 um, cylindrical-clavate, 4-spored. Cheilocystidia 22-53 x 6-11 um, diverticulate, clavate, cylindrical, irregularly cylindrical, fusiform, mostly branched, often multiply branched, hyaline, thin-walled (Fig. 4b). Pileipellis a trichodermium of loosely interwoven hyphae, 2-5 um wide, hyaline, thin-walled. Clamp connections present in all tissues.
Habitat: Solitary to gregarious on branches of Quercus frainetto, Quercus coccifera and Cistus sp. Specimens examined — lake Plastira, Agios Athanasios, Karditsa, forest of Quercus frainetto, on fallen branches of Q. frainetto, 1 Nov. 1998, Delivorias, ATHU-M 5112; lake Plastira, Agios Athanasios, Karditsa, forest of Q. frainetto, on fallen branches of Q. frainetto, 1 Nov. 1998, Delivorias, ATHU-M 5113; lake Plastira, Agios Athanasios, Karditsa, forest of Q. frainetto, on fallen branches of Q. frainetto, 18 Sep. 1999, Delivorias, ATHU-M 5114, Domokos, Fthiotida, maquis vegetation, on branches of Quercus coccifera, 13 Nov. 1999, Delivorias, ATHU-M 5115; Mt. Ymittos, Attiki, maquis vegetation, on twigs of Cistus sp., 7 Dec. 2002, Dimitriadis, ATHU-M 4648.
Remarks: C. variabilis is characterized by the lobed pileal margin (not always distinct, however), the small-sized, cylindrical, punctate-warty basidiospores and the diverticulate cheilocystidia. The lobed pileal margin may be a good distinctive feature for macroscopical identification when clearly formed, but, as in all white species of Crepidotus, careful microscopical examination is essential for identification. The characteristic small-sizéd, cylindrical basidiospores provide a reliable distinguishing feature. C. variabilis var. trichocystis Hesler & A.H. Sm. is reported to have larger basidiospores and longer, narrowly cylindrical to narrowly lageniform cheilocystidia (Senn-Irlet 1995).
C. variabilis seems to be common in Greece, as it is reported a number of times in the literature (Maire & Politis 1940, as Dochmiopus variabilis; Minter 1988; Avtzis & Diamandis 1988; Konstantinidis 2002) and we have collected it a few times ourselves.
oh
O8 JOU a an (SCPE IF a |
Fig. 4. C. variabilis: a. basidiospores, b. cheilocystidia. Scale bars = 10 um.
It has also been recently collected on branches of Alnus glutinosa and Quercus coccifera (Dimou pers. com.). It is newly recorded for Greece on branches of Quercus frainetto, Q. coccifera and twigs of Cistus sp., the last two being representative plants of the maquis vegetation.
Section Crepidotellae Hesler & A.H. Sm., 1965 Subsection Autochthoni Senn-Irlet, 1995
Crepidotus autochthonus J.E. Lange Figs 5a-b, 9f, 11c, e Crepidotus autochthonus J.E. Lange, Dansk bot. Ark. 4 (6): 51 (1938)
Pileus 10-40 mm, semicircular to flabelliform, convex to plano-convex, laterally or almost laterally attached to the substrate, with incurved, later even to undulating margin, surface dry, glabrous to minutely fibrillose-tomentose, dirty whitish, cream to yellowish buff. Lamellae whitish in young specimens, then spotted brownish and finally uniformly cinnamon brown to fulvous, crowded, emarginately adnate, margin even. Stipe absent or rudimentary. Spore print yellowish brown to umber.
Basidiospores 7.0-9.0 x 5.0-6.0 um, Q = 1.27-1.64, ellipsoid, amygdaliform or lemoniform in side view, with a more or less acute apex, smooth, thick-walled, occasionally wall thinning at acute apex, yellowish to yellowish brown in KOH, usually with a large oil drop (Figs 5a, 9f). Basidia 25-30 x 7-9 um, cylindrical-clavate, with 4 sterigmata. Cheilocystidia 17-32 x 7-13 um, cylindrical, clavate, some subcapitate, short lageniform, not branched, rarely septate, thin to thick-walled (Figs 5b, 11c). Basidioles and cheilocystidia sometimes with yellow-golden, smooth content (Fig. lle). Pileipellis a cutis of hyaline hyphae, 3-5 um wide, some ascending. Pileal trama without gelatinous layer. Lamellar trama with few secretory hyphae, usually hyaline, seldom golden-yellow. Clamp connections present in all tissues.
Habitat: Gregarious or in small groups on ground, in forest of Quercus frainetto or mixed Q. frainetto and Abies borisii-regis.
28
Fig. 5. C. autochthonus: a. basidiospores, b. cheilocystidia. Scale bars = 10 um.
Specimens examined — Lake Plastira, Kryoneri, Karditsa, clearing of forest of Quercus frainetto, on ground, 14 Oct. 2000, Delivorias, ATHU-M 5121; lake Plastira, Kastania, Karditsa, mixed forest of Q. frainetto and Abies borisii-regis, on ground, 7 Sep. 2002, Delivorias, ATHU-M 5124.
Remarks: C. autochthonus is rather similar-looking macroscopically to C. mollis and has practically identical basidiospores, which nevertheless can be distinguished by their acute apex, without the characteristic wall curving or apical depression of C. mollis spores. It is also easily identifiable by the terrestrial habit, the lack of a gelatinous layer in the pileal trama, the shape of the cheilocystidia and the presence of clamp-connections. According to Senn-Irlet (1995), it is the only terrestrial species of Crepidotus in Europe.
C. autochthonus is newly recorded from Greece and is reported for the first time in Europe in forests of Quercus spp.
Subsection Pleurotellus (Fayod) Senn-Irlet, 1995
Crepidotus epibryus (Fr.: Fr.) Quél. Figs 6a-b; 10i; 11f Crepidotus epibryus (Fr.: Fr.) Quél., Mém. Soc. Emul. Montbéliard, sér. 2, 5: 138 (1872); misdet.: Crepidotus perpusillus (Lumn.: Fr.) Maire, Fungi Catal. I: 102 (1937)
Pileus up to 10 mm, rounded flabelliform or campanulate when young, circular with age, spreading out on the substrate, becoming almost resupinate, with white tomentum around the margin where attached, sessile, surface tomentose, white, even when dried. Lamellae rather distant to moderately crowded, adnexed, whitish to pale ochraceous in fresh specimens, remaining so or darkening to fulvous in dried ones, margin concolorous, even to slightly uneven, often browning at places, especially when dried. Flesh very thin, white. Stipe absent.
Basidiospores 6.0-9.0 x 2.5-3.0 um, cylindrical, somewhat fusoid to narrowly amygdaliform or pip-shaped, some slightly curved, smooth, hyaline or pale yellow in KOH, yellow in the commonly formed masses (of two, four or more) (Figs 6a, 10i). Basidia 15-20 x 5-6 um, clavate, 4-spored, usually hyaline; some disintegrating basidioles and
£9
basidia with granular, golden brown content, larger in size (Fig. 11f). Cheilocystidia up to 50 x 7 um, cylindrical to narrowly lageniform, flexuous, sometimes with strongly curved or whirled apex, rarely branched, often difficult to be observed (Fig. 6b). Parts of the hymenium covered with golden-brown granular material. Pileipellis a transition between a cutis of interwoven hyphae and a trichodermium of erect, straight, filiform, hyaline hyphae. Clamp connections absent in all tissues.
Habitat: Solitary on stalks and laminas of fallen leaves of Castanea sativa.
Specimens examined: Mt. Tymfristos, Agios Nikolaos, Evrytania, mixed forest of Castanea sativa, Abies borisii-regis, Quercus sp. and Corylus avelana, on fallen leaves of C. sativa, 14 Nov. 1998, Gonou, ATHU-M 5106.
Remarks: Distinctive microscopical characters of C. epibryus are the size and shape of the basidiospores as well as the narrowly lageniform, flexuous and/or curled at the apex cheilocystidia. The cheilocystidia were scarce and difficult to find, possibly because the lamellar edge had been injured. The golden-brown material covering parts of the hymenium is probably the result of the excretion of necropigments from the concolorous disintegrating basidia and basidioles and corresponds macroscopically to the browning spots of the lamellae. Similar pigmented and amorphous aggregations on the hymenium are reported from species of Lyophyllum as well as pigmented basidia from species of Inocybe, Cortinarius and Pholiota (Clémencon 1997).
C. epibryus has been twice reported from Greece, on leaves of Fagus sylvatica (as Crepidotus perpusillus, Maire & Politis 1940) and on leaves of Quercus cerris (Polemis et al. 2002). Our collection is the first recording of C. epibryus on leaves of Castanea sativa in Greece and one of the very few in Europe (Senn-Irlet 1995).
BON (72 O( ASO
Fig. 6. C. epibryus: a. basidiospores, b. cheilocystidia. Scale bars = 10 um.
ae
30
Subsection Fibulatini Singer, 1947
Crepidotus lundellii Pilat Figs 7a-b; 10a, d Crepidotus lundellii Pilat, Fungi Exsiccati Suecici fasc. V-VI: 10 (1936)
Pileus 3-20 mm, circular to semicircular or roundedly flabelliform, convex to plano- convex, eccentrically to almost laterally attached to the substrate, with incurved and later even margin, surface dry, felty, pure white to pale cream. Lamellae whitish in young specimens, then clay buff and finally cinnamon brown to fulvous, moderately crowded to crowded, emarginately adnate, margin minutely fimbriate, remaining whitish. Stipe absent or rudimentary.
Basidiospores 6.5-8.5 (—9.0) x 4.5-5.5 (-6.0) um, Q = 1.40-1.80, in frontal view broadly ovoid to ellipsoid, in side view ellipsoid to slightly amygdaliform, yellowish in KOH, wall slightly roughened, almost smooth, frequently seemingly smooth even under oil immersion but never actually completely smooth (Figs 7a, 10a). Basidia 21-30 x 6-8 um, cylindrical-clavate, 4-spored. Cheilocystidia 32-56 x 7-12 um, clavate, cylindrical, fusiform, with an obtuse, sometimes subcapitate apex, rarely branched, hyaline, thin- walled (Figs 7b, 10d). Pileipellis a trichodermium of loosely interwoven hyphae 3-5 um wide, hyaline, thin-walled. Clamp connections present in all tissues.
Habitat: Gregarious on fallen branches of Platanus orientalis.
Fig. 7. C. lundellii: a. basidiospores, b. cheilocystidia. Scale bars = 10 um.
Specimens examined — Mt. Katachloro, Kedros, Karditsa, riparian vegetation, on fallen branches of Platanus orientalis, 13 Nov. 1999, Delivorias, ATHU-M 5119; Mt. Tymfristos, Agios Nikolaos, Evrytania, mixed forest of P. orientalis, Quercus frainetto, Castanea sativa and Abies borisii-regis, on branches of P. orientalis, 23 Oct. 2004, Gonou, ATHU-M 5171.
Remarks: C. lundellii is characterized by the unique ornamentation of the basidiospores, consisting of very low warts and ridges. The spores seem smooth in low magnification
eM
and the ornamentation is revealed only under oil immersion. The basidiospores are ellipsoid to slightly amygdaliform and faint yellowish in KOH. In the macroscopically similar C. subverrucisporus, the spore ornamentation is much more distinct, the spore colour is darker and the cheilocystidia are more consistently narrowly lageniform (apically tapered) (Senn-Irlet 1995; Bandala et al. 1999).
C. lundellii is newly recorded for Greece and is reported for the first time on Platanus in Europe (Senn-Irlet 1995). It has also been recently collected on branches of Alnus glutinosa (Dimou pers. com.).
Crepidotus luteolus (Lambotte) Sacc. Figs 8a-b; 10b, e Crepidotus luteolus (Lambotte) Sacc., Syll. Fung. (Abellini) 5: 888 (1887)
Pileus 2-15 mm, sessile, young ungulate, campanulate, later convex to plano-convex, flabelliform, reniform or semicircular when seen from above, laterally or dorsally attached, often spread out over the substrate, almost resupinate, margin even, straight, remaining so or becoming undate, lobate, white-yellowish to yellowish-cream when wet, straw to buff when dried, surface first tomentose-hirsute with a smooth margin, later smooth throughout or hirsute only near the point of attachment, usually with a rich tomentum on the substrate. Lamellae adnexed to narrowly adnate, moderately crowded, whitish or pale yellowish at first, later buff to buffish brown, fulvous when dried, margin whitish, fimbriate when young, concolorous and almost smooth when mature. Flesh white. Stipe absent or rarely observed in very young fruit bodies.
Basidiospores 8.0-9.5 (—10.5) x 4.5-5.5 (-6.0) um, Q = 1.55-2.10, ellipsoid in frontal view, ellipsoid to usually amygdaliform in side view, yellowish brown in KOH, minutely roughened (Figs 8a, 10b). Basidia 20-30 x 7-9 um, clavate, usually strongly granular, 4-spored. Cheilocystidia 45-60 x 5-8 um, cylindrical to narrowly lageniform, strongly flexuous, often branched or rarely angular, hyaline, thin walled (Figs 8b, 10e). Lamellar edge in some sections somewhat gelatinous, holding cheilocystidia rather packed. Pileipellis either a cutis of interwoven hyphae with transitions to a trichodermium bearing bundles of exerted hyphal ends, or a real trichodermium with erect hyphae, hyphae straight, flexuous or winding, thin to slightly thick walled, hyaline or pale yellow, some of the latter slightly encrusted. Clamp connections present in all tissues.
Habitat: Solitary or in small groups, rather gregarious, on fallen twigs of Platanus orientalis.
Specimens examined — Mt. Tymfristos, Agios Nikolaos, Evrytania, mixed deciduous forest of Platanus orientalis, Quercus sp. and Castanea sativa, on fallen twigs of P. orientalis, 8 Nov. 2003, Gonou, ATHU-M 5110.
Remarks: C. luteolus is characterized macroscopically by the yellowing colors of the basidiocarps and microscopically by the faintly ornamented, amygdaliform, relatively long and narrow basidiospores as well as the polymorphic, flexuous, frequently branched cheilocystidia. Our specimens of C. subverrucisporus exhibit the same yellowing colors when fresh and could be confused macroscopically with those of C. luteolus. The two species can be distinguished microscopically by their basidiospores and cheilocystidia. The basidiospores of C. subverrucisporus differ in being more ornamented and ellipsoid (broader) rather than amygdaliform in side view, while the cheilocystidia are less flexuous and branched.
ey)
Fig. 8. a-b. C. luteolus: a. basidiospores, b. cheilocystidia; c-d. C. subverrucisporus: c. basidiospores, d. cheilocystidia. Scale bars = 10 um.
It is worth mentioning that besides C. luteolus, specimens of C. cesatii were collected on the same day from the same locality, on nearby fallen twigs of Platanus orientalis. Basidiocarps of C. luteolus exhibited a pale yellow color on the pileus in contrast to the whitish color of C. cesatii.
C. luteolus is newly recorded from Greece and is reported for the first time on Platanus in Europe. It has also been recently collected on Nerium oleander (Dimou pers. com.)
Crepidotus subverrucisporus Pilat Figs 8c-d; 10c, f; lla Crepidotus subverrucisporus Pilat, Studia Botanica Cechoslavaca 10: 151 (1949)
Pileus 3-10 mm, circular to semicircular or roundedly flabelliform, convex to plano- convex, eccentrically to almost laterally attached to the substrate, where usually with a whitish or yellowish tomentum, margin incurved and later even, surface dry, felty, white
33
or dirty whitish to pale yellowish cream. Lamellae whitish in young specimens, later buff and finally cinnamon brown to fulvous, moderately crowded, emarginately adnate, margin minutely fimbriate, remaining whitish. Stipe absent.
Basidiospores (7.0—) 7.5-10.0 (—11.0) x (4.5-) 5.0-6.0 (—7.0) um, Q = 1.45-1.78, ovoid to ellipsoid, slightly amygdaliform in side view, yellowish brown in KOH, minutely but distinctly rugulose (Figs 8c, 10c). Basidia 20-25 x 7-8 um, cylindrical-clavate, 4-spored, some 2-spored. Cheilocystidia 28-65 x 4-10 x 3-5 um, cylindrical, broadly lageniform to narrowly, elongate lageniform, often angular or sometimes branched towards the apex or apex subcapitate, sometimes septate at upper 1/3, hyaline, thin-walled (Figs 8d, 10f, 11a). Pleurocystidia absent but seldom lageniform pleurocystidioid bodies present. Basidioles and pleurocystidioid bodies sometimes with golden-yellow content. Pileipellis a trichodermium with transitions to a cutis, hyphae loosely interwoven, 3-6 tum wide, hyaline, thin-walled. Clamp connections present in all tissues.
Habitat: Gregarious on fallen branches of Platanus orientalis. Specimens examined — Mt. Katachloro, Kedros, Karditsa, riparian vegetation, on fallen branches of Platanus orientalis, 13 Nov. 1999, Delivorias, ATHU-M 5120; Mt. Tymfristos, Agios Nikolaos, Evrytania, mixed forest of P. orientalis, Quercus frainetto,
Castanea sativa and Abies borisii-regis, on mossy branches of P. orientalis, 23 Oct. 2004, Gonou, ATHU-M 5172.
Remarks: The fresh fruit bodies from both collections exhibited a pale yellowish color in the pileus which was retained in the exsiccata. C. subverrucisporus is characterized by the minutely but distinctly rugulose, slightly amygdaliform, moderately dark coloured basidiospores and the rather simple lageniform cheilocystidia. The cheilocystidia are reported to be generally unbranched (Watling & Gregory 1989; Senn-Irlet 1995; Breitenbach & Kranzlin 2000). Type studies performed by Senn-Irlet (1993) showed that the cheilocystidia in the holotype (found on Robinia pseudoacacia) are often septate (also observed by Bandala et al. 1999) and sometimes branched. The same observation was made by Senn-Irlet (1995) on material collected from Italy, also on Robinia. We have encountered in our specimens both septate and branched at the tips cystidia.
We have also occasionally observed pleurocystidioid-like bodies, some with a golden-yellow content, but they are probably of no taxonomic significance. Bandala et al. (1999) also report sterile basidiole-like elements rarely present on the sides of the lamellae and consider them probably to be abnormal basidia or basidioles.
C. subverrucisporus is newly recorded from Greece and is reported for the first time on Platanus in Europe.
Discussion
With few exceptions, such as C. calolepis or C. cinnabarinus, the species of Crepidotus are ~ more or less macroscopically similar, i.e. whitish, small to medium sized basidiocarps, with whitish to pale brownish lamellae and without a distinctive smell or taste. Hence, careful microscopic examination is absolutely essential for determination at a species level. The most important microscopic features from a taxonomical standpoint are the morphology of basidiospores, cheilocystidia and, to a lesser degree, pileipellis, and the
34
presence or absence of clamp connections (Nordstein 1990; Senn-Irlet 1995; Bandala & Montoya 2004). In few species other features are also of taxonomical importance, such as the presence of a gelatinous layer in the pileal trama in C. calolepis and C. mollis, the distant, pinkish lamellae in C. cesatii, or some striking habitat preferences, such as the growth on stems, leaves or litter in C. epibryus, or directly on soil in C. autochthonus. In general, however, habitat preferences are an indicative and not a decisive taxonomical feature. C. calolepis and C. mollis are considered by most authors to grow exclusively on deciduous trees, but we have collected both species on conifers in more than a few occasions. C. cesatii var. cesatii is considered to prefer deciduous trees whereas var. subsphaerosporus coniferous trees, but the decisive distinctive feature between the two varieties is the shape of the basidiospores and not the habitat. We have collected specimens of C. cesatii on both deciduous and coniferous trees, but without any notable microscopic differences between them.
We noted many deviant features in our specimens, such as a callus-like structure at the apex of the basidiospores in C. calolepis and C. mollis, pleurocystidioid-like bodies in C. calolepis, C. applanatus var. subglobiger and C. subverrucisporus, septate cheilocystidia in C. mollis, C. applanatus var. subglobiger, C. autochthonus and C. subverrucisporus, thick-walled cheilocystidia in C. applanatus var. subglobiger, basidia, basidioles and cheilocystidia with pigmented content in C. mollis, C. autochthonus and C. subverrucisporus, amorphous material on the hymenium in C. epibryus, gelatinized lamellar trama in C. calolepis and C. mollis, and secretory hyphae in many species. The presence of the callus-like structure in C. calolepis and C. mollis, although sporadic, is constant and should be considered characteristic for the two species. Singer (1973) also mentioned such features. All the other features have an inconstant appearance and are due most probably to environmental conditions or different stages of maturity, and therefore no taxonomic value can be attributed to them.
The determination of the diversity and distribution of Crepidotus species is not an easy task, as they usually form small-sized basidiocarps that may be overlooked. Prior to this work, 7 taxa of Crepidotus were reported from Greece: C. applanatus (Diamandis & Perlerou 1990; Diamandis 1992), C. cesatii (Maire & Politis 1940, as Dochmiopus sphaerosporus, Dimou et al. 2002a, as Crepidotus sphaerosporus), C. calolepis (Maire & Politis 1940), C. cinnabarinus (Dimou et al. 2002b), C. epibryus (Maire & Politis 1940, as Crepidotus perpusillus; Polemis et al. 2002), C. mollis (Diapoulis 1939; Maire & Politis 1940; Pantidou 1991; Zervakis et al. 1998; Dimou et al. 2002a) and C. variabilis (Maire & Politis 1940; Minter 1988; Avtzis & Diamandis 1988; Diamandis 1992; Konstantinidis 2002). Most of these taxa were reported only once or twice, with the exceptions of the seemingly common C. variabilis and C. mollis. Concerning the latter, as stated in this work, it is possible that some collections attributed to C. mollis may in fact represent collections of C. calolepis. Some older collections (Diapoulis 1939; Maire & Politis 1940) cannot be accounted for and probably have not survived to this day. We have examined collection ATHU-M 1071 identified as C. mollis (Pantidou 1991) and have attributed it to C. calolepis, and have also cross-examined two specimens of C. calolepis and one of C. mollis, kindly provided to us by Dimou. We are convinced that C. calolepis is much more common in Greece than C. mollis.
35
Crepidotus species never form the dominant element in any European vegetation unit (Senn-Irlet 1995), and this obviously applies for Greece as well. Most of the gathered collections consist of rather few individuals. Few of the collected species were found to grow gregariously, i.e. C. calolepis, C. mollis, C. autochthonus and, to a lesser degree, C. luteolus and C. subverrucisporus. However, in some occasions we collected more than a few different species from the same location. Seven taxa: C. calolepis, C. mollis, C. lundellii, C. subverrucisporus, C. cesatii var. cesatii, C. luteolus and C. epibryus, were collected from a small area on Mt. Tymfristos (Agios Nikolaos), the first five on the same day. In addition, C. calolepis and C. cesatii were collected in two successive years from that area. Three of the similar-looking white species, C. cesatii, C. lundellii and C. subverrucisporus, were collected on the same day from the same location (Mt. Katachloro). In one case, basidiocarps of two species, C. cesatii and C. calolepis, were found on nearby branches of the same Platanus orientalis, a few meters apart from a second Platanus colonized by C. mollis (Mt. Liakoura). We have not, however, encountered more than one species on the same branch. Senn-Irlet (1995) states that in no case had she encountered basidiocarps of more than one Crepidotus species on the same substrate.
Floristic studies in other countries of the Mediterranean region reveal a similar diversity of Crepidotus species as in Greece. Senn-Irlet (1995) provides data for most countries surrounding the Mediterranean, such as Bulgaria, Jugoslavia, Turkey, Italy, France and Spain. A recent checklist of Crepidotus in Croatia includes eight species in total, all of which have also been found in Greece (Tkalcec & Mesi¢é 2003). The same applies for the seven species of Crepidotus reported from Turkey (Selsi & Denchev 2005). The diversity of Crepidotus in Spain (Ortega & Buendia 1989) is also similar to that of Greece. Lonati (2000) presented a paper concerning the diversity of Crepidotus in the Mediterranean area, in which he reports sixteen species (twelve, if synonymy is considered) collected from various provinces of Italy. Malencon & Bertault (1975) report eight species from Morocco, Algeria and Tunisia.
In conclusion, the biodiversity of Crepidotus in Greece includes, to date, 12 taxa in total. A survey of all species recorded in Greece is given in Table 1, where both published and unpublished data are included. Five taxa are newly recorded from Greece: C. applanatus var. subglobiger, C. autochthonus, C. lundellii, C. luteolus and C. subverrucisporus. Other five taxa are recorded on new substrates for Greece: C. calolepis on trunks and branches of Abies cephalonica, Abies borisii-regis and Pinus nigra, C. cesatii var. cesatii on twigs and branches of Platanus orientalis and Pinus nigra, C. epibryus on leaves of Castanea sativa, and C. variabilis on twigs and branches of Cistus sp., Quercus frainetto and Q. coccifera. Moreover, C. calolepis and C. mollis are encountered for the first time in Europe on conifers, C. calolepis, C. lundellii, C. luteolus and C. subverrucisporus on Platanus, C. autochthonus in Quercus forests and C. variabilis on Cistus.
Acknowledgments
We wish to thank Drs. Beatrice Senn-Irlet and Victor Bandala for their thorough revision of the paper, valuable comments and constructive correspondence and Dr. Shaun Pennycook for his nomenclatural review. We thank D. Dimou and S. Diamandis for giving us useful information from their data on the distribution of Crepidotus species in Greece and especially the former for the provision of dried specimens for microscopic examination.
36
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Fig. 9. a-b. Thick-walled basidiospores with apical wall curving and small depression (arrows); c. pale yellow hyphae of the pileipellis with granular (g) or minutely encrusting (e) pigment; d. yellow-brown hyphae of the pileipellis with strongly encrusting zebra-like pigment (eke a,c. C. mollis (ATHU-M 5123); b, d. C. calolepis (Dimou 731). e-f. Basidiospores: e. C. cesatii var. cesatii (ATHU-M 5109, 5125); f. C. autochthonus (ATHU-M 5121). Scale bars = 10 um.
39
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40
Fig. 11. ac. Cheilocystidia with septae (arrows): a. C. subverrucisporus (ATHU-M 5120); b. C. mollis (ATHU-M 5123); c. C. autochthonus (ATHU-M 5121). d. Yellowing basidiole: C. mollis (ATHU-M 5123). e. Yellowing, thick-walled cheilocystidia: C. autochthonus (ATHU-M 5121). f. Disintegrating basidiole and basidium with yellow-brown, strongly granular content: C. epibryus (ATHU-M 5106). g. Pleurocystidioid body: C. calolepis (ATHU-M 5105). h-j. Septate, branched and thick-walled cheilocystidia: C. applanatus var. subglobiger (ATHU-M 5332). k. Secretory hyphae in the lamellar trama: C. mollis (ATHU-M 5123). Scale bars = 10 um.
4]
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Kiihner R. 1980. Les Hyménomycetes agaricoides (Agaricales, Tricholomatales, Pluteales, Russulales). Etude générale et classification. Bull. Soc. Linn. Lyon 49: 1-1027, numero speciale.
Lonati G. 1993. Fungi Mediterranei rariores. Crepidotus mollis var squamulosus Coutinho. Mic. Veg. Med. 8: 83-84.
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Moncalvo J-M, Vilgalys R, Redhead SA, Johnson JE, James TY, Aime MC, Hofstetter V, Verduin SJW, Larsson E, Baroni TJ, Thorn RG, Jakobsson $, Clémencon H, Miller OK. 2002. One hundred and seventeen clades of euagarics. Mol. Phylogenet. Evol. 23: 357-400.
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Nordstein S. 1990. The genus Crepidotus (Basidiomycotina, Agaricales) in Norway. Synopsis Fungorum 2, Fungiflora, Oslo. :
Ortega A, Buendia AG. 1989. Notas sobre el genero Crepidotus (Fr.) Staude en Espafia peninsular. Int. J. Myc. Lich. 4 (1-2): 93-105.
Oztiirk C, Kasik G, Dogan HH, Aktas S. 2003. Macrofungi of Alanya District. Turk. J. Bot. 27: 303-312.
Pantidou ME. 1991. Mushrooms in the forests of Greece. The Goulandris National History Museum, Athens.
Pilat A. 1948. Monographie des espéces européenes du genre Crepidotus Fr. Atl. Champ. Eur. 6. Praga.
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Senn-Irlet B. 1993. Type studies in Crepidotus II. Persoonia 15: 155-167.
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Senn-Irlet B, De Meijer A. 1998. The genus Crepidotus from the State of Parana, Brazil. Mycotaxon 66: 165-199.
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MYCOTAXON
Volume 94, pp. 43-46 October-December 2005
The lichen flora of the Termessos National Park in Southwestern Turkey
OzcE TUFAN, HiseyIn SUMBUL & AYSEN OZDEMIR TURK
ozgetufan@akdeniz.edu.tr Akdeniz University, Faculty of Arts and Sciences, Biology Department, TR-07058 Antalya, Turkey
Abstract - Between March 2002 and 2003, the lichen flora of the Termossos National Park was studied for the first time. In all, 161 taxa (152 species, 4 subspecies, 5 varieties) were determined from 1114 lichen samples, of which 86 were new to Antalya Province and 5 were new to Turkey. The complete checklist can be downloaded as PDF file from www.mycotaxon.com/resources/weblists/html.
Key words - lichens, Giilliik Mountain, Antalya
Introduction
The number of studies on the lichen flora of Turkey has increased significantly over the last two decades (e.g., Aslan 2000, Breuss & John 2004, Cicek & Ozdemir Tiirk 1998, Giiveng et al. 1996, Ozdemir Tiirk & Giiner 1998, Oztiirk & Giiven¢ 2003). Although the lichen flora of the Mediterranean phytogeographical region of Turkey has received more attention than the other regions of Turkey (John 1996, 2003; John & Nimis 1998; Nimis & John 1998; John et al. 2000), even this area needs additional research. To determine the lichen flora of the region, floristic studies that focus on small areas with high biodiversity are needed.
Throughout the Mediterranean region of Turkey, there are ruins and cities from the ancient civilizations, but until now no lichen floristic or biodeterioration study has been published from such places. Termessos (Giilliik Mountain) National Park, located on the West side of the Taurus Mountains in Antalya province, southwestern Turkey, is such a site. It is famous for its ancient city, Termessos, which is situated on a natural platform at the top of Gilliik Mountain, which has been formed by chemical erosion and tectonic movements. It includes a canyon with very steep walls as high as 500-600 m.
Material and Methods
The research is based on 1114 lichen samples, which were collected from 54 localities (Table 1) in Termessos National Park between March 2002 and September 2003. In every locality coordinates and altitude were measured by GPS (Garmin 12X) and all lichen samples were taken together with their substratum. The samples were brought the laboratory and air-dried under room conditions (25 + 2 °C, RH 60+10).
44
For identification, macroscopic and microscopic characters were examined with stereo- and light microscopes and by reference to recent literature (e.g. Wirth 1995, Purvis et al. 1992, Clauzade & Roux 1985, Giordani et al. 2002, Jorgensen 1997, Zeybek et al. 1993, Breuss 1990, Moberg 1977). Following identification, the lichens were deposited in Akdeniz University Herbarium (AKDU).
Results
A PDF file containing the list of lichen species (including locality numbers and substrata) encountered in Termessos National Park, table of localities, and map of the study area can be downloaded from http://www.mycotaxon.com/resources/ weblists.html. The abbreviations of authors are in accordance with Brummitt & Powell (1992). Lichen taxa new to Turkey are indicated by *, those new to Antalya province by #.
Discussion
This study reports 161 taxa from Termessos National Park, of which Collema conglomeratum, Lecania inundata, Leptogium furfuraceum, Peltigera monticola and Physconia servitii are new to Turkey and 86 are new to Antalya province. Although the lichen flora of the Mediterranean region of Turkey is reasonably well studied, it is quite remarkable to find still so many new lichen records for the region as well as some new to the country, emphasizing that much more explorative effort should be made on its lichen flora.
In the study area, in addition to common lichen species for the Mediterranean Region, such as Lecanora bolcana and Diploschistes ocellatus, we determined “manna” lichens, such as Aspicilia desertorum and A. hispida, which usually grow in steppes, suboceanic species such as Degelia plumbea and Staurolemma omphalarioides, and oceanic species, such as Collema furfuraceum and C. nigrescens. Although the study area is relatively small (ca. 6702 ha), the wide variation in its topology, formed by high mountains, valleys and a deep canyon, evidently provides habitats for a rich lichen biodiversity.
Calcareous species are dominant due to the widespread occurrence of calcareous . rocks throughout the study area, and because of the frequency of trees with acidic bark, such as Pinus nigra and Quercus coccifera, most of the epiphytic species are acidophytic.
Of particular note are Anthracocarpon virescens, Caloplaca adriatica, Hypocenomyce anthracophila, Neocatapyrenium rhizinosum, Pertusaria hymenea, Placidium pilosellum, Solenopsora liparina and Staurolemma omphalarioides, for which there are only one or two records from Turkey (Pisut 1970; Breuss 1998; Nimis & John 1998; John et al. 2000; John 1996, 2003; Breuss & John 2004).
On the ruins of the ancient city of Termessos, not only species with a wide ecological amplitude, such as Aspicilia calcarea, Caloplaca aurantia, Lecanora muralis, Lobothallia radiosa, Placynthium nigrum and Xanthoria elegans were found, but also species mainly found in the Boreal-Mediterranean region and between the south of Central Europe and the Mediterranean Region such as Aspicilia farinosa, Caloplaca chrysodeta, C. xantholyta, Collema cristatum, Diploschistes ocellatus, Lepraria nivalis, Solenopsora candicans and
45
Solenopsora liparina (Wirth 1995). Because the ancient city was built from local stones, the species on the ruins are similar to the lichen flora found elsewhere in the study area.
SES ARAL tex “ < yA ra ra x ~
&
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oe OSI +o
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eat
bey
yn aA Sener
ot:
. > < a dae — . < . Zi ae 5 ~~ e
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;
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Figure 1: Map of Termessos National Park
Acknowledgements
The authors thank Prof. Mark R. D. SEAWARD (England) and Dr. Harrie SIPMAN (Germany) for suggestions and comments that improved the manuscript. The lichen species were collected during the project “The comparison of the lichen floras of the Termessos National Park and Diizler¢ami Region damaged in the July 1997 fire’, funded by Akdeniz University Scientific Research Projects Unit (Project number 2002.02.0121.02). We are indebted to Akdeniz University Scientific Research Projects Unit for financial support, to Prof. Dr Per. Magnus JORGENSEN (Norway), Prof. Dr Helmut MAyRHOEER (Austaria), Prof. Dr Teuvo AuTI (Finland), Assoc. Prof. Dr Roland MoBerG (Sweden), Dr Othmar Breuss (Austaria), Dr Orvo VITIKAINEN (Finland), Dr Brian Copprns (Edinburg), Dr Volker JoHN (Germany) and Alan ORANGE (Britain) for their identification of lichens, and to the Antalya Directorship of National Parks for granting permission for this study in Termessos National Park.
46
Literature Cited
Aslan A. 2000. Lichens from the regions of Artvin, Erzurum, and Kars (Turkey). Israel Journal of Plant Sciences 48: 143-155.
Breuss O. 1990. Die Flechtengattung Catapyrenium in Europa. Stapfia 23, 110 pp.
Breuss O. 1998. Catapyrenium und verwandte Gattungen (lichenisierte Ascomyceten, Verrucariaceae) in Asien—eine erste Ubersicht. Annalen des Naturhistorischen Museum in Wien, B, 100: 656-669.
Breuss O, John V. 2004. New and interesting records of lichens from Turkey. Osterreichische Zeitschrif fiir Pilzkunde 13: 281-294.
Brummitt RK, Powell CE. 1992. Authors of plant names. Royal Botanical Gardens, Kew, 732 pp.
Clauzade G, Roux C. 1985. Likenoj de Okcidenta Eurupo. Bulletin de la Societé Botanique du Centre-Ouest, Nouvelle série, 893 pp.
Cicek A, Ozdemir Tiirk A..1998. Lichen flora of Sakarya province (Turkey). Doga-Turkish Journal of Botany 22: 99-119.
Giordani P, Nicora P, Rellini I, Brunialti G, Elix JA. 2002,. The lichen genus Xanthoparmelia (Ascomycotina, Parmeliaceae) in Italy. Lichenologist 34 (3): 189-198.
Giivenc $, Aslan A, Oztiirk $. 1996. The lichen flora of Kapidag Peninsula.- In: Oztiirk, M. A., Secmen, O. & Gérk, G. (eds.) Plant life in southwest and central Asia. Proceedings of the 4 th plant life in southwest Asia symposium held in Izmir 21 - 28 may 1995, Ege Univ. Press, Bornova-Izmir, pp. 472-478.
John V. 1996. Preliminary catalogue of lichenized and lichenicolous fungi of Mediterranean Turkey. Bocconea 6: 173-216.
John V. 2003. Flechten aus der Tiirkei, von G. Ernst gesammelt. Herzogia 16: 167-171.
John V, Nimis PL. 1998. Lichen flora of Amanos mountain and the province of Hatay. Doga-Turkish Journal of Botany 22: 257-267.
John V, Seaward MRD, Beatty JW. 2000. A neglected lichen collection from Turkey: Berkhamsted School expedition 1971. Doga-Turkish Journal of Botany 24: 239-248.
Jorgensen PM. 1997. Further notes on hairy Leptogium species. Symbolae Botanicae Upsalienses 32 (1): 113-130.
Moberg R. 1977. The lichen genus Physcia and allied genera in Fennoscandia. Symbolae Botanicae Upsalienses 22: 1-108.
Nimis PL, John V. 1998. A contribution to the lichen flora of Mediterranean Turkey. Cryptogamie, - Bryologie et Lichénologie 19: 35-58.
Ozdemir Tiirk A, Giiner H. 1998, Lichens of the Thrace region of Turkey. Doga-Turkish Journal of Botany 22: 397-407.
Oztiirk $, Gitveng $. 2003. Lichens from the western part of the Black Sea Region of Turkey. Acta Botanica Hungarica 45: 169-182.
Pisut, I. 1970. Interessante Flechtenfunde aus der Tiirkei. Preslia (Praha) 42: 379-383.
Purvis OW, Coppins BJ, Hawksworth DL, James PW, Moore DM. 1992. The lichen flora of Great Britain and Ireland. Edmundsbury Press, London, 719 pp.
Wirth V. 1995. Die Flechten Baden—Wirttembergs. Teil: 1-2, Eugen GmbH & Co., Stuttgart, 1006 pp.Zeybek U., John V, Lumbsch HT. 1993. Turkiye likenlerinden Hypogymnia (Nyl.) Nyl. cinsi iizerinde taksonomik arastirma. Doga-Turkish Journal of Botany 17: 109-116.
Zeybek U, John V, Lumbsch HT. 1993. Tiirkiye likenlerinden Hypogymnia (Nyl.) Nyl. cinsi tizerinde taksonomik arastirma. Doga-Turkish Journal of Botany 17: 109-116.
MYCOTAXON
Volume 94, pp. 47-50 October-December 2005
Two new species of Anthracoidea (Ustilaginales) from China
LIN Guo
guol@sun.im.ac.cn Key Laboratory of Systematic Mycology e& Lichenology Institute of Microbiology, Chinese Academy of Sciences Beijing 100080, China
Abstract—Two new species, Anthracoidea setosae on Carex setosa and A. xizangensis on Kobresia duthiei, are described and compared with A. misandrae and related species.
Key words—smut fungi, Ustilaginomycetes, taxonomy
A new species of Anthracoidea on Carex setosa (Subgen. Carex, Sec. Frigidae) was discovered from our herbarium (HMAS 67908, 34929, 34930). These specimens were wrongly identified by the author (Guo 1994) as A. misandrae. The new species of Anthracoidea is similar to A. misandrae and A. sempervirentis Vanky on host plants in the same section of Carex in having ustilospores of the same size. It differs mainly from A. misandrae by ustilospores with minute warts measuring 0.125-0.3 um in diam., while A. misandrae has ustilospores with larger warts [(0.2-)0.4-0.8(-1) um in diam. (Kukkonen 1963: 83)]. It differs mainly from A. sempervirentis by ustilospores with regularly distributed warts as seen by SEM (scanning electron microscopy), while A. sempervirentis has ustilospores with a “surface by SEM partly with sparse, 0.1-0.2 um high knobs, partly with abundant and dense, up to 0.5 um high, irregular, often confluent, rounded warts.” (Vanky 1979: 226). The new species is described as:
Anthracoidea setosae L. Guo, sp. nov. Figs. 1-2
Sori in ovariis, subglobosi vel ovoidei, 1.2-2 mm longi, 1-1.8 mm lati, primum membrana cinerascenti, fungali cooperti, deinde expositi. Massa sporarum nigra, semiagglutinata. Ustilosporae a fronte globosae, ellipsoideae, leviter irregulares vel irregulares, 17.5-25(-27)
x 12.5-20 um, ab acie 10-15 um latae, flavidobrunneae vel atrobrunneae; pariete aequaliter vel inaequaliter incrassato, 1-2.5(-3) wm crasso, tumores interni desunt, regiones lucem repercutientes desunt, superficie minute et dense verruculoso sub SEM.
Sori in ovaries, subglobose or ovoid, 1.2-2 mm long and 1-1.8 mm wide, at first covered by a grayish, fungal membrane, later becoming exposed. Spore mass black, _ semi-agglutinated. Ustilospores in plane view globose, ellipsoidal, slightly irregular or irregular, 17.5-25(-27) x 12.5-20 um, in side view 10-15 um wide, yellowish-brown or blackish-brown; wall evenly or unevenly thickened, 1-2.5(-3) um, no internal swellings, no light reflective areas, surface minutely and densely verruculose as seen by SEM.
On Carex setosa Boott (Cyperaceae, Subgen. Carex, Sect. Frigidae), Gansu: Zhouqu, Shatanlinchang, alt. 3050 m, 4 IX 1992, L. Guo 1276, HMAS 67908 (holotypus hic
48
designatus); Sichuan: Emei Shan, Leidongping, alt. 2500 m, 10 VII 1969, C. M. Wang, Y. X. Han & Q. M. Ma 314, HMAS 34930 (paratypus); Emei Shan, Jindingsi, alt. 3150 m, 9 VII 1969, C. M. Wang, Y. X. Han & Q. M. Ma 296, HMAS 34929 (paratypus).
Etymology: Refers to the host plant Carex setosa.
Anthracoidea misandrae was discovered in the Herbarium of the Institute of Botany, Chinese Academy of Sciences (PE) and collected by Prof. Kuan Kechien from Xinjiang Uygur Autonomous Region in 1957. It is described as:
Anthracoidea misandrae Kukkonen, Ann. Bot. Soc. Zool.-Bot. Fenn. ,Vanamo* 34(3): 82, 1963. ve
Sori in ovaries, ellipsoidal, 2.5-3.5 mm long and 1.5-2.5 mm wide, at first covered by a grayish, fungal membrane, later becoming exposed. Spore mass black, semi-agglutinated. Ustilospores in plane view subglobose, ellipsoidal or ovoid, 17.5-25(-26) x 15-20(-22) um, in side view 10-14 um wide, dark reddish-brown; wall evenly thickened, ca. 1 um, no internal swellings, no light reflective areas, surface verrucose.
On Carex stenocarpa Turcz. ex V. Krecz. (Cyperaceae, Subgen. Carex, Sect. Frigidae), Xinjiang: Nilka Xian, 60 Km N of Wulasitai (in the Borohoro Mountains) 31 VIII 1957, K. C. Kuan 3991, HMAS 132710.
Another new species of Anthracoidea on Kobresia duthiei (Sec. Elyna) was discovered from our herbarium (HMAS 67973) and collected by Prof. Zhuang Jianyun in 1990. The specimen was wrongly identified by the author as A. filifoliae L. Guo (1995-1996) on the section Kobresia. The new species differs from A. filifoliae by minute warts on the surface of the ustilospore as seen by SEM and host plants in different sections of the genus Kobresia, while A. filifoliae has dense and minute warts between the larger warts on the surface of the ustilospores. Only A. elynae (Syd.) Kukkonen (1963: 65) has been recorded previously on the section Elyna. The new species differs from A. elynae by having warts on the surface of the ustilospores, while the surface of the ustilospores of A. elynae is smooth. The new species is described as:
Anthracoidea xizangensis L. Guo, sp. nov. | Figs. 5-6
Sori in ovariis, ellipsoidei vel ovoidei, 1-2 mm longi, 0.7-1 mm lati, primum membrana cinerascenti, fungali cooperti, deinde expositi. Massa sporarum nigra, semiagglutinata. Ustilosporae a fronte globosae, ellipsoideae vel ovoideae 17-22.5 x 15-18 wm, ab acie 10-15 um latae, atrobrunneae; pariete aequaliter incrassato, 1.5-2 um crasso, tumores interni desunt, regions lucem repercutientes desunt, superficie minute et dense verruculoso sub SEM.
Sori in ovaries, ellipsoidal or ovoid, 1-2 mm long and 0.7-1 mm wide, at first covered by a grayish, fungal membrane, later becoming exposed. Spore mass black, semi-
Figs. 1-2. Ustilospores of Anthracoidea setosae on Carex setosa as seen by LM (light microscopy) and SEM (HMAS 67908, holotypus).
Figs. 3-4. Ustilospores of Anthracoidea misandrae on Carex stenocarpa as seen by LM and SEM (HMAS 132710).
Figs. 5-6. Ustilospores of Anthracoidea xizangensis on Kobresia duthiei as seen by LM and SEM (HMAS 67973, holotypus). Bars = 10 um
50
agglutinated. Ustilospores in plane view globose, ellipsoidal, or ovoid, 17-22.5 x 15-18 um, in side view 10-15 um wide, blackish-brown; wall evenly thickened, 1.5-2 um, no internal swellings, no light reflective areas, surface minutely and densely verruculose as seen by SEM.
On Kobresia duthiei C. B. Clarke (Cyperaceae, Sect. Elyna), Xizang: Dinggye, Yala Shan, alt. 4950 m, 15 VIII 1990, J. Y. Zhuang 2919, HMAS 67973 (holotypus hic designatus).
Etymology: Refers to the locality Xizang Autonomous Region (Tibet).
Since 2000 (Guo 2000), eight new species and three new records of the genus Anthracoidea have been recorded in China (Guo 2002, 2004, Guo & Wang 2005, Guo & Zhang 2004, Wang & Piepenbring 2002, Zhang & Guo 2004), including A. setosae and A. xizangensis (in this paper).
Acknowledgements
The author would like to express her deep thanks to Drs. Vanky and Chlebicki for reading the manuscript and serving as pre-submission reviewers, to Dr. Pennycook for nomenclature review, to Profs. Liang Songjun and Zhang Shuren (Institute of Botany, Chinese Academy of Sciences) for identifying the host plants, and to Mrs. Xie Jiayi for assistance with SEM photographs. ‘This study was supported by the National Natural Science Foundation of China (No. 30230020 and No. 30270016).
Literature Cited
Guo L. 1994. Anthracoidea and allied genera in China (Ustilaginales). Mycosystema 7: 89-104.
Guo L. 1995-1996. Anthracoidea filifoliae sp. nov. and four smut species new to China. Mycosystema 8-9: 163-168.
Guo L. 2000. Flora Fungorum Sinicorum, Ustilaginaceae Vol. 12, Science Press, Beijing (China). 124 pp.
Guo L. 2002. Two new species of Ustilaginomycetes and a species new to China. Mycotaxon 82: 147-150.
Guo L. 2004. Anthracoidea shaanxiensis sp. nov. (Ustilaginales) from China. Nova Hedwigia 79: 507-509.
Guo L, Wang SR. 2005. A new species and a new record of Anthracoidea (Ustilaginales) from China. Mycotaxon 93: 159-162.
Guo L, Zhang HC. 2004. A new species and two new records of Ustilaginomycetes from China. Mycotaxon 90: 387-390.
Kukkonen I. 1963. Taxonomic studies on the genus Anthracoidea (Ustilaginales). Ann. Bot. Soc. Zool.-Bot. Fenn. “Vanamo” 34(3): 1-122.
Vanky K. 1979. Species concept in Anthracoidea (Ustilaginales) and some new species. Bot. Not. U32: 221-23
Wang SR, Piepenbring M. 2002. New species and new records of smut fungi from China. Mycol. Progr. 1: 399-408.
Zhang HC, Guo L. 2004. Two new species and a new record of Anthracoidea (Ustilaginales) from China. Mycotaxon 89: 307-310. j
MYCOTAXON
Volume 94, pp. 51-54 October-December 2005
A new species of Lepiota (Agaricaceae, Basidiomycetes) from China
HAN-CHEN WANG
higherfungi2005@yahoo.com.cn Kunming Institute of Botany, Chinese Academy of Sciences Kunming 650204, Yunnan Province, P. R. China Institute of Applied Ecology, Chinese Academy of Sciences Shenyang 110016, Liaoning Province, P. R. China Graduate School of the Chinese Academy of Sciences, P. R. China
ZHU-LIANG YANG*
fungi@mail.kib.ac.cn Kunming Institute of Botany, Chinese Academy of Sciences Kunming 650204, Yunnan Province, P. R. China
Abstract—A new species, Lepiota catenariocystidiata, is described and illustrated. It is compared with similar species.
Key words—Agaricales, taxonomy
Introduction
During our study of lepiotaceous fungi, we came across an undescribed species of the genus Lepiota (Pers.: Fr.) Gray. It is described and illustrated herein. In descriptions of the basidiomata, color designations (e.g., 1A1) are from Kornerup & Wanscher (1981), and color names with first letters capitalized (e.g., Pale Smoke Gray) are from Ridgway (1912). In descriptions of basidiospores, the notation [n/m/p] shall mean n basidiospores measured from m basidiomata of p collections in Melzer’s reagent. Q is used to mean quotient of length and width of a spore in side view; Q means average Q of all basidiospores + sample standard deviation. Herbarium code HKAS = Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences.
Taxonomy
_ Lepiota catenariocystidiata Han C. Wang & Zhu L. Yang, sp. nov. Figs. 1-4
Pileus 3-5 cm latus, initio subcampanulatus, deinde convexus vel applanatus, albidus vel griseolus, squamulis tomentosis, griseis vel obscure griseis. Lamellae liberae, albidae vel cremeae. Stipes 4-6 x 0.3-0.6 cm, subcylindricus, albidus, annulatus, squamulis confertis,
* Corresponding author
ad
tomentosis, griseis infra annulum. Annulus superus, membranaceus. Basidiosporae (7.0) 7.5-9.0 (10.0) x (3.5) 4.0-4.5 (5.0) um, oblongae vel subcylindricae, incoloratae, hyalinae, dextrinoideae. Basidia 20-30 x 7.5-10 um, subclavata, 4-sporigera, raro 2- sporigera. Pleurocystidia nulla. Cheilocystidia 10-20 x 8-12 um, subglobosa, ovata vel breviclavata, catenaria, incolorata. Squamulae pilei ex hyphis subcylindricis terminalibus compositae. Fibulae praesentes.
Holotype: CHINA, Yunnan Prov., Mengla County, Menglun, 2. XI. 1989, Z.L. Yang 918 (HKAS 22145).
Etymology: Named because of the cheilocystidia often in chains.
Basidiomata (Fig. 1) scattered. Pileus 3-5 cm in diam, at first subcampanulate, then convex to applanate, with an obtuse umbo or non-umbonate; pileal surface dry, whitish to grayish (1A1-1B1; Pale Smoke Gray) but with pinkish tinge (11A2-11B2; Light Brownish Drab) at center, densely covered with minute, dark gray (11C1 + 11D1 + 11E1; Dark Neutral Gray to Blackish Slate) tomentose squamules over disc, with small, concentrically arranged, grey to dark grey squamules towards the margin, margin often slightly exceeding lamellae. Lamellae free, whitish to cream-coloured, moderately crowded, in 2-3 ranks, up to 0.7 cm broad, with white to concolorous eroded edge. Stipe 4-6 x 0.3-0.6 cm, central, subcylindrical, hollow, slightly enlarged near base, surface whitish, lower part covered with grey (11C1 + 11D1; Dark Neutral Gray to Hair Brown) tomentose squamules often in belts. Annulus superior, membranous, upper surface white and glabrous, lower surface covered with grey tomentose squamules, persistent or fugacious. Context whitish, unchanging; odor indistinct; taste slightly hot.
Basidiospores (Fig. 2) [42/2/2] (7.0) 7.5-9.0 (10.0) x (3.5) 4.0-4.5 (5.0) um [Q = (1.67) 1.75-2.25, Q=2.01 + 0.18], oblong to subcylindrical, with a distinct suprahilar depression in side view, water-drop-shaped in front view, broader at apical part, attenuate towards the base, slightly thick-walled, colorless, hyaline, dextrinoid, reddish in Congo Red, not metachromatic in Cresyl Blue. Basidia 20-30 x 7.5-10 um, subclavate, hyaline, thin- walled, 4-spored, rarely 2-spored. Pleurocystidia absent. Cheilocystidia (Fig. 3) abundant, 10-20 x 8-12 um, subglobose, ovoid to short clavate, often in chains, colorless and hyaline. Squamules (Fig. 4) on pileus a disrupted trichodermium consisting of loose fascicles of long, more or less erect, subcylindrical, terminal elements (45-300 x 10-17 um) with. tapering or round apex and pale yellow-brown to dark yellow-brown intracellular pigments; with short clavate cells at the base of these long elements; the repent hyphae at the base of erect elements sometimes with incrusting pigments. Squamules on surface of stipe similar to those on pileus. Clamp connections abundant in basidiomata.
Habitat: On well-rotten wood with soil in limestone monsoon forests; fruiting in summer and autumn in southwestern China at 600-700 m elev.
Known distribution: Known from tropical Yunnan only.
Additional material examined: CHINA, Yunnan Prov., Mengla County, Menglun Nature Reserve, 22. X. 1988, Zang 11515 (HKAS 20358).
Notes: Lepiota catenariocystidiata is well characterized by its whitish to grayish pileus with a pinkish center, gray to dark gray squamules on the pileus made up of a disrupted trichodermium with long subcylindrical terminal elements and short clavate cells at the base of these long elements, water-drop-shaped basidiospores in front view, catenulate cheilocystidia and the common presence of clamp connections in the basidiomata.
53
Figs. 1-4. Lepiota catenariocystidiata (from holotype) 1. Basidiomata. 2. Basidiospores. 3. Cheilocystidia. 4. Squamules on pileus
Due to the trichodermium type of the squamules on the pileus and the oblong to subcylindrical basidiospores, L. catenariocystidiata may belong to Lepiota sect. Ovisporae (J.E. Lange) Kuhner. Species with pileal squamules made up of long, erect elements and short, clavate elements in between were put in the subsect. Felininae Bon within sect. Ovisporae (Vellinga 2001). Because L. catenariocystidiata has short, clavate elements at the base of long, erect ones, it should be placed in the subsection. However, according to recent molecular phylogenetic studies, the ITS data set does not support sect. Ovisporae, and thus a re-evaluation of this section is needed (Vellinga 2003). The present species may be clustered within the Clade 1 of Lepiota s. |. (Vellinga 2003).
Lepiota catenariocystidiata may be related to L. felina (Pers.) P. Karst., L. pseudolilacea Huijsman and L. pseudohelveola Kiihner ex Hora. However, the latter two species have differently coloured basidiomata, ellipsoid to oblong basidiospores in front view and rarely with a suprahilar depression in side view, and differently shaped cheilocystidia (Kithner 1936; Huijsman 1947; Hora 1960; Enderle & Krieglsteiner 1989; Bon 1981, 1996; Candusso & Lanzoni 1990). According to Vellinga (2001), L. pseudohelveola should be regarded as a synonym of L. pseudolilacea. Lepiota felina differs from L. catenariocystidiata in the colour of the basidiomata, the shape of the cheilocystidia and
54
basidiospores (Kithner 1936; Huijsman 1947; Hora 1960; Bon 1981, 1996; Enderle & Krieglsteiner 1989; Candusso & Lanzoni 1990; Vellinga 2001).
The basidiospores of L. catenariocystidiata are very similar to those of L. plumbicolor (Berk. & Broome) Sacc., originally described from Sri Lanka. However, the latter has blackish purple squamules on pileus with elongate clavate terminal elements with an obtusely rounded apex and clavate-cylindrical cheilocystidia (Pegler 1972, 1986).
Acknowledgements
Weare very grateful to Dr. E.C. Vellinga for having sent us her valuable publications on Agaricaceae, and to Drs. E.C. Vellinga and D.E. Desjardin for their critical reviewing the manuscript. This project was financed by the Natural Science Foundation of Chinas Yunnan Province (No. 2002C0059M) and the National Natural Science Foundation of China (No. 30270017).
Literature cited
Bon M. 1981. Clé monographique des “Lépiotes” d'Europe (= Agaricaceae, Tribus Lepioteae et Leucocoprineae). Doc. Mycol. 11 (43): pl-p77.
Bon M. 1996. Die Grofpilzflora von Europa 3. Lepiotaceae (iibersetzt und bearbeitet von F. Medjebeur-Thrun & W. U. Thrun). IHW- Verlag: Eching (Germany). 141 pp.
Candusso M, Lanzoni G. 1990. Lepiota s. |. Fungi Europaei 4. Giovanna Biella: Saronno (Italy). 743pp.
Enderle M, Krieglsteiner GJ. 1989. Die Gattung Lepiota (Pers.) S. EF Gray emend. Pat. in der Bundesrepublik Deutschland (Mitteleuropa). Z. Mykol. 55: p43-p104.
Hora FB. 1960. New check list of British Agarics and Boleti. Transactions British Mycol. Soc. 43: p440-p459.
Huijsman HSC. (1947). Lepiota pseudolilacea nov. spec. Bull. Soc. Linn. Lyon 16: p180-p183.
Kornerup A, Wanscher JH. 1981. Taschenlexikon der Farben. 3. Aufl. Muster-Schmidt Verlag: Zurich (Switzerland). 242pp.
Kiihner MR. 1936. Recherches sur le genre Lepiota. Bull. Soc. Mycol. France 52: p175-p238.
Pegler DN. 1972. A revision of the genus Lepiota from Ceylon. Kew Bull. 27: p155-p202.
Pegler DN. 1986. Agaric Flora of Sri Lanka. Kew Bull. Add. Ser. 12: p1-p519.
Ridgway R. 1912. Color Standards and Color Nomenclature. R. Ridgway, Washington, D.C. (USA). 43pp.
Vellinga EC. 2001. Lepiota (Pers. : Fr.) S. FE Gray. In: Flora Agaricina Neerlandica 5 (eds. M.E. Noordeloos, Th.W. Kuyper and E.C. Vellinga). A. A. Balkema Publishers, Holland: p109-p151.
Vellinga EC. 2003. Phylogeny of Lepiota (Agaricaeae) - Evidence from nrITS and nrLSU sequences. Mycol. Progr. 2: p305-p322.
MYCOTAXON
Volume 94, pp. 55-73 October-December 2005
ITS sequence analysis and ascomatal development of Pseudogymnoascus roseus
Y. JIANG & Y. -J. YAo*
yaoyj@sun.im.ac.cn Systematic Mycology and Lichenology Laboratory, Institute of Microbiology Chinese Academy of Sciences, Beijing 100080, China
Abstract—ITS sequence analysis and ascomatal development of Pseudogymnoascus roseus strains isolated from sclerotia of Cordyceps sinensis collected from the Tibetan Plateau, China, are reported in this paper. The ITS sequences of three strains from different locations were identical and were compared with sequences obtained from the BLAST search in GenBank. The strains display the same morphology as the reference collection deposited in K, matching the species description of P roseus. Ascomatal development of the P roseus strains is described. Ascomata of P. roseus were found to comprise an aggregation of asci from several different ascomatal initials enveloped by a loose, thick-walled hyphal network. In the parsimony analysis, ITS sequences of P. roseus and other Myxotrichaceae grouped outside the Onygenales and clustered with those of discoid fungi. Members of Myxotrichaceae were considered closely related to discomycetes, but greatly diverged from onygenalean fungi. Myxotrichaceae did not form a monophyletic group in the ITS tree.
Key words—DNA, fungal culture, taxonomy
Introduction
Pseudogymnoascus Raillo, a genus of Ascomycetes established with two species in 1929, has been referred to either Gymnoascaceae Baran. (e.g. Kuehn 1958, Arx 1971, Alexopoulos & Mims 1979, Orr 1979, Benny & Kimbrough 1980, Eriksson & Hawksworth 1986, 1993) or to Onygenaceae Berk. (Arx 1987). However, it was placed by Currah (1985) in Myxotrichaceae Locq. ex Currah, based on cellulose degradation capacity, smooth ascospores and rhexolytically dehiscing conidia. The latter taxonomic treatment has been widely accepted (Alexopoulos et al. 1996, Kirk et al. 2001). Species of this genus have yellow or rose, globose to subglobose, discrete or confluent ascomata; ascomatal peridium composed of a network of slightly thick-walled hyphae; appendages simple and not distinct; asci globose to ellipsoid, normally 8-spored; ascospores ellipsoid - to fusoid, smooth, hyaline, yellow, orange to pink (Cejp & Milko 1966, Orr 1979, Currah 1985).
In addition to the original two species of Pseudogymnoascus, P. roseus and P. vinaceus Raillo, several more species have since been described, e.g. P. caucasicus Cejp & Milko,
* Author for correspondence
56
P. bhattii Samson, P. alpinus E. Mill. & Arx and P. dendroideus Locq.-Lin. However, Samson (1972) considered P. roseus and P. vinaceus identical and lectotypified the genus with P. roseus. Orr (1979) discussed the status of the genus and recognised two species, P. vinaceus and P. roseus, listing P. bhattii as a synonym of P. vinaceus. In his monographic study of Onygenales, Currah (1985) treated both P. vinaceus and P. bhattii as synonyms of P. roseus. Although Samson's (1972) choice of P. roseus as the type species has been followed by others (Arx 1974, 1981, 1987, Currah 1985, Sigler et al. 2000), the genus was also typified by P. vinaceus early (Kuehn 1958, Orr 1979).
As more species of Pseudogymnoascus were described, the range of morphological characters became more diverse. For example, P alpinus was described as having navicular-fusoid ascospores, and ascomata with branched and recurved appendages (Miller & Arx 1982); P. dendroideus with ramified ascomatal appendages and striated ascospores (Locquin-Linard 1982); and P. roseus var. ornatus Udagawa & Uchiy. with irregularly lobate-reticulate ascospores (Udagawa & Uchiyama 1999). Further, a weakly cellulolytic species having fusoid ascospores with a longitudinal sigmoid crest was assigned to the genus as Pseudogymnoascus sp. (Lumley et al. 2000).
Anamorphs of Pseudogymnoascus have been referred to Geomyces Traaen (Orr 1979, Currah 1985). Geomyces pannorum var. vinaceus (Dal Vesco) Oorschot is considered as the anamorph of P. roseus (Sigler & Carmichael 1976, Oorschot 1980).
Systematically, Myxotrichaceae was placed in the order Onygenales, along with Arthrodermataceae Locq. ex Currah, Gymnoascaceae and Onygenaceae, by Currah (1985). Species of Myxotrichaceae are saprobic, cellulose-degrading, and usually inhabit forest soils and decay plant material. Members of Gymnoascaceae do not exhibit strong substrate preferences, and are neither keratinolytic nor cellulolytic. The Arthrodermataceae and the Onygenaceae degrade keratin and usually inhabit soils enriched with keratin or dung. Currah (1994) further restricted the Onygenales to keratinolytic genera and suggested that the Myxotrichaceae might represent a distinct evolutionary line from typical members of Onygenales and derive from the inoperculate discomycetes and that it merited a placement in its own order. The distant relationship between the Myxotrichaceae and other members of Onygenales has been confirmed by recent studies (Sugiyama et al. 1999, Mori et al. 2000, Kirk et al. 2001). :
Recently, molecular approaches have been introduced to the systematic study of Myxotrichaceae and related fungi (Bowman & Taylor 1993, LeClerc et al. 1994, Hambleton et al. 1998, Sugiyama et al. 1999, Sugiyama & Mikawa 2001). Some molecular evidence indicates that Myxotrichaceae are distantly related to other members of Onygenales (Sugiyama et al. 1999, Mori et al. 2000). Sugiyama et al. (1999) examined the molecular systemtatics of onygenalean taxa (including P roseus and members of Amauroascaceae Arx, Arthrodermataceae, Gymnoascaceae and Onygenaceae) based on 18S rDNA sequences and suggested that the Myxotrichaceae should be placed in an independent position among the Helotiales and the Erysiphales, on a different lineage from the keratin-degrading fungi. Based on sequences of 18S rDNA and partial sequences of 28S rDNA, Mori et al. (2000) demonstrated that the Myxotrichaceae was distantly related to the other onygenalean families, as a sister group to the Erysiphales and that the Erysiphales/Myxotrichaceae clade was also closely related to some discomycetous fungi, e.g. Helotiales and Thelebolaceae (Brumm.) Eckblad. Pseudogymnoascus has also been used as the outgroup in other molecular systematic studies, e.g. Hambleton et al. (1998)
oi
and Sugiyama & Mikawa (2001). The molecular research has provided more evidence to support the idea that the Myxotrichaceae is not closely related to onygenalean fungi.
Ascomatal development of Pseudogymnoascus has been described in some taxonomic studies (Samson 1972, Locquin-Linard 1982, Miiller & Arx 1982, Tsuneda 1982, Ito & Yokoyama 1987). Samson (1972) described the ascomatal initials as borne on the vegetative hyphae, consisting of coiled ascogonia and producing loose wefts of ascogenous hyphae inside hyphal tufts, and illustrated the ascomatal initials and the ascus formation. When describing the new species P. alpinus, Miller & Arx (1982) indicated that ascomatal initials grew as aerial branches of vegetative hyphae, loosely interwoven and sympodially branched once or twice. Locquin-Linard (1982) provided three drawings to describe the different stages of ascomatal develolpment of P. dendroideus. Tsuneda (1982) illustrated the development of ascomata in a scanning electron microscopic study of P. roseus and Ito & Yokoyama (1987) provided a photograph of the ascomatal initial but without description. The process of ascomatal development in Pseudogymnoascus has not yet been described in detail.
Pseudogymnoascus roseus is a species of worldwide distribution frequently found in soil, usually from alpine or forest areas (Christensen et al. 1962, Orr 1979, Ito & Yokoyama 1985, 1987, Currah 1985, Yokoyama et al. 1989, Udagawa & Uchiyama 1999), and occasionally on dung (Ellis & Ellis 1988). It has been also reported from tropical regions (Farrow 1954, Siddiqi 1964, as P vinaceus). Pseudogymnoascus roseus was also reported to form mycorrhizal associations with Vaccinium angustifolium Aiton in the laboratory (Dalpé 1989), and typical ericoid mycorrhiza with salal (Gaultheria shallon Pursh), and also to degrade cellulose and to use organic forms of nitrogen (Xiao & Berch 1995),
During fieldwork undertaken on the Tibetan Plateau for investigation of the Chinese Caterpillar Fungus (Cordyceps sinensis, a well-known fungus used as a tonic in traditional Chinese medicine), some other fungi were also isolated from sclerotia of the fungus. Among them, three strains displayed different characters from those of C. sinensis in culture. Sequences of internal transcribed spacers (ITS) in the nuclear ribosomal DNA (nrDNA) were obtained for molecular systematic analysis and observations on the cultures were made to elucidate the development of ascomata in these strains. The taxonomic position of the strains was determined as P. roseus based on both molecular data and morphological observation. The results are reported here to provide further information on the biology of this fungus.
Materials and Methods
Fungal cultures
Fungal cultures used in this study were isolated from C. sinensis specimens, collected from Xiaojin County, Sichuan Province, and Yulong Snow Mountain, Yunnan Province - of China, on the southeast of the Tibetan Plateau. Soil and plant debris on the fresh specimens were removed and the surface of the specimens was sterilised with 70% ethanol before isolation. The exoskeleton of the host larva was peeled off by using a scalpel and small pieces of the inner tissue of sclerotium of C. sinensis were transferred to fresh slopes of bran agar-peptone medium (potato dextrose agar (PDA) supplemented with 5 % wheat bran and 0.5 % peptone). A few pieces of the exoskeleton were also used
58
as inocula for comparison. Pure cultures were obtained by sub-culturing hyphal tips of primary isolates. Living cultures were maintained at the Institute of Microbiology, Chinese Academy of Sciences, and dried voucher specimens are deposited in HAMS (Chinese Academy of Sciences, Beijing, China) and K (Royal Botanic Gardens, Kew, UK). The details of strains used in this study are listed in Table 1. The cultures were kept at 4 °C for 3-16 weeks for morphological observation and molecular experiments.
DNA extraction
Samples of fresh mycelium were obtained by scraping the culture from the surface of the nutrient slopes after 6 weeks of incubation. DNA extraction was carried out following a modification of Yao et al. (1999). About 0.1 g of fresh mycelium (including some agar) was ground into powder in liquid nitrogen and transferred to a 1.5 ml tube. The lysis buffer of 600 ul 2% CTAB was added, followed by incubation in a water bath at 65 °C for 1h or more. An equal volume of phenol/chloroform/isoamylol (25:24:1) was added and mixed, then centrifuged at 13000 rpm for 10 min. The supernatant was transferred to a fresh 1.5 ml tube, followed by extraction of the chloroform/isoamylol alcohol (24:1). After centrifugation, the supernatant was transferred to a fresh tube and 250 ul isopropanol was added to precipitate the DNA at —20 °C for 4 h or overnight. The precipitate was centrifuged at 13000 rpm for 10 min, then the liquid was drained off and the tube dried at room temperature for more than 2 h. The DNA preparation was resuspended in 40 ul of sterile deionised water. The crude extracts containing unquantified DNA amounts were used as templates for PCR amplification. Dilution of these extracts 2-10 times was sometimes required for successful DNA amplification.
PCR amplification and sequencing
The entire ITS region of nrDNA, including ITS1, ITS2 and 5.88 gene, was amplified by polymerase chain reaction (PCR) utilizing the ITS5/4 primers (White et al. 1990). The amplification was performed in 25 ul volumes of reaction mixture containing: 10 mM Tris/HCl (pH 8.3), 2.5mM MgCl, 0.2 mM of each of the four deoxyribonucleotide triphosphates, 0.4 uM of each of the two primers, 24 U ml' Taq polymerase (Sino- American Biotechnology Co.), 1 ul of DNA template (some of them were diluted from. the crude DNA extracts). The PCR was performed with an initial denaturation of 97 °C for 2.5 min and 35 cycles of 97 °C for 30 sec, 50 °C for 1 min, 72 °C for 1.5 min and final 72 °C for 10 min.
Products were purified using Watson's PCR Purification Kit (Watson Ltd). Sequencing was performed by the cyclic reaction termination method using fluorescently labelled dideoxyribonucleotide triphosphates, according to the manufacturer’s protocols on the Geneamp PCR System 2400 or 9700 (Perkin-Elmer). The sequencing products were purified by ethanol precipitation according to the sequencing kit protocol (ABI Prism® BigDye™ Terminator Cycle Sequencing Ready Reaction Kit, Original and Version 2.0, ABI). Sequencing was performed on an ABI Prism® 3100 Genetic Analyzer (Applera Corporation) and data collected on a Dell computer with the DNA Sequencing Analysis programme (ABI Prism®’DNA Sequencing Analysis Software™, Version 3.7). Each fragment was sequenced in both directions for confirmation and the two strands of sequences were assembled with Seqscape programme (ABI Prism’ SeqScape Software™, Version 1.1).
59
Table 1. Test strains of Pseudogymnoascus roseus and Cordyceps sinensis used in this
study. GenBank eee Fungus Location | Elev. Colt Voucher | Access. ca date 4
Xiaojin Sclerotium 8 Pseudogymnoascus HMAS : County, 3700m of Cordyceps June, AY608923 roseus Raillo ‘ 79435 Sichuan sinensis 2000 ee Yulong Snow Exoskeleton of 31 are seudogymnoascus 8y Mountain, 4060m C. sinensis host May, AY608924 roseus 79436 Yunnan larva 2001 Yulong Snow : 31 aS Pseudogymnoascus ‘ Sclerotium of C. . 79438 rors! Mountain, 4060m ; é May, K(M AY608922 Yunnan giiasier 2001 (M) 108601 oes pe Sclerotium of C. oh HMAS Mountain, 3 , May, 79439 AY608925 Yunnan eshte 2001
DNA sequence analysis and molecular identification of the strains
ITS sequences obtained from this study were compared with existing sequences in GenBank by BLAST database search (Altschul et al. 1997). Additional ITS sequences from Myxotrichaceae (Myxotrichum arcticum) and from families in the Onygenales (including Gymnoascus Baran. of Gymnoascaceae, Ajellomyces McDonough & A. L. Lewis of Onygenaceae, Amauroascus J. Schrot. of Onygenaceae and Arthroderma Curr. of Arthrodermataceae), two representatives of pyrenomycetes, Neurospora crassa Shear & B. O. Dodge and Erysiphe cichoracearum, and two species of basidiomycetes were also retrieved from GenBank. One of the C. sinensis strains among the isolates obtained from this study was also included for ITS sequence analysis. All the sequences from GenBank are listed in Table 2. Sequences were initially aligned with BioEdit 5.0.6 (Hall 1999) and analysed in PAUP 4.0b10 for Macintosh (Swofford 2001). Extra bases of the ITS fragment in several sequences from Genbank were edited. The alignment was further manually adjusted to reduce some obvious mismatch of sequences created by computer alignment.
A total of 29 sequences was included in the analysis and a data matrix containing 761 base pairs of nucleotides was established. A few dozen bases at both ends were excluded from the analysis owing to uncertainty in determining the sequence. Heuristic searches (Swofford & Olsen 1990, Maddison 1991), including TBR (tree bisection-reconnection) swapping for 1000 replicates of random taxon addition using equal weights were used to explore the set of possible trees from many starting points. Ten trees were saved at each replicate. Nucleotide substitutions were treated as unordered and alignment gaps as missing. Relative supports were assessed by bootstrapping (Felsenstein 1985) using equally weighted characters for 1000 replicates. The tree was rooted with the two basidiomycetes, Agaricus bisporus and Ustilago maydis.
Isolation source
Cordyceps sinensis (Berk.) Sacc.
60
Table 2. ITS sequences from GenBank used for sequence analysis.
Fungus
Taxonomic position
Accession #
Ajellomyces capsulatus Se _ McGinnis & Katz ©
Arthroderma persicolor (Stockdale) Weitzman et al. Bisporella citrina (Batsch) Korf & S.E. _Carp.
oe viburni aay Groves
Erysiphe cichoracearum DC.
Otani Geomyces asperulatus Sigler & J. W. Carmich. Geomyces pannorum (Link) Sigler & J Ww. _ Carmich. _
Geomyces pannorum
_ Carmich. var. pannorum co Gymnostellatospora japonica Udagawa et al.
Gymnoascus petalosporus (G. FE Orr et al. ) Gymnoascus punctatus (B. G. Dutta & G. oR, Ghosh) Arx.
Neofabraea malicorticis H. S. Jacks. _ Neurospora tetrasperma Shear & B. O. Dodge Pezicula ocellata (Pers.) Seaver Pseudogymnoascus roseus
Pseudogymnoascus roseus
Scleromitrula spiraeicola (Dennis) er Schumach. & Holst-Jensen
Sclerotinia trifoliorum Erikss.
Ustilago maydis (DC.) Corda
Agaricus bisporus (J. E. Lange) Imbach
Amauroascus mutatus (Quél.) Rammeloo
ae deformans Oita DNS
Gelatinipulvinella astraeicola Hosoya KY.
Myxotrichum arcticum Udagawa et al.
Arthrodermataceae;
_ Geomyces pannorum (Link) Sigler & J.W. |.
ad
Dermateaceae; Helotiales
Agaricaceae; Agaricales Onygenaceae; Onygenales Onygenaceae; Onygenales Onygenales
Helotiaceae; Helotiales
Dermateaceae; Helotials
ROM Rhytismatales |
Erysiphaceae; Erysiphales |
Helotiaceae; Helotiales nae as Anamorph Anamorph Myxotrichaceae
Gymnoascaceae; Onygenales
DU ie Sages
JSR, Dcainaiccceee OES
Sordariaceae; Sordariales
Myxotrichaceae Myxotrichaceae LOOM SELE Helotiales
SAL Loess oa
AF465404
AF038353
AJ271565
AJ000614
AF335454
AF141163
AF203469 AF011295
U72611
AJ390390
AJ509872
AF015789
AF307760
AF062818
AJ315829
AJ315825
AF062810
AF141189
AF388929 AF141199 ye Neier
Z81448
Z99676
Ustilaginaceae; Ustilaginales AF038826 ee tes g see orl
61
Morphological observation
The cultivated strains were examined frequently over 12 weeks to observe the growth of the colony. When ascomata were visible, daily observation was carried out to distinguish the different stages of ascomatal development. For ascomatal initial stages, minute tufts of hyphae were removed and placed in a drop of water on a slide for microscopic observation. For later ascomatal development stages, the ascomata were removed under the dissecting microscope and sectioned by using a freezing microtome or dissected on the slide to spread the asci and ascospores. Most preparations were mounted in water and observed immediately under the microscope. Some of the slides were stained with cotton blue in lactic acid to preserve the structure for later observation and photographing.
Results Molecular analysis
The complete sequences of the ITS region of the strains CS20, CS22, CS6-61 and CS18 were 521-543 bp long. The ITS sequences from CS20, CS22 and CS6-61 were identical and different from that of CS18, which was identified as C. sinensis. The sequences have been submitted to GenBank with accession numbers from AY608922 to AY608925.
The sequences obtained from the BLAST search could be divided into several groups based on the taxonomic position of the fungi. They were named Pesudogymnoascus and its related fungi, including P. roseus, Geomyces, Gymnostellatospora Udagawa et al. and Chrysosporium Corda. The majority of the sequences were of discomycetes, including Helotiales and Rhytismatales, and of other ascomycetes, including Dothideales and Erysiphales. The sequences selected for analysis represented the major groups found in the BLAST search. To clarify the systematic relationship of CS20, CS22 and CS6-61 with Onygenales species, sequences of Gymnoascus, Myxotrichum Kunze, Amauroascus, Arthroderma and Ajellomyces were included in the analysis.
A total of 675 bp of the ITS regions was used in the analyses. Among the nucleotides, 185 were constant. Of the remaining variable bases, 329 were potentially parsimony informative. Ten most parsimonious trees were obtained with this alignment. One of them is shown in Fig. 1. The sequences formed four groups marked as pseudogymnoascean, discomycetous, pyrenomycetous and onygenalean according to the species within each group. The other nine trees differed from Fig. 1 in the positions of AF081431- Pseudogymnoascus roseus and AF062818-Gymnostellatospora japonica within the pseudogymnoascean group; the position of AF203469-Elytroderma deformans being a sister group to both the pseudogymnoascean group and the major clade of the discomycetous group in some other trees; and the positions of AF335454-Bisporella citrina and U72611-Gelatinipulvinella astraeicola (as ‘astraoeca’ in Genbank) within the discomycetous group. | _ The pseudogymnoascean group comprised sequences of P. roseus, Geomyces and Gymnostellatospora and was supported by bootstrap analysis at 88% (Fig. 1). The sequences of CS20, CS22, CS6-61 and of AF062819, named P. roseus in GenBank, were identical. AF081431-P. roseus is an incomplete ITS sequence containing only ITS2 and partial 5.8S gene, almost identical to the same part of ITS sequences of AF062819-P roseus, CS20, CS22 and CS6-61. Although it was shown with no change from others
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of these sequences in Fig. 1, it was sometimes placed as a sister group to AF062819- P. roseus, CS20, CS22 and CS6-61 in the other parsimony trees. The grouping of these sequences in a terminal clade received 92% bootstrap support. Sequences of AJ509872 and AF015789, both named as Geomyces pannorum in Genbank, grouped together with 83% bootstrap support. They formed a clade with the P. roseus sequences having bootstrap support of 74%. The other two sequences of Geomyces, AF307760-G. pannorum and AJ390390-G. asperulatus, are almost identical with only two nucleotide substitutions. The two sequences clustered with AF062818-Gymnostellatospora japonica having 69% support in Fig. 1, but the latter was grouped with the P. roseus and Geomyces pannorum clade in some other trees. Several records of named Geomyces pannorum in GenBank (accession numbers from AJ509866 to AJ509871) have similar sequences to AJ509872 and AF015789 and were represented by the latter two in this analysis.
Thediscomycetous group mainly contained Helotialesand Rhytismatales. Myxotrichum arcticum (Myxotrichaceae) and Erysiphe cichoracearum (Erysiphaceae) were also included in this group. The discomycetous group was the sister group to the pseudogymnoascean group, but it was polyphyletic because one of the discomycetes, Elytroderma deformans, was placed as an immediate sister group to the pseudogymnoascean group (Fig. 1). In fact, the pseudogymnoascean group is imbedded within the discomycete taxa.
Neurospora tetrasperma (AF388929) and Cordyceps sinensis (CS18) were clustered together to form the pyrenomycetous group, which was supported by 100% in bootstrap analysis. The pyrenomycetous group is the sister group to the clade containing pseudogymnoascean and discomycetous groups.
Five species from four families of Onygenales were clustered together forming the onygenalean group. The support for this group was very strong, reaching 95% in bootstrap analysis although they demonstrated many variations in ITS sequences.
Morphological descriptions
Morphological characters of the strains, including colony, anamorph and teleomorph, were observed and described from culture. The developmental sequence of ascomata is described and illustrated in detail to demonstrate the formation of a massive ascoma. Culture: Isolates of CS20, CS22 and CS6-6lon bran-agar medium formed white colonies with thick aerial mycelium covering the sclerotium tissue. Colonies of the cultures were flocculant with woolly aerial hyphae, white at first but later becoming pinkish brown to purple, with pigmentation varying in the same colony. Reverse of the colony was red-brown to purplish brown. Ascomata appear in 6-8 weeks. Mature ascomata were pale yellowish-brown to pinkish brown and were either scattered upon the surface of the colony or aggregated into dense clusters. Finally the woolly aerial hyphae disappear and the colonies are covered with clay-pink, farinaceous granules. Microscopic observation: Vegetative hyphae were hyaline, 1.0-2.5 um diam. Ascomata were discrete or confluent, globose to subglobose, 52-320 um diam., at first white, finally pale pink to pinkish brown under the dissecting microscope. The outer part of the ascoma formed a defined layer of hyphae, which was usually regarded as a peridium. The peridium was composed of pale yellow to yellow-brown, septate, thick- walled hyphae, 2.0-3.0 um diam., sometimes thickening at the node reaching 3.5 um diam. Thin-walled hyphae arising from the thick-walled hyphae in the peridium were regarded as appendages, which were simple and up to 40 um long. Asci were hyaline,
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C&22 CS6-61 = S20 AF062819 Psezdogpmmoascus roseus oF AFO81431 Pseudogymmoascus roseus 7 } As509872 Geompoes parmorum ] - 33 8 AF015789Gecmpoes pernorum $0 al AJS3SSO0390 Geompoes asperulatus 2 38 " AF SU? 760 Geompees permorum var, pamnorien : io AFUE28 1 8Gymastellatospora japonica 38 AF 203459 Biptraderm a deformnans f4( = AP141163Dered vitor 5t 16 AF141199Pexicula ocellata 2 = AF1A11S9heofabraca malicorficrs 73 22 __ J99B7EStlerotvia frifaliorzan 23 1448 Sieromitide sniracicola ry 89 re te AP335454 Bisporelia citing 63 AFOG26 10 Mprotricizan ancticum AFO1 1295 Bivsiphe cichoraceaum 77611 Gelatinipedvinelia astraceicola ne 430 2 AF 338929 Neurospora isfrasperma 3 100 a C318 Cordycens sinensis a 28 _ A. 1915829 Gymmaascus petalospons % BS 38. A1915825 Grrmoascus purciates 6 52 AFOSESES Aeliompces capsud ahes 4 144 33 Bg Potent 95 AJOD0B 1 4 Arthroderma persicolor ks AJ27 1565 Amaeoascus mutates 183 AP ASSAM Agaricus bisnorus EL AF(IG8926 Ustilago maydis 50 changes
Fig. 1. One of the ten most parsimonious trees obtained from the analysis of nucleotide sequences of ITS regions (nrDNA). The upper and lower numbers on each branch denote the number of estimated substitutions and the percentage of bootstrap replicates respectively. Only bootstraps higher than 50% are shown. The length of the tree is 1607 steps, with consistency index=0.5083 and retention index=0.5401. Group 1=pseudogymnoascean, 2=discomycetous, 3=pyrenomycetous and 4=onygenalean.
_subglobose to oval, stalked, 8-spored, (6.0—) 7.0-9.0 um diam. Ascospores were hyaline, ellipsoid to fusoid-ellipsoid or fusoid, pale olivaceous to pink, smooth, yellowish brown in mass, 3.5-5.5x1.8-3.0 um.
Anamorph: Hyphae were hyaline, smooth and thin-walled, 0.8-2.5 um wide. Conidiophores were hyaline and dendroid, or sometimes absent. Conidia formed terminally or were intercalary, thin-walled, smooth, 3.0-6.0x2.0-3.5 um. The terminal conidia had a truncate base (about 1.8 um wide) and were cuneiform, ellipsoid or ampullaceous. The intercalary conidia were barrel-shaped. Numerous conidia sometimes
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Figs 2-9. Pseudogymnoascus roseus. Fig. 2. Anamorphic stage. Terminal and intercalary conidia were borne on dendroid conidiophores. Fig. 3. Ascoma initial. The ascoma initial arose as a short branch (arrow) from a vegetative hypha. Fig. 4. Two pairs of adjacent ascoma initials. The right two initials curved towards each other whilst the left two curved away to the opposite direction. Fig. 5. Ascoma initial coil. The other part of the initial is out of focus. Fig. 6. The same ascoma initial in Fig. 5 observed at a different focus. Hyphal branches emerge from the coil. Fig. 7. Close-up of two ascoma initials. The initials have complex coiling hyphae and branches. Fig. 8. Multiple ascoma initials from one site. Two tight ascoma initial coils (arrow heads) and two initial branches (arrows) are visible. Fig. 9. Young asci produced in groups. There is no ascospore delimitation at this stage. Bar=10 um, except for Fig. 2, where Bar=20 um.
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‘Figs 10-17. Pseudogymnoascus roseus. Fig. 10. Young ascospores formed within asci. Ascospores (arrow) are clearly visible at this stage and there is no envelopment of the asci, which were produced from the same ascoma initial. Fig. 11. Groups of asci. The groups of asci lack a defined envelope separating them from the surrounding hyphae and may merge together to form an ascus aggregation. Ascospores are visible within asci. Fig. 12. A small and immature globose ascoma. There is a released ascus to the upper right of the ascoma. Fig, 13. Part of a section of an ascoma. A large number of asci are enwrapped by a network of thick-walled hyphae (peridium). Fig. 14. Close-up of peridium in a section of an ascoma. The peridium hyphae are thick-walled with many branches. Fig. 15. Dissection of an ascoma. The asci in the ascoma are released at the upper right from the peridium, which is at the lower left. Fig. 16. Dissection of an ascoma. Asci are formed on stalks (arrow). Fig. 17. Released ascospores.
Bar=10 um in Figs 10-12, 14 and 17; Bar=20 um in Fig. 16 and Bar=30 wm in Fig. 13.
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aggregated together to form a subglobose conglobation. If the strains were kept for long enough, ascomata of P roseus appeared and the colonies became pinkish coloured in 6-8 weeks.
Development of ascomata: Ascomatal initials are scattered among, and formed from, vegetative hyphae. Initially, ascomatal initial branches, usually 2.5 um diam. wide, arise from vegetative hyphae. The short branch with thicker cytoplasm than surrounding vegetative hyphae (Fig. 3) was the earliest stage of ascomatal initial observed. As ascomatal initials grew, they became curved. Adjacent ascomatal initials might sometimes grow toward each other or apart from each other (Fig. 4). Figure 4 shows two pairs of adjacent initials: the right two initials curved towards each other while the left two curved away to the opposite direction. The conjunction of two initials and differentiation of gametangia were not detected. Ascomatal initials continued to grow and formed coils (Figs 5, 6). Figures 5 and 6 show an ascomatal initial coil observed at different focuses. Some thick branch structure emerged from the coil. The initials coiled further to form helical loops (Figs 5-8). Several initials arising nearby were at the same or different developing stages (Figs 7, 8). Figure 8 shows two tightly coiled ascomatal initials (arrow heads) and two ascomatal initial branches (arrows) which arose from the same hypha. Figures 5-8 also show irregular branches stretched out from the coiled initials. These branches can continue to grow and form new coils, or interweave to form an enveloping network in the later stages.
As the ascomatal initial develops, asci are produced. Asci are globose and produced in groups (Fig. 9). Asci begin to enlarge and ascospores are delimitated and form inside the asci (Figs 10, 11). The asci aggregated to form large structures without any defined envelope separating them from the surrounding hyphal components (Figs 10, 11). Figure 11 demonstrates groups of asci can be produced close to each other, and asci from different initials can result in single ascus aggregations. As the number of asci increases, ascomata also increase in size and appear as dark spheres under the microscope owing to the thickness preventing light transmission (Fig. 12). The early ascomata appear as small white points attached to white vegetative hyphae under the dissecting microscope.
As ascus aggregation develops and merges from different ascomatal initials, a network of thick-walled hyphae also envelops the asci. The thick-walled hyphae interweave to form a loose network, which is pale yellow to yellow in colour. Pigmentation of these hyphae increases as the ascomata enlarge. Eventually, a specified layer of the hyphal network, i.e. the peridium, is formed. Figures 13 and 14 are sections of ascomata showing a large number of asci surrounded by the hyphal network (peridium). The peridial network comprises thick-walled hyphae which are richly branched and anastomosed (Figs 14, 15). Some thin-walled, short branches from the hyphal network extend from the globose ascoma. Figure 15 demonstrates that the peridium is composed of loosely interwoven hyphae, which forms a net enveloping the densely packed asci. The whole ascoma appeared dark coloured under the transmission microscope, but as spheres with some shades of pink to pale purple under the dissecting microscope.
Figure 16 shows the attachment of asci to hyphal stalks squeezed out of an ascoma which had a hyphal network (peridium) enveloping the ascus aggregation. When the ascoma matures, a large number of ascospores are released from asci spreading out of the ascoma and among the hyphal network. The ascospores were fusoid to ellipsoid (Fig nl):
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Discussion
The sequence comparison revealed that the ITS sequences of CS20, CS22 and CS6-61 were identical to AF062819-Pseudogymnoascus roseus in GenBank. The DNA sequence analysis showed that these sequences and AF081431-P roseus, the partial sequence of ITS region included in this study, formed a unique terminal group. The strains CS20, CS22 and CS6-61 should be regarded as the same species as AF062819-P. roseus, which was submitted by Hambleton et al. in 1998 from strain UAMH 9163, isolated from roots of Abies lasiocarpa (Hook.) Nutt. in Alberta, Canada.
The morphological characters of CS20, CS22 and CS6-61 matched those described for P roseus by Cejp & Milko (1966), Orr (1979) and Currah (1985). A reference collection of P. roseus deposited at K (K(M) 116466: Pseudogymnoascus roseus, isolated from Eucalyptus, Australia, G. Johnson, 1975) was also examined for confirmation and they showed the same morphological features. Based on the molecular and morphological data, CS20, CS22 and CS6-61 were identified as P. roseus. The strains also exhibit an anamorphic stage with dendroid conidiophores, and cuneiform to ellipsoid and terminal or intercalary conidia. Compared with the morphological characters of Geomyces pannorum var. vinaceus (Sigler & Carmichael 1976, Oorschot 1980), the anamorphic stage of these strains should be classified in the genus Geomyces and might be the same as G. pannorum var. vinaceus.
The two G. pannorum sequences (AF509872 and AF015789) demonstrated only a few variations from that of P. roseus and formed a clade with P roseus in the ITS analysis. This result suggested that the two strains of G. pannorum were likely to be related to P roseus. The sequence variation between G. pannorum and P. roseus was nearly the same as within the G. pannorum clade.
Four sequences of Geomyces, i.e. three G. pannorum and one G. asperulatus, were separated into two clades. It is possible that more than one taxon has been involved in the strains named as G. pannorum. The sequence of AF307760-G. pannorum vat. pannorum is closer to AJ390390-G. asperulatus than to the other two G. pannorum isolates. Geomyces asperulatus and G. pannorum are similar in morphology. Geomyces asperulatus has hyaline, narrow, sometimes branching conidiophores 0.5-1.0 um long, and yellow, barrel-shaped (or cuneiform if terminal), long-chained arthroconidia whist G. pannorum differs from G. asperulatus in forming conidia in shorter chains and in having aleurioconidia formed laterally on the hypha. It is not easy to distinguish the two taxa based on morphology. It appears that molecular systematic analysis based on the DNA sequence data may be a reliable method to avoid misidentification. Gymnostellatospora japonica was clustered with two of the four Geomyces sequences (AJ390390 and AF307760), and is possibly related to P. roseus and Geomyces species, but they may not be the same species judged from the sequence variations and from the parsimony analysis. Gymnostellatospora was published and placed in Myxotrichaceae by Udagawa et al. (1993), based on gross morphological characteristics. Species of Gymnostellatospora possess yellowish or reddish brown ascomata; hyphal network of peridium; indistinct ‘appendages’; 8-spored, globose or ovoid, evanescent asci; and fusoid, hyaline to pale yellow ascospores (Udagawa et al. 1993). These features are similar to those of Pseudogymnoascus. However, the peridial hyphae of Gymnostellatospora are incompositoperidium-type (see Currah 1985) at maturity and the ascospore wall has
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narrow longitudinal crests and a convex surface, which is clearly different from that of Pseudogymnoascus. If the two genera are more widely sampled for investigation of molecular sytematics, it may be possible to find relationships between them.
The systematic position of the genera Gymnostellatospora, Myxotrichum and Pseudogymnoascus were classified in the same family, Myxotrichaceae. Members of these genera are similar in morphology, having brown shaded ascomata; hyaline, globose or subglobose asci; and hyaline, ellipsoid or fusoid ascospores. In the present ITS sequence analysis, Myxotrichum arcticum was distantly related to P roseus and Gymnostellatospora japonica. Myxotrichum arcticum was placed in the discomycetous group, and P. roseus and Gymnostellatospora japonica formed a separate clade with the anamorphic Geomyces. Members of Myxotrichaceae in this analysis were not in a monophyletic group, as speculated by Currah (1994) based on ascospore morphology. They were separated and embedded among discomycete taxa. Based on 18S rDNA analysis, Sugiyama et al. (1999) described two separate lineages within Myxotrichaceae, in which Myxotrichum was separated from Pseudogymnoascus. Hambleton et al. (1998) also reported that Myxotricum and Byssoascus Arx diverged significantly from Gymnostellatospora japonica and P. roseus in ITS sequence analysis. The result of the present study coincides with those reports by Sugiyama et al. (1999) and Hambleton et al. (1998). Although the close relationship between members of Myxotrichaceae and discomycetes has been revealed in molecular studies, their ascomatal structure and morphology differ remarkably. Asci of discomycetes are formed from a hymenium within the apothecium, but a hymenium or apothecium are absent in P. roseus and other species of Myxotrichaceae.
Pseudogymnoascus was formerly placed in Onygenales (e.g. Currah 1985, 1988). The onygenalean fungi produce evanescent asci in the mycelium or cleistothecial ascomata, and unicellular ascospores. To confirm their molecular relationship, several ITS sequences of onygenalean fungi were included in this analysis. The sequences of onygenalean fungi greatly diverged from those of P roseus, Geomyces pannorum and other selected Myxotrichaceae. Pseudogymnoascus roseus and Myxotrichum arcticum are separated from the onygenalean group by the pyrenomycetous group, and closely related to the discomycetes. The results of the present study reveal that the onygenalean group is distantly related to the Myxotrichaceae in ITS sequence and shows the independent evolutionary line of P. roseus and Myxotrichum from other onygenalean taxa as suggested by Currah (1994). Recent molecular systematic studies (Sugiyama et al. 1999, Mori et al. 2000) based on a different region of rDNA also obtained the same results.
The onygenalean fungi were rather isolated from other ascomycetes tested. The discomycete and pyrenomycete species were more closely related to each other than to the onygenalean fungi (Fig. 1). Furthermore, many variations in ITS sequences were also observed among the onygenalean fungi. It seems that there is a complex relationship in Onygenales and great divergence may exist within the order.
Morphologically, Pseudogymnoascus is similar to members of Onygenales in many aspects. The ascomatal stucture and brown colour of the reticulate hyphal network (peridium) in Pseudogymnoascus is very close to that of Gymnoascus. The characters of the peridium were very much emphasised in previous classifications and Pseudogymnoascus was considered to be related to Gymnoascus, which possesses oblate ascospores with an acute rim. Pseudogymnoascus differs from Gymnoascus only in the
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absence of peridial appendages and ascospore morphology. Currah (1985) noted that characters of ascospore morphology, enzymatic capacities, and nature and the occurrence of anamorphs provide significant information for classification. Based on ascospore morphology and cellulolytic capacities, Currah (1985) placed Pseudogymnoascus in Myxotrichaceae and Gymnoascus in Gymnoascaceae, thus separating these two genera in different families. Molecular investigations (Sugiyama et al. 1999, Mori et al. 2000, and this study) also consider that Pseudogymnoascus is widely separated from members of Gymnoascus and other onygenalean fungi.
Different peridial types evolved within the prototunicate ascomycetes in response to selective pressures affecting spore dispersal from enclosed areas (Currah, 1994). The loose hyphal network of the peridium exposes the asci and ascospores. Along with the evanescent asci, this may contribute to the release and dispersal of ascospores and increase the chance of propagation. The similar peridium occurring in quite different lineages of genera may be the result of convergent evolution.
The close relationship of P roseus with the discomycetes has been suggested by molecular investigations (Sugiyama et al. 1999, Mori et al. 2000, and this study). Pseudogymnoascus roseus and other members of Myxotrichaceae share some characters in common: unicellular ascospores; scattered (without a hymenium), thin-walled and evanescent asci; cleistothecial ascomata; and ascomatal peridium of a thin hyphal weft. These characters are very different from those of typical discomycetes. Mori et al. (2000) considered that the close relationship between Erysiphales, Myxotrichaceae and some discomycetous fungi (mainly Helotiales and Thelebolaceae) suggested a novel evolutionary pathway from cleistothecial discomycetous fungi to Erysiphales and Myxotrichaceae. The present study also revealed a close relationship between P. roseus, other members of Myxotrichaceae and discomycete fungi.
Pseudogymnoascus roseus shares a similar structure of ascoma and peridium with members of Onygenales. The ascomatal structure and loose hyphal network of the peridium found in P. roseus and other species of Myxotrichaceae may have been reversed from the advanced apothecium to primitive ascoma in Onygenales, if the latter is considered to have diverged earlier in the ascomycetes. This reversion may also have occurred more then once (at least in the pseudogymnoascean group and Myxotrichum arcticum (Fig. 1) in the course of evolution.
Based on the observation of ascomatal development in this study, the formation of ascoma in P. roseus was initiated from short branches of vegetative hyphae. Currah (1985) generalised that the fertile ascomata of Onygenales were initiated by the formation of paired gametangial hyphae. Ascomatal development in P roseus may be similar to that in Onygenales, but the microscopic observation made in this study did not detect the contact of two gametangia and the migration of nuclei or cytoplasm. ‘The structure of hyphae in the ascomatal initial coils was complex and it proved difficult to distinguish their constituents under the light microscope.
Several ascomatal initials often appeared very close to each other from the same site in the colony of P roseus. Asci from one or several initials might grow together without a defined envelope at early stages. Many asci grouped to form a large globose aggregation. A loose weft of hyphae (peridium) developed later, enveloping the aggregation. From the observation in this study, a large ascoma should be regarded as the ascus aggregation
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from several different ascomatal initials because it occupied a large space covering the area where different initials were formed. The peridium is only a loose network of thick-walled hyphae, and was termed a ‘reticuloperidium’ by Currah (1985). Like the ascoma, the peridium may also come from several different ascomatal initials. Although initial stages of ascomatal development and the structure of mature ascomata have been described (Cejp & Milko 1966, Orr 1979, Currah 1985), detailed observation of ascomatal formation has not been reported elsewhere. It is revealed for the first time that the ascomata in Pseudogymnoascus are formed by the aggregation of asci.
The morphology of ascospores and of peridial appendages are important taxonomic characters in recognising species of Pseudogymnoascus. The ascospores are generally considered smooth and the appendages not distinct in Pseudogymnoascus. Two species of Pseudogymnoascus have rather different features from P. roseus: P. dendroideus has ramified appendages and striated ascospores with slight thickening at the equator (Locquin-Linard 1982) and P. alpinus has navicular-fusoid ascospores, and branched and recurved appendages (Miller & Arx 1982). Either the taxonomic position of these two species is in need of reconsideration or the generic concept of Pseudogymnoascus must be revised. The species that Lumley et al. (2000) described as ‘“Pseudogymnoascus sp. also requires further investigation because the longitudinal sigmoid crest on the ascospores is not characteristic of Pseudogymnoascus. Molecular research may elucidate the relationships of these morphologically different fungi.
Pseudogymnoascus roseus is distributed worldwide and has often been isolated from alpine regions. It is interesting that the fungus was also isolated from sclerotia of C. sinensis in this study. Because of its medicinal value, C. sinensis has been the target of efforts to obtain pure isolates for massive production in industrial fermentation. Various fungi, about 22 names in 13 different genera, have been related in the literature to the anamorph of C. sinensis, either for isolates from material of the fungus or for postulated asexual states (Jiang & Yao 2002). Although many of the isolates are not true C. sinensis, they may have similar chemical properties and medicinal effects, e.g. Paecilomyces sinensis Q. T. Chen et al. and Tolypocladium sinense C. Lan Li (Gui & Chen 1983, Li 1988, Liu et al. 1991). Some of those isolates are even widely used in manufacture referring to the products of C. sinensis (Jiang & Yao 2002, 2003). It may be ascribed to the interaction of fungi or selective pressure in the same ecological environment that different fungi produce similar medicinal properties. Probably the strains of Pseudogymnoascus roseus were present in the alpine soil of the Tibetan Plateau and contaminated the Cordyceps isolates. As another micro-organism living in the same niche with C. sinensis, whether P. roseus has similar chemical properties that can be used for medicinal purpose may deserve further investigation. Currah (1985) suggested that the pale rose reticuloperidium and ascospores of P. roseus probably indicated a reliance on burrowing animals for dispersal. The host of C. sinensis, the caterpillar larvae of Hepialus armoricanus Obertheir, living in the same microcosm may also be one of the animals helping the dispersal of P. roseus ascospores.
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Acknowledgements
The authors are grateful to Drs B. M. Spooner and P. D. Bridge for serving as pre-submission reviewers and for their valuable comments and suggestions. This project is supported by the Chinese Academy of Sciences and the National Natural Science Foundation of China through the ‘Key Research Direction of Renovation Program (KSCX2-SW-101C)’ and the scheme of ‘Introduction of Overseas Outstanding Talents, and ‘National Science Fund for Distinguished Young Scholars’ (30025002) respectively.
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MYCOTAXON Volume 94, pp. 75-84 October-December 2005
Morphological and molecular characterization of the mycorrhizas of Inocybe rufuloides and I. splendens
Mirco [ottT1, MAURO MARCHETTI, ENRICO BONUSO & ALESSANDRA ZAMBONELLI
zambonel@agrsci.unibo.it Dipartimento di Protezione e Valorizzazione Agroalimentare via Fanin 46, 40127 Bologna, Italy
Abstract—The mycorrhizas of Inocybe rufuloides on Pinus pinea and of Inocybe splendens on Ostrya carpinifolia were characterized by morphological and molecular methods. Molecular identification was performed by comparing the rDNA ITS sequences obtained from Inocybe fruiting bodies with those of the mycorrhizas collected from the same area. Morphological characterization was carried out by a simplified Agerer protocol. This study provides sets of characters which can be used in further studies for the identification of these two Inocybe species found contaminating Tuber infected plants in experimental truffieres.
Key words—ectomycorrhizas, ribosomal DNA sequences, morphological description, truffle cultivation
Introduction
Ectomycorrhizas are dominant in forests of temperate and boreal regions of the Northern Hemisphere (Brundrett et al. 1996). It has been estimated that 6000 or more species of fungi form ectomycorrhizal associations with approximately 10% of the Angiosperms and many Gymnosperms (Trappe 1987). The ectomycorrhizal communities are dynamic and changes in their population structure occur depending on the age of the host plants, season and environmental factors (Molina et al. 1992, Deacon & Fleming 1992). In the past research into ectomycorrhizal fungal diversity were almost exclusively carried out by identifying and counting fruiting bodies that appeared above the ground or by inspecting the roots of trees and grouping their ectomycorrhizas into morphotypes (Selosse 2001). While the first method is relatively quick and easy the number of species of ectomycorrhizal fungi that fruit above the ground is generally considered to be a gross underestimate of the number of ectomycorrhizal fungi present in an environment (Yamada & Katsuya 2001, Selosse 2001). For example, ectomycorrhizal fungi such as Cenococcum geophilum and many members of Telephoraceae, which are some of the most common mycorrhizas (Richard et al. 2005, Horton & Bruns 2001), do not produce fruiting bodies or form cryptic fruiting structures. In contrast, although morphotyping of ectomycorrhizas give a better picture of fungal diversity and subtle changes that can occur during competition in ectomycorrhizal communities, it is often impossible to relate the morphotypes to a known fungal species (Horton & Bruns 2001).
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The recent application of molecular methods has revolutionised our ability to identify ectomycorrhizal fungi. They are useful either for the certification of plants that have been inoculated with commercially valuable species such as Tuber and also allow us to carry out more ambitious studies on ectomycorrhizal community structures and competition within them (Amicucci et al. 2002, Dahlberg, 2001). A comparison of ITS sequences obtained, for example, from traditionally identified fruiting bodies with those obtained from mycorrhizas can provide a means for identifying the fungi that form mycorrhizas (Horton & Bruns 2001). However ITS sequences for many ectomycorrhizal fungi have yet to be determined and so their identification remains impossible.
The aim of the work presented here was to provide a set of molecular and morphological characters for the identification of Inocybe rufuloides and Inocybe splendens by ITS sequencing of the mycorrhizas and of their fruiting bodies and the morphological description of their mycorrhizas which were found as contaminants in cultivated truffieres.
Materials and methods Sampling
The fruiting bodies of the two Inocybe species used in this study were collected from two different experimental trufhéres of Emilia Romagna, Italy. I. rufuloides was found in October 2003 in a Pinus pinea - Tuber borchii productive trufheére established in 1990. The plantation is located in Marina di Ravenna on the Adriatic coast (49° 30’ 24” N, 17° 60’ 70” E) on littoral sandy soil (Zambonelli et al., 2000) while I. splendens was collected in March 2004 in a Tuber aestivum trufhere established in 1997 with Corylus avellana, Ostrya carpinifolia and Quercus pubescens infected seedlings in the park of S. Giulia, Palagano, Modena (44° 23’ 50°N, 10° 39° 40°E, elevation 932 m) (Zambonelli et al. 2005). I. rufuloides was found near P. pinea in the T: borchii trufhiére and I. splendens close to the trunks of the O. carpinifolia seedlings in the T. aestivum truffiere. Dried specimens of each species were deposited in the herbarium of the “Centro di Micologia” of Bologna (I. rufuloides n.1994, I. splendens n. 5033).
Root samples were also collected from the truffieres under fruiting bodies by removing 6 cm diameter soil cores between 0-30 cm. The soil cores were stored overnight in refrigerator at 5 °C and the roots then removed by carefully washing over an 0.8 mm mesh sieve.
Morphological characterisation
The I. rufuloides and I. splendens basidiomes were identified on the basis of their macro- and microscopic morphological characters (Heim 1931; Kuyper 1986; Stangl 1989; Moser et al. 1994, 1996; Bon 1997; Franchi et al. 2001). The microscopic features of the basidiomes were examined in both fresh and dried material previous rehydrated in 10% KOH and stained with Congo red.
Samples of mycorrhizas putatively formed by Inocybe sp. were selected from the root samples of Pinus pinea and Ostrya carpinifolia on the basis of their morphological features following Agerer’s descriptions of Inocybe mycorrhizas (Agerer 1987-2002). Their morphological features were then described using sets of characters suggested
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by Agerer (1987-2002, 1991). The colour of the fresh mycorrhizas was recorded under a stereomicroscope and compared with the Royal Botanic Garden colour identification chart (RBG chart) (1969). Mycorrhizal tips were then fixed in FAA bleached by heating to 90 °C in 10% w/v KOH and treated with a few drops of H,O, for 20-30 seconds in order to better examine the anatomy of the mantle under the microscope (Giomaro et al. 2000). Ectomycorrhizal tips were fixed in glutaraldehyde (25%), embedded in Tissue Tek OCT (Sakura, Zoeterwounde, Netherlands) compound and then cut with a rotary cryomicrotome (Tissue Tek® II, Miles, Elkhart, IN, USA) (8-10 um thickness). Serial sections were stained with cotton blue, mounted in lactic acid and observed under a ECLIPSE TE 2000-E microscope (Nikon, Tokyo, Japan).
Mean dimensions of fruiting body and mycorrhiza characters were determined using Axio Vision 2.05 software (Carl Zeiss Vision GmbH, Hallbergmoos, Germany) from images captured with a DXM1200F digital camera (Nikon, Tokyo, Japan) and standard deviations then calculated. Each biometrical character was the mean of at least 50 measurements.
Molecular characterisation
Molecular identification of the mycorrhizas and fruiting bodies was performed using sequence data of the ITS regions of the ribosomal DNA. Total genomic DNA was isolated by DNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and then eluted in 50 ul of sterile water. Ten mycorrhizas of each morphotype were pooled and 100 ug of fruiting body tissue were used. ITS-1, 5.88 and ITS-2 regions were amplified in a 50 ul volume reaction containing 1-10 ng of genomic DNA, using the primers pair ITS1 and ITS4 (White et al., 1990) in a T gradient Thermal Cycler (BIOMETRA, Gottingen, Germany) according to Amicucci et al. (1996). PCRs were performed using 1.5 units of Taq DNA polymerase (Fermentas, Vilnius, Lithuania).
The amplified products were first purified by Gene Clean II kit (BIO 101, Vista, CA, USA) and then sequenced using both the primers mentioned above. Sequence reaction was performed using the ABI PRISM 3700 DNA Analyzer (Applied Biosystem, Foster City, CA, USA). The obtained ITS sequences of fruiting bodies and mycorrhizas were compared each other and with those on the GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) and UNITE (http://unite.zbi.ee/analysis.php3) databases using BLASTN search (Altschul et al., 1997). The ITS sequences of the fruiting bodies obtained in this study have been deposited in GenBank with the following accession numbers: I. rufuloides (DQ067579) and I. splendens (DQ067580).
Results
Morphological characterization
-Inocybe rufuloides Bon
The I. rufuloides basidiomes had pileus 15-35 mm broad, campanulate, conico-convex, then plano-convex, at maturity applanate, with or without umbo, with margin inflexed when young, dark brown, orange-brown, tomentose around disc, outwards fibrillose, with fibrils diverging and sometimes squamulose-subsquarrose. Lamellae were adnexed
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Fig. 1 - Caulocystidia (a), spores (b), cheilocystidia (c) and pleurocystidia (d) of I. rufuloides (Bars = 10 um).
to adnate, L = 28-45, | = 1-3, moderately crowded, ventricose, 4-8 mm broad, pale greyish buff, then brown, dark brown, edge fimbriate, whitish. The stipe was 25-50 x 3-5 (6) mm, equal to clavate, subbulbous, brown to orange brown, extreme apex hairy- pruinose, near base whitish, downwards longitudinally white-fibrillose. The context was white in pileus, red-brown in stipe; smell and taste spermatic. The spores were (8) 10.5 + 1.4 (13) x (4.7) 5.9 + 0.7 (7) um, Q = (1.5) 1.8 + 0.2 (2.1), smooth, ovoid to amygdaliform, with subobtuse to indistinctly conical apex (Fig. 1b). The basidia were 28-38 x 9-12 um, 2-4-spored. The hymenial cystidia were 50-70 x 10-20 um, cylindrico-clavate, clavate, fusiform, lageniform, sometimes subcapitate, with a thick wall 1.5-2.5 um, slightly yellow with ammoniac (Figs 1c & 1d). Caulocystidia were absent or present only at stipe apex (to 1/10"), similar to hymenial cystidia and mixed with cauloparacystidia (Fig. 1a), downwards are present caulocystidioid hairs.
I. rufuloides mycorrhizas were simple, ramified or with limited dichotomous branching involving a few lateral root tips, rarely coralloid. The unramified ends were short, straight or slightly twisted, 594.4 + 167.3 um long and 419.1 + 79.0 um in diameter. The structure of the surface was woolly with whitish emanating hyphae. The mycorrhizal tips were white (RBG chart n. 7) or greyish-cream (RBG chart n. 1) and orange-cream (RBG chart n. 44) at the base. When viewed with a microscope the mantle was plectenchymatous and had three distinct layers. The outermost was composed of partially bundled, loosely woven hyphae which were without a gelatinous matrix (Fig. 3a) whereas the middle and inner layers were made of tightly appressed hyphae (Fig. 3b & 3c). The mantle was 68.5
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Fig. 2 - Caulocystidia (a), pleurocystidia (b) and spores (c) of I. spendens (Bars = 10 um).
+ 13.1 um thick and the Hartig net was one, rarely two cells deep, 3.5 + 1.1 wm thick and composed of a single hyphal row. In cross sections the mantle was composed of loosely arranged cells 4.7 + 0.8 um x 2.6 + 0.5 um in the external layers and 4.4 + 0.9 um x 2.8 + 0.5 um cells in the inner layer. The root cortical cells were tangentially oval 43.0 + 9.1 um x 19.6 + 4.4 um. The root cortical cells with Hartig net were radially oval 33.0 + 7.4 um x 18.0 + 4.1 um. In longitudinal section the mantle was composed of loosely arranged hyphal cells 5.4 + 1.2 um x 3.4 + 0.9 um in the external layer and of 5.0 + 1.2 um x 3.0 + 0.6 um in the inner layer. The shape of the root cortical cells was oval 58.3 + 15.7 um (tangentially) x 24.7 + 5.5 um. The root cortical cells adjacent to the Hartig net were 38.9 + 9.8 um (tangentially) x 19.7 + 4.6 um. The emanating hyphae (2.1 + 0.4 um thick) possessed clamp connections with a distinct hole and had a characteristically constricted fusion point between the arched part of the clamp and the parental hypha (Fig. 3d). Rhizomorphs and cystidia were absent.
Inocybe splendens R. Heim
I. splendens basidiomes had pileus 30-50 (70) mm broad, convex, soon plano-convex, finally applanate, with inflexed margin, later straight, often with conspicuous but low broad umbo, brownish-ochraceous to dark brown, sericeous-fibrillose, with fibrils not or diverging, sometimes radially rimulose. Lamellae were adnate to almost free, L=45-65, l=1-3, crowded, ventricose, 3-9 mm broad, whitish then greyish-yellow, finally yellow- brown, sometimes with olivaceous tinge, edge whitish, fimbriate. The stipe was 25-90
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Fig. 3 - Mantle of I. rufuloides mycorrhizas (a external, b middle, c internal layers) and of I. splendens mycorrhizas (f external, g internal layers) under a light microscope. Clamp connections of I. rufuloides (d) and of I. splendens (e) (Bars = 5 um).
x 6-15 mm, sometimes equal, but often marginately bulbous or subbulbous, whitish, finally ochraceous-yellow to pale brownish, pruinose all over. The context was white in pileus, ochraceous-yellow in stipe; smell subspermatic or as Amanita phalloides, taste not distinct. The spores, were (8) 10.7 + 1.513) x (4.8) 5.9 + 0.9 (7) um, Q = 1.7.0.2 (20), smooth, amygdaliform, with suprahilar depression, with subconical apex (Fig. 2c). The basidia were 26-40 x 8-12 um, 4-spored. The hymenial cystidia were (45) 50-80 x 14-20 (27) um, clavate, fusiform to utriform, sometimes sublageniform, with a thick wall 1.5-3.0 um, colourless or slightly yellow with ammoniac, with crystalliferous at apex (Fig. 2b); paracystidia pyriform, thin-walled, colourless. The caulocystidia were similar to hymenial cystidia, descending almost to base of stipe, mixed with cauloparacystidia throughout (Fig. 2a).
I. splendens mycorrhizas were simple or with monopodial ramification with few lateral root tips. The unramified ends were straight or slightly twisted 965.2 + 353.2 mm long and 222.4 + 29.4 mm in diameter. The structure of the surface was woolly with whitish
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emanating hyphae. The colour of tips was white (RBG chart n. 7) or greyish-cream (RBG chart n. 1) and orange-cream at the base (RBG chart n. 44). Under a microscope the mantle was plectenchymatous, with two distinct layers, formed of closely appressed hyphae and without a gelatinous matrix (Fig. 3f & 3g). The thickness of the mantle was 27.2 + 5.3 um. The Hartig net was one, rarely two cells deep, 2.4 + 0.5 um thick, and one cell wide. In cross section the mantle was composed of appressed hyphal cells 4.2 + 1.3 um x 2.0 + 0.6 um in the external layer and of 4.1 + 1.3 um x 2.0 + 0.7 um in the inner layer. The root cortical cells were approximately tangentially oval 31.7 + 6.1 um x 20.9 + 5.0 um. The root cortical cells adjacent to the Hartig net were radially oval 27.7 + 4.5 um x 14.0 + 2.7 um. In longitudinal section the mantle was composed of appressed hyphal cells 4.4 + 0.8 um x 2.5 + 0.6 um in the external layer and 4.3 + 1.0 um x 2.5 + 0.6 um in the inner layer. The shape of the root cortical cells was oval 40.0 + 7.5 um (tangentially) x 19.9 + 4.5 um. The root cortical cells with Hartig net were 37.9 + 7.4 um (tangentially) x 13.1 + 3.5 um. The emanating hyphae (2.0 + 0.3 um thick) possessed clamp connections with a small hole and with a characteristically constricted fusion point where the arched part of the clamp met the parental hypha (Fig. 3e). Rhizomorphs and cystidia were lacking.
Molecular characterisation
The amplicons of I. rufuloides and I. splendens resulting from ITS1/ITS4 amplification showed length of 690 pb and 710 pb respectively. No differences were found between sequences obtained from mycorrhizas and from the respective fruiting body of each Inocybe species. The sequences resulting from ectomycorrhizae and fruiting body of I. rufuloides showed 93% level of similarity (510/544 nt) with a nearly complete ITS1- 5.8S-ITS2 sequence of an uncultured ectomycorrhiza of Inocybe (accession number AY825514) described by Richard et al. (2005). Instead, the sequences resulting from ectomycorrhizae and fruiting body of I. splendens showed 90% level of similarity (580/640 nt) with a nearly complete ITS1-5.8S-ITS2 sequence of an uncultured fungus from ectomycorrhizal root isolate (accession number AY702725) described by Izzo et al. (2005). Lower similarities were obtained with the Inocybe sequences in the UNITE database.
Discussion
The molecular and morphological characterization of I. rufuloides and I. splendens mycorrhizas provide reliable instruments for the identification of these two fungi that are potential contaminants in natural and cultivated truffieres.
Many species of ectomycorrhizal fungi are able to contaminate cultivated truffieres such as less valuable Tuber species, Cenococcum spp., Hymenogaster spp., Tomentella spp., Scleroderma spp., Hebeloma spp., and several, as yet, unidentified fungi known only as morphotypes (Donnini & Bencivenga 1995, De Miguel & Saez 2005). This replacement of inoculant fungi in truffieres by more aggressive competing fungi is one of the most important problems during the cultivation of truffles (Sourzat 2001). These ectomycorrhizal species usually either have a broad range of adaptability to different ecological conditions or ecological requirements similar to cultivated truffles (Dalberg 2001, Giovannetti 1983). I. rufuloides is reported to grow in association with Pinus
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maritima on sandy soil along the Mediterranean coasts while I. splendens is associated with broad-leaf trees on calcareous sand and clay soils in Europe (Kuyper 1986). These are the same habitats where T. borchii and T. aestivum grow respectively. The discovery of Inocybe spp. in cultivated trufhéres and natural Tuber magnatum (Giovannetti 1983, Pirazzi 2001), T. aestivum (Chevalier & Frochot 1997) and T: borchii (Zambonelli et al. 2002) truffiéres, and their ecological requirements seems to confirm that members of this genus, might be important competitors.
The morphological characterization of I. rufuloides and I. splendens mycorrhizas confirms that some characters such as the plectenchymatus mantle structure, the form of the fusion point of clamp connections can be useful to distinguish the mycorrhizas formed by the species belonging to the genus Inocybe (Agerer 1987-2002). However, the only morphological characters don't allow identification at species level. Moreover, colour and form of the mycorrhizas can vary with their age; the form of the mantle cells, which is a quite important taxonomic character, can show some differences depending on the fungal strain and the host plant (Giomaro et al. 2000, 2002; Sisti et al. 2003).
The early identification of the presence of contaminant fungi in cultivated trufheres offers growers the possibility to promptly introduce cultivation procedures that favour the establishment and development of Tuber ectomycorrhizas such as correct irrigation rates and mulching (Zambonelli et al. 2005).
Molecular analyses have allowed us to unequivocally identify I. rufuloides and I. splendens mycorrhizas by comparing their ITS sequences in the symbiotic and reproductive phases. The ITS sequence of I. rufuloides and I. splendens deposited in genbank will be a useful tool for other researchers who need to confirm the morphological identification of these fungi by the comparison of their sequences and, for example, will permit them to quantify the extent of problems caused by these fungi and develop specific strategies for their control.
Acknowledgements
The authors thank Dr. Ian Hall and Prof. Ursula Peintner for their manuscript presubmission review and for the help in the arrangement of the manuscript.
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Giomaro G, Zambonelli A, Sisti D, Cecchini M, Evangelista V, Stocchi V. 2000. Anatomical and morphological characterisation of mycorrhizas obtained in vitro using Tilia platyphyllos Scop. plantlets and five different strains of Tuber borchii Vittad. Mycorrhiza 10: 107-114.
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MYCOTAXON
Volume 94, pp. 85-88 October-December 2005
Russula in Himalaya 1: A new species of subgenus Amoenula
K. Das’, S.L. MILLER’, J.R. SHARMA, P. SHARMA4, & R.P. BHATT*
daskanad@yahoo.co.in ' Mycology and Plant Pathology Group, Agharkar Research Institute G.G. Agarkar Road, Pune 411 004, India
?Botany Department, University of Wyoming Laramie, Wyoming 82071 USA
Botanical Survey of India, 192, Kaulagarh Road Dehradun 248195, India
*Department of Botany, H.N. Bahuguna Garhwal University Srinagar (Garhwal) 246174, India
Abstract—Russula mukteshwarica, a species closely related to R. violeipes, is proposed here as new to science. Macro- and micromorphological characters of this species are described and illustrated in detail.
Key words—macrofungi, Russulaceae, taxonomy, phylogeny, India
Introduction
Extensive investigation on Himalayan Russulaceae has been carried out by the authors (Das & Sharma 2003, Das et al. 2003, Das & Sharma 2004, Das et al. 2004, Das & Sharma 2005, Das et al. 2005) for the last eight years. During macrofungal surveys of Kumaon and Garhwal Himalaya, the authors came across a species of Russula which after thorough macro- and microscopic studies followed by molecular analysis appeared to be an undescribed taxon and is proposed here as Russula mukteshwarica.
Materials and Methods
Macroscopic characters were noted from fresh material. Microscopic characterization was done from dried material by mounting free hand basidiome sections in 5% KOH, Melzer’s reagent, Congo red, Lactophenol-cotton blue and Carbol Fuchsin. Colour terms follow Kelly & Judd (1955). Microscopic line drawings were made with the aid of a camera lucida at original magnification of 1500x for basidiospores, 1000x for other microstructures. Density of lamellae (L/cm) was measured at the margin of the pileus excluding lamellulae. Spore print colour follows Romagnesi (1967). Basidiospores length excludes the length of ornamentation. Basidium length excludes the length of sterigmata.
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Quotient (Q = L/W) was calculated considering the mean value of length and width of 25 basidiospores. Herbarium names used follow Holmgren et al. (1990). Materials and methods for rDNA sequencing followed those of Miller & Henkel (2004).
Description of the species
Russula mukteshwarica K. Das, S.L. Mill. J.R. Sharma & R.P. Bhatt sp. nov. Figure 1 Etymology: From Mukteshwar, referring to the type locality.
Pileus 65-130 mm diam., planoconvexus, leviter depressus in centro, purpureus ad violaceus. Lamellae adnatae, confertae, luteae. Stipes 45-87 x 14-27 mm, cylindricus, purpureus. Sporae in cumulo albae, 7.6-9.3 x 7.3-8.2 um, subglobosae vel globosae, amyloideae, verrucosae et incomplete reticulatae. Pleurocystidia 80-110 x 11-17 um, fusiformia. Cheilocystidia 70-100 x 11-16 um. Pileipellis bistrata. Typus: INDIA, Uttaranchal, Nainital, Mukteshwar, August 2003, leg. K. Das, KD2120 (HOLOTYPUS, BSD; ISOTYPUS, TUR-A) ;
Pileus 65-130 mm diam., planoconvex, becoming umbilicate with depressed center at maturity; pileipellis dry, viscid when moist, pruinose to subvelvety, dark purple, deep to very deep purple or deep to dark violet, light to brilliant or very greenish yellow at the center; margin slightly decurved, almost plain at maturity, irregularly lobed, splitted, peeling up to 1/4" of the radius. Lamellae broadly adnate to adnexed, close (ca 7-9 per cm), forked from the base, brittle, cream to pale yellow or light greenish yellow; lamellulae present; edges even. Stipe 45-87 x 14-27 mm, cylindric to subclavate, dry, pruinose, light reddish purple at the top, gradually with white areas downwards but always with a white rim between the juncture of lamellae and stipe, pale greenish yellow (lemon yellow) at the base. Context stuffed, cream, changing light brown with phenol. Taste mild. Spore print white (Ia).
Basidiospores 7.6-9.3 x 7.3-8.2 um, subglobose to globose (Q = 1-1.15, av. 1.05-1.10); ornamentation amyloid, composed of warts (=0.75 um long) and ridges forming incomplete reticulum. Basidia 30-45 x 7-9 um, subclavate to clavate, 4-spored; sterigmata =6.5—7 um. Pleurocystidia 80-110 x 11-17 tm, abundant, emergent up to 40 um, ventricose, subfusiform to fusiform, thick walled, content dense; wall up to 1.3 um thick. Lamellae edge fertile, composed of basidia and cystidia. Cheilocystidia 70-100 x 11-16 um, thick walled, same as pleurocystidia. Subhymenium layer narrow, up to 12 um thick, cellular. Pileipellis two layered; upper layer composed of suberect to erect subulate, septate hyphae, 5-11 um broad, pileocystidia absent; subpellis cellular.
Ecology - Russula mukteshwarica grows in close association with species of Quercus, Rhododendron and Myrica in moist deciduous to mixed subtropical to temperate (1800-2200 m) forests.
OTHER SPECIMENS EXAMINED: INDIA, Uttaranchal: NarniTAL, Mukteshwar, August 2003, leg. K. Das, KD2127; PirHoRAGARH, Dafia Dhura, September 2001, leg. K. Das & J.R. Sharma, KD4082; Pauri, Nagdev forest on path to Snake Temple, June 2003, leg. P. Sharma & S.L. Miller, PR310; ibid., June 2003, leg. S.L. Miller & P. Sharma, SLM 03-04, . SLM 03-07; ibid., Khirsu, June 2003, leg. S.L. Miller & P. Sharma, SLM 03-22.
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Fig. 1. Russula mukteshwarica (from holotype). a. Basidiomes. b. Basidiospores. c. Pleurocystidia. d. Cheilocystidia. e. Cross-section of pileipellis. Bars: a = 10 mm; b-e = 10 um.
Comments - Typically subulate, up to 70 um long hyphal ends of pileipellis and absence of dermatocystidia undoubtedly place the taxon in the subgenus Amoenula Sarnari. This species resembles Russula amoenicolor Romagn., R. violeipes Quél. and R. amoena Queél. from Europe. However, all these three species have a different spore print colour which varies from Ila—IIc (Romagnesi 1996), or from Ia-IIa (Sarnari 1998). Moreover, the red to purple colour of stipe base in R. amoenicolor; presence of distinctly globose lower cells of the pilear hyphae in R. violeipes and the comparatively narrower pleurocystidia and reddish stipe base in R. amoena further separate these three species from R. mukteshwarica. Molecular analysis gathered from rDNA sequencing of the ITS gene
é
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region (not shown) has also confirmed that the new species belongs to the subgenus Amoenula and is closely related to R. violeipes.
Acknowledgements
We wish to express our gratitude to Dr. M. Sanjappa, Director and Dr. D.K. Singh, Joint Director, Botanical Survey of India, Kolkata and Dr. V.S. Rao, Director, Agharkar Research Institute, Pune for providing facilities during the present study. We are thankful to Mr. Jukka Vauras, Abo akademi University (Turku, Finland) and Dr. L.L. Norvell (USA) for critically reviewing the manuscript and checking the Latin diagnosis. This research was also partially supported by funding to S.L. Miller from the National Science Foundation (DEB-0315607), USDA (2003-01542) and EPSCoR (04- 47681). We gratefully acknowledge Terry McClean from the Nucleic Acid Exploration Facility at the University of Wyoming for sequencing these specimens.
Literature Cited
Das K, Sharma JR. 2003. New and interesting species of Lactarius from India. Mycotaxon 88: 7 I-38).
Das K, Sharma JR. 2003. New records of Russula from Kumaon Himalaya. Indian Journal of Forestry 26 (3): 320-326.
Das K, Sharma JR. 2004. Lactarius in Kumaon Himalaya 2: New and interesting species of subgenus Plinthogali. Mycotaxon 89 (2): 289-296.
Das K, Sharma JR. 2005. New records of Lactarius from India. Annals of Forestry 13 (1): 1-8.
Das K, Sharma JR, Verbeken A. 2003. New species of Lactarius from Kumaon Himalaya, India. Mycotaxon 88: 333-342.
Das K, Sharma JR, Montoya L. 2004. Lactarius (Russulaceae) in Kamaon Himalaya. 1. New species of subgenus Russularia. Fungal Diversity 16: 23-33.
Das K, Sharma JR, Basso MT, Bhatt, RP. 2005. Lactarius (Russulaceae) in Kumaon Himalaya 4: A new species of subgenus Piperites. Mycotaxon 91: 1-7.
Holmgren PK, Holmgren NH, Barnett LC. 1990. Index Herbariorum. Part 1: Herbaria of the world, 86" ed. Bronx: New York Botanical Garden.
Kelly KL, Judd DB. 1955. The ISCC-NBS Method of Designating Colors and a Dictionary of Colour Names. ISCC-NBS Color-Name Charts Illustrated with Centroid Colors. National Bureau of Standards Circular 553. U.S. Government Printing Office, Washington, DC.
Miller SL, Henkel TW. 2004. Biology and molecular ecology of subiculate Lactarius species from Guyana. In C.L. Cripps, ed., Fungi in forest ecosystems - systematics, diversity and ecology. New York Botanic Garden, vol. 89, pp. 297-313.
Romagnesi H. 1996. Les russues d’Europe et d'Afrique du nord. Bordas, Paris.
Sarnari M. 1998. Monografia illustrata del genere Russula in Europa. Tomo Primo. Italia. 799 pp.
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MYCOTAXON
Volume 94, pp. 89-92 October-December 2005
Streptopodium passiflorae comb. nov. on Passiflora rubra
J. R. LIBERATO * & R. W. BARRETO ?
jose.liberato@dpi.qld.gov.au ' Department of Primary Industries & Fisheries, Plant Pathology Herbarium 80 Meiers Rd, Indooroopilly, Qld 4068, Australia
? Universidade Federal de Vicosa, Departamento de Fitopatologia Vicosa, MG, 36571-000, Brazil
Abstract — Reexamination of Ovulariopsis passiflorae, the causal agent of a powdery mildew on Passiflora rubra, revealed that the fungus has dimorphic conidia and conidiophores formed from the external mycelium, a combination of features typical of Streptopodium. An emended description of this species is provided and the new combination Streptopodium passiflorae is proposed.
Key words — Erysiphaceae, Phyllactinieae
Introduction
Ovulariopsis passiflorae Syd. (Erysiphaceae) was described on Passiflora rubra L. based on a specimen collected in Venezuela by Sydow (1930). Its published description suggested that its maintenance in Ovulariopsis Pat. & Har. under the present concept for the genera in the Phyllactinieae (Braun et al. 2002) might be inadequate. A re-examination of the type specimen was made, confirming this suspicion. This paper reports the results of this new study of the type material and the nomenclatural change that resulted from it.
Material and Methods
In order to elucidate whether the conidiophores were produced from external or internal mycelium, a critical feature for separating genera in the Phyllactinieae, a whole leaf clearing and staining technique was used. Leaf pieces were immersed in solution of 50 g chloral hydrate in 20 mL of distilled water and left in stoppered glass vials at room temperature, for 24 h. The leaf pieces were then mounted on microscope slides in 85% lactic acid with aniline blue 1 g/L (Liberato et al. 2005). Only turgid and mature conidia (those unattached to conidiophores) were measured.
Results
Dimorphic conidia were found, feature that excludes the possibility of this fungus belonging to Ovulariopsis. The leaf clearing and staining technique enabled the visualization of superficial hyphae entering the leaves through stomata, an indication
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that the fungus has hemiendophytic mycelium (partly external and partly internal). With a single exception [species belonging to Cystotheca (Braun 1987)], this feature is exclusive to the tribe Phyllactinieae (Braun et al 2002). This technique also enabled the visualization of conidiophores arising from external mycelium. This combination of characters clearly indicates that the fungus belongs in the genus Streptopodium R.Y. Zheng & G.Q. Chen emend. (Liberato et al. 2004).
Taxonomic Description
Streptopodium passiflorae (Syd.) Liberato & R.W. Barreto comb. nov. (emended) Figs. 1-8 = Ovulariopsis passiflorae Syd. Ann. mycol. 28(1-2): 199, 1930. On living leaves. Mycelium hypophyllous. Superficial hyphae branched, septate, hyaline, flexuous, 4-6 um wide, mycelial appressoria indistinct. Conidiophores produced from the external mycelium, cylindrical, hyaline, smooth, unbranched, septate, up to 264 x 5-7 um, foot-cells mostly straight or somewhat slightly sinuous, very long, followed by 1-2 shorter cells. Conidia single, dimorphic: primary conidia lanceolate, apically pointed, 54-84 x 14-28 um, I/w ratio 1.9-5.1; secondary conidia cylindrical to clavate with apically rounded, basally subtruncate ends, 50-76 x 16-26 um, I/w ratio 1.9-4.8, aseptate, hyaline, smooth. One germ tube per conidium, up to 2 x the length of the conidium, with indistinct appressoria. Primary conidium with germ tube at base or at apex, secondary conidium with germ tube arising from the conidial shoulder. Teleomorph: not found.
SPECIMENS EXAMINED - VENEZUELA, La Victoria, Aragua, on Passiflora rubra L., 31 Jan 1928, H. Sydow (HOLOTYPE: BPI 414143; ISOTYPE: K(m) 131785).
Discussion
There are few reports of powdery mildew on Passiflora. Oidium passiflorae Politis was described from Greece (Politis 1938) and also reported in Germany (Braun 1998) and Australia (Liberato 2006). Anamorphic Leveillula taurica (Lév.) G. Arnaud was reported in Australia (Liberato 2006). Amano (1986) listed Passiflora spp. as hosts of these three’ species in some countries, although this author did not provide the original references from such records. ‘The status of the two South African specimens, listed as Ovulariopsis passiflorae (Doidge 1950), and of Ovulariopsis sp. reported in Colombia (Tamayo & Pardo Cardona 2000) remain to be clarified. Misplacement of members of Streptopodium in Ovulariopsis appears to have occurred with some frequency in the past. For instance the powdery mildew of Tabebuia serratifolia (Vahl) G. Nicholson, originally identified by Ferreira (1989) as Ovulariopsis sp. was recently recollected and described as the new species Streptopodium tabebuiae Liberato & R.W. Barreto (Liberato & Barreto, 2005). This work represents part of an ongoing study aimed at elucidating the status of some dubious Ovulariopsis.
Now, there are five species included in Streptopodium: S. bonariense (Speg.) R.Y. Zheng & G.Q. Chen (teleom.: Pleochaeta polychaeta (Berk. & M.A. Curtis) Kimbr. & Korf) (Zheng & Chen 1978), S. caricae Liberato & R.W. Barreto (Liberato et al. 2004), S. diospyri G.J.M. Gorter (Gorter 1988), S. passiflorae and S. tabebuiae (Liberato &
Figs 1-8. Streptopodium passiflorae (from isotype) (bar = 20 um). Figs 1-4. Conidiophores. Figs 5-6. Primary conidia. Figs 7-8. Secondary conidia.
Barreto 2005). Moreover, three other species of Pleochaeta Sacc. & Speg. emend. have unnamed anamorphs belonging in Streptopodium: P. indica N. Ahmad, A.K. Sarbhoy & Kamal (Ahmad et al. 1995), P. prosopidis (Speg.) U. Braun (Braun 1987) and P. shiraiana (Henn.) Kimbr. & Korf (Zheng & Chen 1978, Gorter & Eicker 1983).
Acknowledgements
The author acknowledges the herbaria BPI and K(m) for specimen loans and thank Dr Uwe Braun (Martin-Luther-Universitat, Germany), Dr Hyeon-Dong Shin (Korea University) and Dr M. Havrylenko (Universidad Nacional del Comahue, Argentina), who kindly reviewed the manuscript. J. R. Liberato acknowledges financial support from the Brazilian Fundacao Coordena¢ao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES).
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Literature Cited
Ahmad N, Sarbhoy AK, Kamal. 1995. New powdery mildews from India. Mycological Research 99; 374-376.
Amano K. 1986. Host range and geographical distribution of the powdery mildew fungi. Japan Scientific Societies Press, Tokyo.
Braun U. 1987. A monograph