THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE
VOLUME 130(1) JANUARY-MARCH 2015
Lactarius olivaceoglutinus sp. nov. (Das, Verbeken & Nuytinck— PLareE 4, p. #115) Kanab Das, artist
ISSN (PRINT) 0093-4666 http://dx.doi.org/10.5248/130 | ISSN (ONLINE) 2154-8889 MYXNAE 130(1): 1-306 (2015)
EDITORIAL ADVISORY BOARD
SABINE HUHNDORE (2011-2016) , Chair Chicago, Illinois, U.S.A. Scott A. REDHEAD (2010-2015), Past Chair Ottawa, Ontario, Canada PETER BUCHANAN (2011-2017) Auckland, New Zealand
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KAREN HANSEN (2014-2019) Stockholm, Sweden
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MY COTA XON
THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE
VOLUME 130(1)
JANUARY-MARCH 2015
EDITOR-IN-CHIEF
LORELEI L. NORVELL
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NOMENCLATURE EDITOR
SHAUN R. PENNYCOOK
PennycookS@LandcareResearch.co.nz Manaaki Whenua Landcare Research Auckland, New Zealand
CONSISTING OF I-X + 306 PAGES INCLUDING FIGURES
ISSN 0093-4666 (PRINT) http://dx.doi.org/10.5248/130(1).cvr ISSN 2154-8889 (ONLINE)
© 2015. MycoTAxon, LTD.
Iv ... MYCOTAXON 130(1)
MY COTAXON
VOLUME ONE HUNDRED THIRTY (1) — TABLE OF CONTENTS
COVER SECTION
EPA inch on okt il sna eRe Meebo oy Aicch OORT AL Sey Dente cA Mea ina At eae RR apna vale 4 vi ROVICWICTS ENE ry oh OE eee cE EEE Rh Eth Ae hee ee EAS Oke ete es vii SOI TSSIOMDTOCCHUMES 3 25 vi Peurizh date ths Red ng er a Ma meri decease 8 Ie Viii ERO TE PEEL Mosty BF Soy inn Pie de On ay wan, A hey nme MERE ix
RESEARCH ARTICLES
Pseudoplectania lignicola sp. nov. described from central Europe S. GLEJDURA, V. KUCERA, P. LIZON, & V. KUNCA Additions to rust and chytrid pathogens of Turkey CuMALI OzasLAN, MAKBULE ERDOGDU, ELsAD HUSEYIN, & ZEKIYE SULUDER Dentipellicula austroafricana sp. nov. supported by morphological and phylogenetic analyses —‘Jta-JIA CHEN, Lu-Lu SHEN, & YU-CHENG Dal A new species of Terriera (Rhytismatales, Ascomycota) on Photinia villosa Quine Li, YuaN Wu, Dan-Dan Lu, YA-Fer Xu, & YING-REN LIN New records of one Amygdalaria and three Porpidia species (Lecideaceae) from China Lu-Lu ZHANG, XIN ZHAO & Line Hu
Anungitea guangxiensis and Ellisembia longchiensis, two new species from southern China JI-WEN X1A, YING-Ru1 Ma, & X1u-GUo ZHANG Fistulina subhepatica sp. nov. from China inferred from morphological and sequence analyses JE SonG, MEI-LING HAN, & BAo-Kat CuI Antherospora sukhomlyniae, a new species of smut fungi on Hyacinthella in Crimea (Ukraine) KyryLo G. SAVCHENKO Tuber xanthomonosporum, a new Paradoxa-like species from China YUAN QING, SHU-HONG LI, CHENG-YI Liu, LIN Li, MEI YANG, XIAO-LEI ZHANG, XIAO-LIN LI, LIN-YONG ZHENG, & YUN WANG Distance1D - a protein profile analytical program designed for fungal taxonomy DuSAN MATERIC, BILJANA KUKAVICA, & JELENA VUKOJEVIC Coccomyces prominens sp. nov. (Rhytismataceae) on Rhododendron coeloneurum in China Ya-FeI Xu, YUAN Wu, YAN-QIONG MENG, SHI-JUAN WANG, & YING-REN LIN
Type studies on Amauroderma species described by J.D. Zhao et al. and the phylogeny of species in China MENG-Jrz Li & Hal-SHENG YUAN
1
11
7.
27
33
4]
47
57
61
69
73
79
JANUARY-MARCH 2015... V
Cladonia corymbescens consists of two species TEUVO AHTI, RAQUEL PINO-Bopas, & SOILISTENROOS 91 Morphology and phylogeny of four new Lactarius species from Himalayan India KANAD Das, ANNEMIEKE VERBEKEN, & JORINDE NUYTINCK 105
Synonymy of two species of Bipolaris from aquatic crops of Poaceae ZI-LAN XIAO, KEVIN D. HYDE, & JING-ZE ZHANG 131 Myxomycetes of Sonora (Mexico) 6. Central plains of the Sonoran Desert Marcos LIZARRAGA, GABRIEL MORENO, MARTIN ESQUEDA, CYNTHIA SALAZAR-MARQUEZ, & MARTHA L. CORONADO 145
New taxa of Ambomucor (Mucorales, Mucoromycotina) from China X1A0-YONG Liu & RU-YONG ZHENG 165
Perenniporia koreana, a new wood-rotting basidiomycete from South Korea YEONGSEON JANG, SEOKYOON JANG, YOUNG WOON LIM, CHANGMU KIM, & JAE-JIN Kim 173 New records of corticolous myxomycetes from Turkey R. BATUR ORAN & C. CEM ErGut 181 A new species of Lophodermium with variously branched paraphyses Hat-Lin Gu, YA-FEI Xu, DAN-DAN LU, SHI-JUAN WANG, & YING-REN LIN 191 Bertia hainanensis sp. nov. (Coronophorales) from southern China LARISSA N. VASILYEVA, HaI-X1A Ma, ALEKSEY V. CHERNYSHEV, & STEVEN L. STEPHENSON 197 Distribution of Alternaria species among sections. 1. Section Porri PHILIpp B. GANNIBAL 207 Morchella galilaea, an autumn morel from Turkey HATIRA TASKIN, HASAN HUtsryvIn DoGAN, & SAADET BUYUKALACA 215 Hyphoderma hallenbergii, a new corticioid species from India MANINDER Kaur, AVNEET P. SINGH, & G.S. DHINGRA 223 Xylaria thailandica- a new species from southern Thailand NATTHAPACH SRIHANANT, VASUN PETCHARAT, & LARISSA N. VASILYEVA 227 Rosellinia brunneola sp. nov. and R. beccariana new to China WEI LI & Lin Guo 233 Phyllachora hainanensis sp. nov. from China Na Liu, LE Wana, Gu Huang, & LIN Guo 237 Two new species of Zasmidium from Nepal RAVINDRA NATH KHARWAR, ARCHANA SINGH, RAGHVENDRA SINGH, & SHAMBHU KuMAR 241 The lichen genus Leiorreuma in China X1A0-Hua WANG, LI-LI XU, & ZE-FENG JIA 247
VI ... MYCOTAXON 130(1)
Periconiella liquidambaricola sp. nov. - a new Chinese hyphomycete Uwe BrAuN, STEFFEN BIEN, Lyp1a HONIG, & BETTINA HEUCHERT 253 First record of Erysiphe syringae-japonicae in Turkey ILGaz AKATA & VASYL P. HELUTA 259 AFLP characterization of three argentine Coprotus species ARACELI MARCELA RAMOs, LuIS FRANCO TADIC, NAHUEL POLICELLI, LAURA INES FERREYRA, & ISABEL ESTHER CINTO 265 Verticicladus hainanensis, a new aquatic hyphomycete MiNG-TIAN Guo, MIN QIAO, JIAN- YING Li, WEI WANG, & ZE-FEN YU 275 Geographic distribution of Sarcoporia polyspora and Sarcoporia longitubulata sp. nov. JOSEF VLASAK, JOSEF VLASAK JR., JUHA KINNUNEN, & VIACHESLAV SPIRIN 279 Coccomyces neolitseae sp. nov. from Zhangjiajie, China QinG Li, YU-X1A CHEN, CHUN-TAO ZHENG, DAN-DAN LU, & YING-REN LIN 289 Datronia ustulatiligna sp. nov. (Agaricomycetes) from India HARPREET KAUR, GURPREET Kaur, & G.S. DHINGRA 295 Three new species of Xylaria from China Gu Huana, RuisHa WANG, LIN Guo, & Na Liu 299 NOMENCLATURAL NOVELTIES AND TYPIFICATIONS PROPOSED IN MYCOTAXON 130-1 305
ERRATA FROM PREVIOUS VOLUMES
VOLUME 129
p-293, line 2 FOR: HASAN HUSEYIN DOGAN READ: HASAN HUSEYIN DOGAN
p.296, line 32 FOR: Lado, C. READ: Lado C.
p.455, line 28 FOR: DM100 READ: DM1000
p.457, line 3 FOR: Spores ornamented with long ridges, arranged in an incomplete banded reticulum.
READ: Spores were decorated with long ridges sometimes forming an incomplete banded reticulum. p.457, line 17 FOR: (ca. 9-12 um diam.) and unbranched and thicker READ: (ca. 9-12 um diam.) and thicker p.458, line 9 FOR: National Basic Research Program of China (3140010180) READ: National Basic Research Program of China (3140010180) and National Science and Technology Foundation Project (2014FY210400)
JANUARY-MARCH 2015 ...
REVIEWERS — VOLUME ONE HUNDRED THIRTY (1)
The Editors express their appreciation to the following individuals who have, prior to acceptance for publication, reviewed one or more of the papers
prepared for this volume.
André Aptroot
N.S. Atri
Z.M. Azbukina Gerald L. Benny
Lina Bettucci
Uwe Braun
Ana Rosa Burgaz
Lei Cai
Matteo Carbone
Lori Carris
Rafael FE Castaneda- Ruiz Julia Checa
Gilberto Coelho Roger T.A. Cook Bao-Kai Cui Yu-Cheng Dai Alejandra Teresa Fazio Ricardo Galan
Tatiana Baptista Gibertoni Shouyu Guo
Ian Robert Hall
Nils Hallenberg Tsutomu Hattori Vasyl Heluta
Bo Huang
Santosh Joshi
M. Kakishima Mitko Karadelev Bryce Kendrick Daniel P. Lawrence Hua-jie Liu
Xiao- Yong Liu
Dimuthu S. Manamgoda
Milan Matavulj
Eric H.C. McKenzie V.A. Melnik
David W. Mitchell Wieslaw Mulenko Lorelei L. Norvell Aysun Peksen Shaun R. Pennycook
Liliane Petrini
Marcin Piatek Michele D. Piercey- Normore
Chaninun Pornsuriya Adam Rollins
Daniel J. Royse
B.M. Sharma
Roger G. Shivas Mirjana Stajic
Steven L. Stephenson Guangyu Sun
Joanne E. Taylor Michal TomSovsky Yuri Tykhonenko Larissa Vasilyeva Xiang-Hua Wang Zheng Wang
A.J.S. Whalley
Ming Ye
Alessandra Zambonelli
Xiu-Guo Zhang Li-Wei Zhou
PUBLICATION DATE FOR VOLUME ONE HUNDRED TWENTY-NINE (2)
MYCOTAXON for OcTOBER-DECEMBER 2014, (I-VI + 215-495)
was issued on January 8, 2015
VII
vill ... MYCOTAXON 130(1)
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JANUARY-MARCH 2015 ... IX
FROM THE EDITOR-IN-CHIEF
WELCOME TO THE YEAR OF MyYCOTAXON 130! — For several decades we have determined MycoTaxon volume size by the number of pages (~512/volume). In recent years, however, our condensed format has — for most papers — produced pages containing far more words than those found in our older volumes, so that it has taken longer to fill a volume.
Beginning with Mycoraxon 130, we move to the publication of one volume (issued quarterly) per year. Pagination will run consecutively through all four issues, but we will continue to list expert reviewers, errata, and nomenclatural novelties in each quarterly for immediate reference. Although the subscription period now coincides with the calendar year, those subscribing to our journal only for 2015 will still be able to access MycoTAxoONn 130 papers in future years for no extra access fee. Our new ‘nomenclatural’ change brings us in line with other scholarly journals, most of which also assess annual subscription rates per ‘volume:
We are pleased to announce that with this move, we are FINALLY back on schedule. The 2015 JANUARY-MARCH quarterly (this issue) presents all papers submitted to the Editor-in-Chief as of March 31. No longer tied to a targeted number of pages per volume, we will be able to deliver future quarterlies promptly and on time. Librarians (and authors) rejoice!
MyYcoTAXON 130(1) contains 37 research papers by 132 authors (representing 19 countries) and revised by 63 expert reviewers.
Within its pages are 32 species and one variety new to science representing Ambomucor, Anungitea, Bertia, Coccomyces, Ellisembia, Fistulina, Leiorreuma, Lophodermium, Periconiella, Phyllachora, Rosellinia, Terriera, Tuber, Verticicladus, and Xylaria from China; Datronia, Hyphoderma, and Lactarius from India; Dentipellicula from South Africa; Perenniporia from Korea; Pseudoplectania from Slovakia and the Czech Republic; Sarcoporia from the Canary Islands and the U.S.A.; Xylaria (again) from Thailand, and Zasmidium from Nepal. We also offer one new name in Periconiella, one new Cladonia combination (resulting from the taxonomic clarification of C. corymbescens), and phylogenetic and pathogenicity confirmation of the synonymy of Bipolaris zizaniae with B. oryzae.
In addition to range extensions for previously named taxa of myxomycetes (for Turkey and Mexico) and chytrids, mildews, morels, and rusts (for Turkey), we present a type study of Amauroderma species from China, a key to Chinese Leiorreuma species, and a complete list of Alternaria species assigned to A. sect. Porri. The applicability of Distance 1D protein profile analysis for fungi and AFLP analyses in Coprotus are also evaluated.
Warm regards,
Lorelei L. Norvell (Editor-in-Chief) 13 April 2015
ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/130.1 Volume 130, pp. 1-10 January-March 2015
Pseudoplectania lignicola sp. nov. described from central Europe
S. GLEJDURA™, V. KUCERA?, P. LIZON?, & V. KUNCA!?
'Faculty of Ecology and Environmental Sciences, Technical University in Zvolen, T. G. Masaryka 24, Zvolen 960 53, Slovakia
?Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 845 23, Slovakia
* CORRESPONDENCE TO: glejdura@gmail.com
ABSTRACT — A new species from Slovakia and the Czech Republic, Pseudoplectania lignicola, is described and illustrated. It is distinguished from other members of the genus by a centrally arranged globose membranous sheath surrounding the spores, thick ectal excipulum of oblong cells at the apothecial base, and growth on less specific biotopes. Comparisons with similar species and the diagnostic significance of membranous sheath surrounding the ascospores are also discussed.
Keyworps — biodiversity, Sarcosomataceae, typification
Introduction
The genus Pseudoplectania was established by Fuckel (1870) for two fungi, Peziza nigrella Pers. and Peziza fulgens Pers. Pseudoplectania nigrella was selected by Seaver (1913) as the lectotype of the genus, and the species itself was neotypified by Carbone & Agnello (2012). Peziza fulgens is today treated in the genus Caloscypha Boud.
Eleven species are currently accepted in the genus: Pseudoplectania affinis, P. carranzae, P. ericae, RP. kumaonensis, P. melaena, P. nigrella, P. ryvardenii, P. sphagnophila, P. stygia, PR tasmanica, and P. vogesiaca (Carbone et al. 2013, 2014; Iturriaga et al. 2012).
The first finding of the new Pseudoplectania reported here was associated with Leucobryum glaucum and tentatively identified in situ as P. sphagnophila. The new fungus was later collected on Abies alba wood buried in soil as well as among Sphagnum bogs. The species was mostly collected on conifer wood.
2 ... Glejdura & al.
Materials & methods
The study is based on the morphological examination of 11 collections from three localities in Slovakia and one in the Czech Republic. The macro-morphological characters were observed in fresh material. The micro-morphological structures were studied in fresh and dried material using a light microscope with oil immersion lens (x1000). Vertical sections of material were examined in 5% KOH, tap-water, Melzer’s reagent, and a Congo Red + ammonia solution. Values of micro-morphological characters were estimated as average plus and minus a standard deviation of 30 measurements for each taxon (with 10 and 90 percentiles presented in parentheses). All descriptions are based on studied specimens. Localities are georeferenced and the coordinates are in WGS 84 system. Examined specimens are kept in herbarium BRA, SAV, and HR. The abbreviations of herbaria are cited in accordance with Index Herbariorum (Thiers 2014).
Taxonomy
Pseudoplectania lignicola Glejdura, V. Kucera, Lizon, Kunca, sp. nov. FIGs 1, 2 MycoBank MB 805097 Differs from Pseudoplectania nigrella by ascospores surrounded by a centrally arranged
globose membranous sheath and by elongated cells in the outer ectal excipulum layer forming a palisade perpendicular to the surface.
Type: Slovakia, Nizke Tatry Mts., ca. 3 km N of Hiadefl village, 48°50’7.38”N 19°18’39.84’E, alt. 700 m, SW exposure, 20° slope, dystric cambisol, biotite granodiorite, in a 100-year old managed forest: Picea abies (L.) H. Karst. 77%, Abies alba Mill., 17%, Larix decidua Mill. 6%; on wood of Abies alba, 21.IV.2010, leg. S. Glejdura (Holotype, BRA CR-19347).
ErymMo.oey: lignicola (Latin) = living on wood. Most specimens were collected from
wood. APOTHECIA at first hemispherical, later cup shaped, plane at full maturity, 1-2.7 cm diam, substipitate or sessile. Hymenium smooth, dark brown to blackish brown. Margin composed of thick-walled cylindrical or slightly clavate hairs 15-110 um long, 5.5-6.1 um broad, with 1-3 septa and 1.5-2.3 um thick walls. Receptacle densely tomentose, covered with thick-walled flexuose, coiled, distantly septate (60-185 um between septa), black-brown to black hairs, <600 um x 4.6-5.5 um, walls 0.9-1.5 um thick.
ECTAL EXCIPULUM 180-200 um thick, outermost zone 2-4 cells thick, cells dark brown, thick-walled, subglobose to elongated 20-27 x 14-18 um, inner layer of subhyaline elongated cells forming a palisade perpendicularly oriented to the surface, cells 34-92 x 15-29 um.
MEDULLARY EXCIPULUM embedded in a gelatinous matrix, outer layer a 180-200 um thick hyaline textura intricata, cells cylindrical, allantoid, narrowly pyriform 40-60 x 12-22 um. Inner layer a 500-600 um thick hyaline densely packed textura intricata, hyphae 9-13 um diam. SUBHYMENIUM 30-40 um
Pseudoplectania lignicola sp. nov. (Slovakia) ... 3
FiGurRE 1. Pseudoplectania lignicola (Holotype BRA CR-19347): A. Ectal excipulum, outermost thick-walled cells. B. Inner layer of the ectal excipulum, elongated cells perpendicularly arranged to the surface. C. Distantly arranged cells of medullary excipulum. Scale bar = 50 um.
thick, composed of light brown densely arranged cylindric, globose and ellipsoid cellular hyphae 3-4.6 um diam.
ASCI narrowly cylindrical, tapered below to thin flexuous aporhynchous base (simple-septate), apex J-, operculate, 8-spored, 259-299 x 12.3-13.7 um.
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v o 8
Od
Cc oS
FiGuRE 2. Pseudoplectania lignicola (Holotype BRA CR-19347). A. Ascus with ascospores from living apothecium; cytoplasmic granules concentrated symmetrically in a limited space by membranaceous sheath (arrowed). B. Ascospores with centrally arranged membranaceous sheath, from living apothecium. Scale bar = 10 um.
ASCOSPORES (mature) globose, thick-walled, with granulate or small guttulate non-oleaginous content (negative reaction in SUDAN 4), (10.2-)11-12.2(-12.3)
Pseudoplectania lignicola sp. nov. (Slovakia) ... 5
um. Mature ascospores surrounded by a membranous cover (sheath) 15.5-19.5 um (including the sheath). The spores are visible in the center of the sheath in almost all mature ascospores, and in exsiccatae sheath remnants are at least visible on several spores. Ascospores within membranous sheaths are separated by cytoplasmic granules laid crosswise in the ascus. Spore walls usually swollen in 5% KOH, with the membranous sheaths dissolving in the same medium.
PARAPHYSES cylindric, septate, brownish in the upper part and often agglutinated with an amorphous substance, sometimes branched and anastomosed in basal and medial parts, 2.2-2.8 um diam, apex mostly straight, often lobed or diverticulate, with lateral proliferations in upper part, enlarged to 2.8-3.6 um diam. Irregularly cylindrical paraphyses can be observed in young or senescent apothecia. Light brown aseptate paraphyses with slightly thicker walls (so-called hymenial hairs) also present in all stages of apothecial development.
ECOLOGY & DISTRIBUTION: The species is known from Slovakia and the Czech Republic and possibly also from Norway (Eckblad 1968, see comment below). All available collections come from sub-montane and montane moist coniferous forests (dominated by Picea abies) on acid soils.
ADDITIONAL SPECIMENS EXAMINED: SLOVAKIA, NizkeE Tatry Mrs., ca. 3 km N of Hiade! village, buffer-zone of Nizke Tatry National Park, 48°50’7.38”N 19°18’39.84’E, alt. 700 m, in the forest, SW exposition, 20° slope, sporadically rocky, dystric cambisol, biotite granodiorite, two-storeyed 100-year old production forest, 1“ storey: Picea abies 77%, Abies alba 17%, Larix decidua 6%; 2‘ storey: Fagus sylvatica L. 100%, among moss Leucobryum glaucum Schimp., 20.I1V.2008, leg. P. Stefanovie (SAV F-10755); on buried wood of Picea abies, 23.III.2010, leg. S. Glejdura (SAV F-10756); on wood of P. abies, 29.V.2011, leg. S. Glejdura (SAV F-10757); VEPORSKE VRCHY MTs., ca. 1 km SW of Sihla village, Nature Reserve Vrchslatina, 48°39’18.96”N 19°37’58.50’E, alt. 900 m, in the forest, flat, 0° slope, dystric histosol (organic soil), diluvial organic sediments (upland moor) on biotite tonalites and granodiorites, three-storeyed ca 65-year old protective forest, 1“ storey: Picea abies 100%, 24, 3rd storey: Picea abies 80-90 %, Alnus incana Mill. 20-10%, Sphagno palustris-Piceetum, well-preserved wetland to peatbog biocenoses with occurrence of specific ecotype of spruce (resonance wood), on buried wood of P. abies, 28.V.2010, leg. S. Glejdura & V. Kunca (SAV F-10758); on buried wood of P. abies, leg. S. Glejdura & V. Kunca (SAV F-10759); 2.V1.2010 (SAV F-10760); VYsoKE TAaTRY Mts., Ticha dolina valley, ca. 6 km NNE of Podbanské village, National Nature Reserve Ticha dolina, 49°12’1.20’N 19°55’33.24’E, alt. 1140 m, in the forest, NW exposition, 35° slope, typical podzol soil, diluvial sediments on porphyric granitoids and granites, one-storeyed 55-year old protective forest, Picea abies 65%, Sorbus aucuparia L. 35%, on dead Sphagnum sp. and needles of P. abies, 18.V.2011, leg. S. Glejdura (SAV F-10761); on buried wood of Picea abies, among Sphagnum sp. bog, 18.V.2011, leg. S. Glejdura (SAV F-10762); on cone of Picea abies, 18.V.2011, leg. S. Glejdura (SAV F-10763).
CZECH REPUBLIC, Hraprec KrALove, Orlicka tabule, Chvojnicka plogina, 50°10’45”N 15°54’15”E, alt. 260 m, among moss Leucobryum glaucum in mixed forest of Picea abies and Pinus sylvestris, 1.1V.2011, leg. Z. Egertova & M. Sochor (HR 89756).
6 ... Glejdura & al.
Discussion
Pseudoplectania is a morphologically and genetically well-delimited genus in the Sarcosomataceae Kobayasi (Carbone et al. 2013, 2014). Identification of taxa is based on the paraphysis morphology, ascospore and apothecium size, and substrate preferences. As we have presented in this paper, another character— the shape of the gelatinous (mucilaginous) sheath of the spore—must also be considered an important diagnostic character. Its nature distinguishes similar species: P. nigrella, P. melaena, and P. lignicola. It is easily detected in fresh material, less in dry herbarium specimens, although in the type specimen of P. lignicola the gelatinous sheath was still visible as late as three years after collection. Spores of P. lignicola lie at the centre of a spherical sheath, whereas in P. nigrella spores lie to one side of the surrounding sheath (Fics 2, 3). The hemispherical sheath of spores of P. melaena does not surround the spores but lies laterally adjacent to the spore surface (Fic 4), a phenomenon that has not been reported before.
Aside from the spore sheath morphology, Pseudoplectania nigrella (Pers.) Fuckel differs from P. lignicola by the thickness and textura of the ectal excipulum as well as by habitat and substrate preferences (on soil in coniferous forests, rarely on wooden substrates). Also, the ectal excipulum at the base of its apothecium is thinner (80-100 um) and composed of smaller cells (17-25 um diam) having a textura angularis-globulosa. Eckblad (1968: 119, fig. 65d) illustrated a specimen of P. nigrella from Norway that has centrally positioned spores in a membranous sheath. We suspect that this could well represent P lignicola, so that our new species may have a wider distribution than currently recorded.
Pseudoplectania melaena (Fr.) Sacc., which has bigger (20-60 mm diam) stipitate apothecia and apically hooked paraphyses, grows on Abies alba branches or logs buried in wet soil. Pseudoplectania sphagnophila (Pers.) Kreisel, associated with Sphagnum sp., has smaller apothecia (10-15 mm diam), and its paraphyses, which may or may not have lateral branches on the upper parts, are apically hooked, as in Otidea (Kreisel 1962, Bauer 1999). Pseudoplectania ericae Donadini, associated with Ericaceae in the Mediterranean area, produces astipitate apothecia <10 mm diam. and paraphyses that are 2-3-branched at the apex (Donadini 1987).
Pseudoplectania stygia (Berk. & M.A. Curtis) Sacc. differs mainly by its small (ca 13 mm diam) apothecia and straight cylindrical paraphyses (Carbone 2013), while P. ryvardenii Iturr. et al. is distinguished from all other Pseudoplectania species by its very small (<7 mm diam) apothecia and ornamented ascospores. Finally, PB kumaonensis Sanwal has distinctly agglutinated paraphyses, and P. carranzae (Calonge & M. Mata) M. Carbone et al. is separated from other
Pseudoplectania lignicola sp. nov. (Slovakia) ... 7
FiGuRE 3. Pseudoplectania nigrella (SAV F-10764). A. Ascus with ascospores from living apothecium; cytoplasmic granules concentrated asymmetrically in a limited space by membranaceous sheath (arrowed). B. Ascospores with eccentrically arranged membranaceous sheath, from living apothecium. Scale bar = 10 um.
members of the genus mainly by the presence of a tomentum or subicular hyphae and smaller asci (170-200 x 10-14 um; Calonge & Mata 2002).
8 ... Glejdura & al.
QO; ao ho
FiGuRE 4. Pseudoplectania melaena (SAV F-10516). A. Ascus with ascospores from living apothecium; cytoplasmic granules concentrated asymmetrically in a limited space by membranaceous sheath (arrowed). B. Ascospores with laterally arranged membranaceous sheath, from living apothecium. Scale bar = 10 um.
Based on our comparison of paraphysis tips in herbarium material of Pseudoplectania lignicola, P. nigrella, and P. sphagnophila with available literature data, we conclude that paraphysis shape is not helpful for distinguishing species. We observed that paraphyses are generally cylindrical, straight, and unbranched in young apothecia, whereas paraphyses in mature and senescent apothecia are branched, anastomosed, and often curved at the tip.
Pseudoplectania lignicola sp. nov. (Slovakia) ... 9
The nature of the membranous sheath of the spore, however, is unique for each Pseudoplectania taxon for which this feature has been observed. The spores of P lignicola lie at the center of this sheath, and its apothecia are <27 mm diameter. The new species probably has a wide ecological amplitude, given its growth on Sphagnum sp., wood, Leucobryum glaucum, and bare soil. Pseudoplectania species usually occur on the same site for years (when the conditions allow), and we have registered and collected P lignicola annually from 2009 to 2013.
In Slovakia P lignicola is rare and possibly endangered (due to lodging and biotope changes) and should be included in the next edition of the Red List of Slovak fungi. The fungus has probably been overlooked and incorrectly identified in the past, but its occurrence in suitable habitats is currently not seriously threatened.
Acknowledgements
We would like to thank Peter Stefanovie for help in researching the type site of Pseudoplectania lignicola (Hiadef village), Vaclav Kautman for assistance in the field, Ginter Bauer for providing material of P. sphagnophila, and Gerard Thijsse and the staff of the herbarium at Leiden for the opportunity to study Persoon’s herbarium specimens. Matteo Carbone (Genova, Italy) and Ricardo Galan (Madrid, Spain), who read and commented on the manuscript, and the support of Shaun Pennycook (Auckland, New Zealand) are acknowledged. This work was supported by the VEGA [02/0088/13, 02/0150/12] for Kucera and Lizon and by the VEGA [1/1190/12, 1/0362/13] for Glejdura and Kunca.
Literature cited
Bauer G. 1999. Pseudoplectania sphagnophila (Pers.: Fr.) Kreisel (Ascomycota, Pezizales, Sarcoscyphaceae) erstmals in Bayern nachgewiesen. Mycol. Bavar. 3: 44—49.
Berkeley MJ. 1875. Notices of North American fungi. Grevillea 3: 145-160.
Calonge FD, Mata M. 2002. Plectania carranzae sp. nov. (Ascomycota) from Costa Rica. Mycotaxon 81: 237-241.
Carbone M. 2013. A type study of Pseudoplectania stygia (Pezizales). Ascomycete.org 5(1): 33-38.
Carbone M, Agnello C. 2012. Studio e tipificazione di Pseudoplectania nigrella. Study and typification of Pseudoplectania nigrella. Ascomycete.org 4(4): 79-93.
Carbone M, Agnello C, Alvarado P. 2013. Phylogenetic studies in the family Sarcosomataceae (Ascomycota, Pezizales). Ascomycete.org 5(1): 1-12.
Carbone M, Agnello C, Alvarado P. 2014. Phylogenetic and morphological studies in the genus
Pseudoplectania (Ascomycota, Pezizales). Ascomycete.org 6(1) : 17-33.
Donadini JC. 1987. Etude des Sarcoscyphaceae ss. Le Gal (1). Sarcosomataceae et Sarcoscyphaceae ss. Korf. Le genre Pseudoplectania emend. nov. P. ericae sp. nov. (Pezizales). Mycol. Helv. 2: 217-246.
Eckblad F-E. 1968. The genera of operculate discomycetes. A re-evaluation of their taxonomy, phylogeny and nomenclature. Nytt Mag. Bot. 15. 191 p.
Fuckel L. 1870. Symbolae mycologicae. Beitrage zur Kenntnis der rheinischen Pilze. Jahrb. Nass. Ver. Naturk. 23-24. 459 p.
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Iturriaga T, Mardones M, Urbina H. 2012. A new species of Pseudoplectania (Sarcosomataceae, Pezizales) from Venezuela. Kurtziana 37(1): 73-78.
Kreisel H. 1962. Pilze der Moore und Ufer Norddeutschlands. III. Pseudoplectania sphagnophila (Fr. pro var.) Kreisel nov. comb. Westfal. Pilzbriefe 3(5): 74-78.
Saccardo PA. 1889. Sylloge Discomycetum et Phymatosphaeriacearum. Sylloge Fungorum, vol. 8. Padova. 1143 p.Sanwal BD. 1953. Contributions towards our knowledge of the Indian discomycetes I. Sydowia 7: 191-199.
Seaver FJ. 1913. The genus Pseudoplectania. Mycologia 5(6): 299-302. http://dx.doi.org/10.2307/3753586
Thiers B. 2014 [continuously updated]. Index Herbariorum: a global directory of public herbaria and associated staff. New York Botanical Garden's Virtual Herbarium. Available: http://sweetgum.nybg.org/ih/ [accessed October 2014]
Van Vooren N, Moyne G, Carbone M, Moingeon J-M. 2013. Pseudoplectania melaena (Pezizales): taxonomical and nomenclatural note. Ascomycete.org 5(1): 47-52.
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MYCOTAXON
http://dx.doi.org/10.5248/130.11 Volume 130, pp. 11-15 January-March 2015
Additions to rust and chytrid pathogens of Turkey
CUMALI OZASLAN”, MAKBULE ERDOGDU’, ELSAD HUSEYIN? & ZEKIYE SULUDERE?
'Dicle University, Faculty of Agriculture, Department of Plant Protection, Diyarbakir-Turkey
?Ahi Evran University, Faculty of Science and Literature, Department of Biology, Kursehir-Turkey
°Gazi University, Faculty of Science, Department of Biology, Ankara- Turkey
*CORRESPONDENCE TO: cumali.ozaslan@dicle.edu.tr
Asstract — Uromyces bornmuelleri on Bongardia chrysogonum and Physoderma maculare on Alisma plantago-aquatica have been recently discovered in Turkey. Morphological data obtained by light and scanning electron microscopy of identified fungi are presented.
Key worps — new records, Chytridiomycota, Basidiomycota
Introduction
The Pucciniaceae are the largest family within the Uredinales, parasitising almost all major angiosperm orders, with some primarily herbaceous plant families such as Asteraceae and Poaceae being most commonly infected (van der Merwe et al. 2007). Uromyces (Link) Unger was raised to generic rank by Unger (1832), and is typified by U. appendiculatus on Phaseolus. Seventy-four species of Uromyces have been registered on 227 species of higher plants in Turkey (Bahcecioglu & Kabaktepe 2012).
All known representatives of the genus Physoderma Wallr. (Blastocladiomycetes, Physodermataceae) are obligate parasites of vascular plants (Olson et al. 1980). The generalized life cycle of Physoderma is composed of two distinct and separate phases, and the ephemeral epibiotic sporangium with an endobiotic rhizoidal system is confined to a single host cell (Sparrow & Johns 1965). Physoderma maculare, the type species of the genus, on Alisma confirms Clinton's (1902) account of the epibiotic stage being produced from resting zoospores. The latter on mature host tissue may also give rise to the endobiotic stage, which bears resting spores. On seedlings, however, resting zoospores produce only epibiotic sporangia (Sparrow 1964).
12 ... Ozaslan & al.
Fics 1-7. Uromyces bornmuelleri. 1: Deformation of host plant. 2: Telia on leaf, in situ. 3: Teliospores. 4-6: Telia and teliospores (SEM). 7. Teliospores (SEM).
Materials & methods
Plant specimens infected with microfungi were collected from Adiyaman and Siirt provinces, Turkey and prepared according to established herbarium techniques. Host plants were identified using the Flora of Turkey and East Aegean Islands (Davis 1965-85). The fungal specimens were prepared from the host plants by obtaining thin sections. Measurements were made from tissues mounted in 5% KOH or tap water. Microscopical features were examined and microphotographs were made using a Leica
Uromyces and Physoderma spp. new for Turkey... 13
DM E light microscope. Thirty spores were measured for each sample. The microfungi were identified using relevant literature (De Toni 1888, Saccardo & Sydow 1902, Gonzalez Fragoso 1918, Kuprevich & Ulijanishchev 1975, van der Merwe et al. 2007). Species names follow Index Fungorum (2014). The examined specimens have been deposited in the mycological collection of the Department of Plant Protection, Faculty of Agriculture, Dicle University, Diyarbakir, Turkey (DUF-M).
For scanning electron microscopy (SEM), 8-10 mm squares of infected leaves were mounted on SEM stubs with double-sided adhesive tape, coated with gold using a Polaron SC 502 Sputter Coater, and examined with a Jeol JSM 6060 scanning electron microscope at 5-10 kV in the Electron Microscopy Unit, Faculty of Science, Gazi University (Turkey).
Taxonomy
Uromyces bornmuelleri Magnus, Verh. Ges. Deutsch. Naturf. 65: 151.1893. Fics 1-7 SPERMOGONIA, AECIDIA and UREDINIA unknown. TELIA amphigenous,
chestnut brown, in dense groups or scattered, 0.2-3 mm diam., causing leaf deformation, at first covered by the epidermis, later becoming erumpent, pulverulent. TELrospores yellow or yellowish-brown, ovoid, globoid, sometimes oblong, 21-30 x 18-21.5 um, rounded at the apex, rounded or sometimes attenuate at the base; wall 2-4 um thick, pedicels hyaline, short, fragile.
SPECIMEN EXAMINED — TURKEY, ADIYAMAN PROVINCE, Center, Kahta, Gerger, Sincik,
Celikhan, Samsat, in wheat field ecosystem, on the leaves of Bongardia chrysogonum (L.)
Spach (Berberidaceae), 12.V.2009, C. Ozaslan CO2029 (DUF-M). Uromyces bornmuelleri has been reported from Azerbaijan on Leontice chrysogonum |= Bongardia chrysogonum] (Tranzschel 1939), and from Iraq and Cyprus on Bongardia chrysogonum (Georghiou 1957, Mathur 1972). Uromyces bornmuelleri is reported for the first time from Turkey.
Physoderma maculare Wallr., Fl. Crypt. Germ. 2: 192. 1833. Fries 8-13
RESTING SPORANGIA amphigenous, mostly hypophyllous and on leaf petioles, concentrated on leaves along the nerves, scattered, covered by the unbroken epidermis, ellipsoid, ovoid, sometimes punctiformis, 1-2 mm diam., chestnut brown. RESTING sporEs chestnut brown, globoid, ovoid to ellipsoid, content granular with more refractive globules, (23.5-)25.5-32 x 21.5-29 um; with two distinct walls, the inner wall thin and colorless, the outer wall chestnut brown, 1-2 um thick, smooth. ZoosPorREs not seen.
SPECIMEN EXAMINED — TURKEY, SurtT PROVINCE, Karaca Village, 38°07 09 N 42°01 59 E, in rice field ecosystem, on the leaves of Alisma plantago-aquatica L. (Alismataceae), 8.V1.2012, C. Ozaslan CO2035 (DUF-M). The genus Physoderma and P. maculare are reported for the first time from Turkey. This fungus has been reported from Canada (Conners 1967), Ireland
14 ... Ozaslan & al.
Ae 1 Avr / We.
xSBB SE find ie 41-668.
f
Figs 8-13. Physoderma maculare. 8: Resting sporangia on leaf, in situ. 9: Resting spores. 10: Resting sporangia on leaf (SEM). 11-13: Resting spores (SEM).
(Muskett & Malone 1984, as Cladochytrium alismatis), Poland (Czeczuga et al. 2007), Siberia (De Toni 1888, as Uredo alismatis), Spain (Gonzalez Fragoso 1918, as U. alismatis), and U.S.A. (Farr et al. 1989).
Acknowledgements
The author thanks Prof. Dr. Z.M. Azbukina (Vladivostok, Russia) and Dr. Yuri Tykhonenko (Kiev, Ukraine) for critically reading the manuscript and serving as presubmission reviewers. Especially we are grateful to Dr. Shaun Pennycook (Auckland,
Uromyces and Physoderma spp. new for Turkey... 15
New Zealand) for detailed linguistic help and nomenclatural review of the manuscript. We would like thank DUBAP (Dicle University Research Projects Coordinator) for financial support of this Project (DUBAP 12ZF71). We also thank Prof. Dr. Mecit Vural (Gazi University, Ankara) and Prof. Dr. A. Selcuk Ertekin (Dicle University, Diyarbakir) for his help with the identification of the hosts.
Literature cited
Bahcecioglu Z, Kabaktepe $. 2012. Checklist of rust fungi in Turkey. [Mycotaxon 119: 494.] http://www.mycotaxon.com/resources/checklists/Bahcecioglu-v119-checklist.pdf
Clinton GP. 1902. Cladochytrium alismatis. Botanical Gazette 33: 49-61.
Conners IL. 1967. An annotated index of plant diseases in Canada and fungi recorded on plants in Alaska, Canada and Greenland. Research Branch, Canada Department of Agriculture, Publication 1251. 381 p.
Czeczuga B, Muszynska E, Godlewska A, Mazalska B. 2007. Aquatic fungi and straminipilous organisms on decomposing fragments of wetland plants. Mycologia Balcanica 4: 31-44.
Davis PH (ed.). 1965-85. Flora of Turkey and East Aegean Islands. Vols 1-9. Edinburgh University Press, Edinburgh.
De Toni GB. 1888. Sylloge ustilaginearum et uredinearum. Sylloge Fungorum 7(2): 449-882.
Farr DF, Bills GF, Chamuris GP, Rossman AY. 1989. Fungi on plants and plant products in the United States. APS Press, St Paul MN. 1252 p.
Georghiou GP, Papadopoulos C. 1957. A second list of Cyprus fungi. Government of Cyprus, Department of Agriculture.
Gonzalez Fragoso R. 1918. La roya de los vegetales. Enumeracion y distribucion geografica de los Uredales. Conocidos hasta hoy en la Peninsula Iberica e Islas Baleares. Trabajos del Museo Nacional de Ciencias Naturales, Serie Botanica 15: 1-267.
Index Fungorum. 2014. http://www.indexfungorum.org/names/names.asp. Accessed 26 May 2014.
Kuprevich VF, Ulijanishchev V. 1975. Key to the rust fungi in SSSR. Minsk, Belarus: Nauka i Tekhnika.
Mathur RS. 1972. Checklist of Iraqi Uredinales. Iraq Natural History Museum, Publication 29: 1=32;
Muskett A, Malone J. 1984. Catalogue of Irish fungi - V. Mastigomycotina and Zygomycotina. Proceedings of the Royal Irish Academy, B. 84: 83-102.
Olson LW, Edén UM, Lange L. 1980. The endobiotic thallus of Physoderma maydis, the causal agent of Physoderma disease of maize. Protoplasma 103: 1-16.
Saccardo PA, Sydow P. 1902. Supplementum universale, pars V. Sylloge Fungorum 16. 1291 p.
Sparrow FK. 1964. Observations on chytridiaceous parasites of phanerogams. XIII. Physoderma maculare Wallroth. Archiv fir Mikrobiologie 48: 136-149.
Sparrow FK, Johns RM. 1965. Observation on chytridiaceous parasites of phanerogams XVI. Notes on Physoderma from Scirpeae. Archiv fiir Mikrobiologie 51: 351-364.
Tranzschel VG. 1939. Compendium of rusts of the USSR. Botanicheskii Institut Akademii Nauk SSSR. Moscow. 426 p.
Unger F. 1832 [“1833”]. Die Exantheme der Pflanzen. Wien. 422 p.
van der Merwe M, Ericson L, Walker J, Thrall PH, Burdon JJ. 2007. Evolutionary relationships among species of Puccinia and Uromyces (Pucciniaceae, Uredinales) inferred from protein coding gene phylogenies. Mycological Research 111: 163-175.
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MY COTAXON
http://dx.doi.org/10.5248/130.17 Volume 130, pp. 17-25 January-March 2015
Dentipellicula austroafricana sp. nov. supported by morphological and phylogenetic analyses
J1A-JIA CHEN*, Lu-LU SHEN* & YU-CHENG DalI*
Institute of Microbiology, Beijing Forestry University, RO. Box 61, Beijing 100083, China *CORRESPONDENCE TO: daiyucheng2013@gmail.com
ABSTRACT — Dentipellicula austroafricana sp. nov. is described and illustrated from South Africa based on morphological characters and rDNA sequence data. It is characterized by an annual growth habit, resupinate basidiocarps, dense soft spines, a monomitic hyphal structure with non-amyloid, non-dextrinoid and acyanophilous generative hyphae, absence of cystidia, presence of cystidioles, and tiny rough basidiospores (2.4-2.9 x 2-2.2 um). A molecular study based on the combined ITS (internal transcribed spacer region) and nLSU (the large nuclear ribosomal RNA subunit) dataset supports the new species in Dentipellicula. A key to accepted species of Dentipellicula is provided.
Key worps — Basidiomycota, Hericiaceae, hydnoid fungi, Russulales, taxonomy, wood- inhabiting fungi
Introduction
Dentipellis Donk (Russulales, Basidiomycota), typified by D. fragilis (Pers.) Donk, was introduced for species characterized by an annual growth habit, hydnoid basidiocarps, soft spines, a monomitic hyphal structure with clamp connections and cyanophilous hyphae, and amyloid rough basidiospores (Dai et al. 2009, Ginns 1986, Zhou & Dai 2013). Zhou & Dai (2013), who demonstrated that Dentipellis was polyphyletic, segregated Dentipellis leptodon (Mont.) Maas Geest. and Dentipellis tatwaniana Sheng H. Wu from Dentipellis based on ITS and nLSU rDNA sequences. They proposed Dentipellicula Y.C. Dai & L.W. Zhou as a new genus for these two hydnoid fungal species, which are distinguished from Dentipellis by their lack of cyanophilous hyphae. Dentipellicula taiwaniana (Sheng H. Wu) Y.C. Dai & L.W. Zhou (the generic type), and D. leptodon (Mont.) Y.C. Dai & L.W. Zhou currently comprise the entire genus.
* Jra-JiA CHEN and Lu-Lu SHEN contributed equally to this work and share first-author status.
18 ... Chen, Shen, & Dai
During studies on the hydnoid fungi, an undescribed Dentipellicula species from South Africa was identified based on morphological characters and phylogenetic analysis of ITS and nLSU sequences. We provide an illustrated description and include an identification key to all three species representing Dentipellicula.
Materials & methods
Morphological studies
Sections were studied microscopically according to Dai et al. (2010) at magnifications <1000x using a Nikon Eclipse 80i microscope with phase contrast illumination. Drawings were made with the aid of a drawing tube. Microscopic features, measurements, and drawings were made from sections stained with Cotton Blue and Melzer’s reagent. Spores were measured from sections cut from the tubes. To present spore size variation, the 5% of measurements excluded from each end of the range are given in parentheses. Basidiospore spine lengths were not included in the measurements. Abbreviations include IKI = Melzer’s reagent, IKI- = negative in Melzer’s reagent, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous, CB- = acyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = the L/W ratio, and n = number of spores measured from given number of specimens. Special color terms follow Petersen (1996). The studied specimens are deposited in the herbaria as cited below; collection abbreviations follow Thiers (2014).
DNA extraction and sequencing
A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies, Beijing) was used to obtain PCR products from dried specimens, according to the manufacturer's instructions with some modifications (Chen & Cui 2014). The DNA was amplified with the primers: ITS5 and ITS4 for ITS (White et al. 1990), and LROR and LR7 for nLSU (Vilgalys & Hester 1990). The PCR procedure for ITS was as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles at 94°C for 40 s, 54°C for 45 s and 72°C for 1 min, and a final extension of 72°C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 50°C for 1 min and 72°C for 1.5 min, and a final extension of 72°C for 10 min. The PCR products were purified and sequenced in Beijing Genomics Institute, China with the same primers.
Phylogenetic analysis
New sequences, deposited in GenBank (TaBLE 1), were aligned with additional sequences retrieved from GenBank (TaBLE 1) using BioEdit (Hall 1999) and ClustalX (Thompson et al. 1997). Bondarzewia podocarpi Y.C. Dai & B.K. Cui and B. sp. were used as outgroup (Zhou & Dai 2013). Prior to phylogenetic analysis, ambiguous regions at the start and the end of the alignment were deleted and gaps were manually adjusted to optimize the alignment. The edited alignment was deposited at TreeBase (http://purl.org/phylo/treebase; submission ID 15859).
Dentipellicula austroafricana sp. nov. (China) ... 19
TABLE 1. Specimens used in ITS and nLSU sequence analyses.
GENBANK ACCESSION NO.
SPECIES SAMPLE NO. LOCALITY ITS nLSU B. podocarpi Dai 9261 China KJ583207 KJ583221 Bondarzewia sp. DAOM F-415 Canada DQ200923 DQ234539 Dentipellicula austroafricana Dai 12580 South Africa KJ855274 KJ855275 D. leptodon GB 011123 Uganda EU118625 EU118625 D. taiwaniana Dai 10867 China JQ349115 JQ349101 Cui 8346 China JQ349114 JQ349100 Dentipellis coniferarum Cui 10063 China JQ349106 JQ349092 Yuan 5623 China JQ349107 JQ349093 D. dissita NH 6280 Canada AF506386 AF506386 D. fragilis Dai 12550 China JQ349110 JQ349096 Dai 9009 China JQ349108 JQ349094 D. microspora Cui 10035 China JQ349112 JQ349098 D. parmastoi Cui 8513 China JQ349113 JQ349099 Hericium abietis NH 6990 Canada AF506456 AF506456 H. alpestre NH 13240 Russia AF506457 AF506457 H. americanum DAOM F-21467 Canada AF506458 AF506458 H. cirrhatum Tubingen F794 Germany AF506385 AF506385 H. coralloides NH 282 Sweden AF506459 AF506459 H. erinaceus NH 12163 Russia AF506460 AF506460 Laxitextum bicolor NH 5166 Sweden AF310102 AF310102
Phylogenetic analysis followed Li & Cui (2013). Maximum parsimony (MP) analysis was performed in PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max- trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates (Felsenstein 1985). Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimonious tree (MPT) generated. Phylogenetic trees were visualized using Treeview (Page 1996).
MrModeltest 2.3 (Nylander 2004) was used to determine the best-fit evolution model of the combined dataset for Bayesian inference (BI). BI was calculated with MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003) with a general time reversible (GTR)
20 ... Chen, Shen, & Dai
model of DNA substitution and an invgamma distribution rate variation across sites. Four Markov chains were performed for 2 runs from random starting trees for 1 million generations of the combined ITS and nLSU dataset, and trees were sampled every 100 generations. The burn-in was set to discard the first 25% of the trees. A majority rule consensus tree of all remaining trees was calculated. Nodes that received BT support >75% and Bayesian posterior probabilities (BPP) 20.95 were considered as significantly supported.
To determine if the dataset was significantly conflicted, the partition homogeneity test option in PAUP 4.0b was used between the loci using 1000 replicates and the heuristic general search option. This test randomly shuffles phylogenetically informative sites between two paired loci: if the dataset is compatible, shuffling sites between the loci should not produce summed tree lengths that are significant greater than those produced by the observed data (Farris et al. 1995; Huelsenbeck et al. 1996).
Molecular phylogeny
Partition homogeneity test showed no conflicts for the two-gene combined loci (P 20.01). Therefore, in this study, ITS and nLSU were combined into a single analysis. The combined ITS and nLSU dataset included sequences from 20 fungal collections representing 17 species. The dataset had an aligned length of 1720 characters, of which 1246 characters are constant, 131 are variable and parsimony-uninformative, and 343 are parsimony-informative. MP analysis yielded 2 equally parsimonious trees (TL = 902, CI = 0. 708, RI = 0.786, RC = 0.557, HI = 0.292). The best model for the combined ITS and nLSU sequences dataset estimated and applied in the BI was GTR+I+G. BI resulted in a similar topology with an average standard deviation of split frequencies = 0.005368 to MP analysis, and thus only the MP tree was provided. Both BT values (=>50%) and BPPs (20.95) are shown at the nodes (Fic. 1).
The newly sequenced specimen from South Africa was embedded in the lineage of Dentipellicula (100% MP and 1.00 BPPs) and was closely related to D. taiwaniana.
Taxonomy
Dentipellicula austroafricana Jia J. Chen, L.L. Shen & Y.C. Dai, sp. nov. Fic. 2 MycoBank MB 811108
Differs from other Dentipellicula species by its exclusively resupinate basidiocarps, dense spines, absence of gloeoplerous hyphae and cystidia, presence of cystidioles, and tiny rough basidiospores.
Type — South Africa, KwaZulu-Natal Province, Durban, on charred wood of Ficus, 1.X.2011, Dai 12580 (holotype, BJFC; isotype, IFP; GenBank KJ855274, KJ855275).
ETyMOLoGy — austroafricana (Lat.): referring to South Africa.
FruiTBopy — Basidiocarps annual, resupinate, inseparable, hard corky upon drying, up to 6 cm long, 2 cm wide, 3 mm thick at center, without odor or taste.
Dentipellicula austroafricana sp. nov. (China) ... 21
87/100 Hericium americanum DAOM F-21467
97/1.00| Hericium alpestre NH 13240
a EF Hericium erinaceus NH 12163 Hericium abietis NH 6990 100/1.00 Hericium cirrhatum Tibingen F794 60/1.00
Hericium coralloides NH 282 Laxitextum bicolor NH 5166 100/0.98| Dentipellis fragilis Dai 12550 99/1.00! ° Dentipellis fragilis Dai 9009
67). Dentipellis dissita NH 6280
Dentipellis parmastoi Cui 8513 100/1.00 | Dentipellis coniferarum Cui 10063
Dentipellis coniferarum Yuan 5623 100/1.00 Dentipellis microspora Cui 10035
86/0.96| Dentipellicula taiwaniana Dai 10867
100/0.99 Dentipellicula tatwaniana Cui 8346
100/1.00 Dentipellicula austroafricana Dai 12580
Dentipellicula leptodon GB 011123 Bondarzewia sp. DAOM F-41
Bondarzewia podocarpi Dai 9261
10 KH
Fic. 1. Strict consensus tree illustrating the phylogenetic position of Dentipellicula austroafricana, generated by maximum parsimony method based on ITS+nLSU sequence data. Branches are labeled with parsimony bootstrap values 250% and Bayesian posterior probabilities >0.95.
Fresh spines soft, white to cream, when dry fragile, cream to buff, up to 2 mm long, 8-10 per mm across base. Margin cottony, buff to clay-buff, <1 mm wide. Subiculum very thin, soft corky, buff to reddish brown, <1 mm thick.
HyYPHAL STRUCTURE — Hyphal system monomitic; generative hyphae with clamp connections, IKI-, CB-; tissues unchanged in KOH.
22 ... Chen, Shen, & Dai
SUBICULUM — Generative hyphae colorless, thick-walled, frequently branched, interwoven, 4-6 um in diam. Gloeoplerous hyphae absent.
HYMENOPHORAL TRAMA — Generative hyphae colorless, slightly thick- walled to thick-walled, moderately branched, more or less subparallel along the spines, 2-5.5 um in diam. Gloeoplerous hyphae absent.
HyMENIUM — Cystidia absent; fusoid cystidioles present, hyaline, thin- walled, 16.5-18 x 1-4 um; basidia clavate with four sterigmata and a basal clamp connection, 18-23 x 3.5-5 um; basidioles similar to basidia in shape, but smaller than basidia. Basidiospores ellipsoid to broadly ellipsoid, thick-walled, colorless, minutely rough, strongly amyloid, CB+, 2.4-2.9(-3) x (1.8-)2-2.2 (-2.4) um, L = 2.83 um, W = 2.12 um, Q = 1.29-1.39 (n = 60/1).
OTHER SPECIES EXAMINED — Dentipellicula leptodon: CANADA. ONTARIO PROVINCE, Algonquin Nat. Res., on Betula, 22.X.1966 (DAOM F-158439).
Dentipellicula taiwaniana: CHINA. HAINAN PROVINCE, Ledong County, Jianfengling Nat. Res., on fallen angiosperm trunk, 12.V.2009, Dai 10872 (BJFC 5114).
Key to accepted species of Dentipellicula
PS BasidiOSPOFES = Sior UI LONG cs firs ade deko ech Ase bet lle & wen ke ¥ wereld Berets Baa D. leptodon Ty BasidiospOrese<t5ro7 UY TOME Ae eta acto Wie doles hte A heated tet dha ee aa 4 2 2. Spities! 5—/. per Mins -Cystidia, presents #4 6.0% Fwhctus Warhol Whee! hee D. austroafricana 2. Spines 8-10 per mm; cystidia absent ................. 00... eee eee D. taiwaniana Discussion
Phylogenetically, Dentipellicula austroafricana is recognized in the Dentipellicula clade and distant from Dentipellis, Hericium Pers., and Laxitextum Lentz based on the combined ITS and nLSU sequences (Fie. 1). Morphologically, D. austroafricana is characterized by an annual growth habit, resupinate basidiocarps, soft and dense spines, a monomitic hyphal structure with non-dextrinoid generative hyphae, presence of cystidioles, and tiny rough basidiospores. Both morphology and rDNA sequence data confirmed that D. austroafricana is a new species in Dentipellicula.
According to the ITS and nLSU-based phylogeny (Fic. 1), Dentipellicula austroafricana is closely related to D. taiwaniana, which also has annual basidiocarps and soft spines but which is distinguished by its effused-reflexed basidiocarps, more scattered spines (5-7 per mm), narrower (2-3.5 um) generative hyphae in the subiculum, presence of gloeocystidia, and longer basidiospores (2.8-3.4 x 2.1-2.4 um; Dai et al. 2009, Wu 2007).
Dentipellicula leptodon also has annual basidiocarps and soft spines but differs from D. austroafricana in its narrower (2-4 um) generative hyphae in the subiculum, presence of gloeopleurous hyphae in the subiculum and gloeocystidia, and larger basidiospores (3.6-4.2 x 2.8-3.3 um; Dai et al. 2009,
SSS ——
Lak aa
Wee
QIFR
A OK ial be eT" Go a \
— f we Mm I ng a
Le
24 ... Chen, Shen, & Dai
Ginns 1986). Moreover, the two species are separated in the ITS and nLSU- based phylogenetic analysis (Fie. 1).
Our phylogenetic tree was overall consistent with that presented by Zhou & Dai (2013): for now, Dentipellis remains polyphyletic based on ITS and nLSU rDNA sequences, while the new segregate genus Dentipellicula is monophyletic (Fic. 1). A fully resolved phylogeny for Dentipellis and its related genera requires evolutionary information from wider taxa samplings and more conserved gene markers.
Acknowledgements
We express our gratitude to Drs. Li- Wei Zhou (Institute of Applied Ecology, Chinese Academy of Sciences, China) and Michal TomSovsky (Mendel University in Brno, Czech Republic) who reviewed the manuscript. The research was financed by the Fundamental Research Funds for the Central Universities (No. BLYJ201403).
Literature cited
Chen JJ, Cui BK. 2014. Phlebiporia bubalina gen. et. sp. nov. (Meruliaceae, Polyporales) from southwest China with a preliminary phylogeny based on rDNA sequences. Mycol. Prog. 13: 563-573. http://dx.doi.org/10.1007/s11557-013-0940-4
Cui BK, Zhao CL, Dai YC. 2011. Melanoderma microcarpum gen. et sp. nov. (Basidiomycota) from China. Mycotaxon 116: 295-302. http://dx.doi.org/10.5248/116.295
Dai YC, Xiong HX, Wu SH. 2009. Notes on Dentipellis (Russulales, Basidiomycota). Mycosystema 28: 668-671.
Dai YC, Cui BK, Liu XY. 2010. Bondarzewia podocarpi, a new and remarkable polypore from tropical China. Mycologia 102: 881-886. http://dx.doi.org/10.3852/09-050
Farris JS, Kallersj6 M, Kluge AG, Bult C. 1995. Testing significance of incongruence. Cladistics 10: 315-319. http://dx.doi.org/10.1111/j.1096-0031.1994.tb00181.x
Felsenstein J. 1985. Confidence intervals on phylogenetics: an approach using bootstrap. Evolution 39: 783-791. http://dx.doi.org/10.2307/2408678
Ginns J. 1986. The genus Dentipellis (Hericiaceae). Windahlia 16: 35-45.
Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41: 95-98.
Huelsenbeck JP, Bull JJ, Cunningham E. 1996. Combining data in phylogenetic analysis. Trends Ecol. Evol. 11: 152-158. http://dx.doi.org/10.1016/0169-5347(96)10006-9
Li HJ, Cui BK. 2013. Taxonomy and phylogeny of the genus Megasporoporia and its related genera. Mycologia 105: 368-383. http://dx.doi.org/10.3852/12-114
Nylander JAA. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
Page RDM. 1996. TreeView: application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12: 357-358.
Petersen JH. 1996. The Danish Mycological Societys colour-chart. Foreningen til Svampekundskabens Fremme, Greve. 6 p.
Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574. http://dx.doi.org/10.1093/bioinformatics/btg180
Swofford DL. 2002. PAUP*: Phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sinauer Associates, Sunderland.
Dentipellicula austroafricana sp. nov. (China) ... 25
Thiers, B. 2014 [continuously updated]. Index Herbariorum: A global directory of public herbaria and associated staff. New York Botanical Garden's Virtual Herbarium. http://sweetgum.nybg.org/ih/
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882. http://dx.doi.org/10.1093/nar/25.24.4876
Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172: 4238-4246.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds).PCR Protocols: a guide to methods and applications. Academic Press, San Diego.
Wu SH. 2007. Three new species of corticioid fungi from Taiwan. Bot. Stud. 48: 325-330.
Zhou LW, Dai YC. 2013. Taxonomy and phylogeny of wood-inhabiting hydnoid species in Russulales: two new genera, three new species and two new combinations. Mycologia 105: 636-649. http://dx.doi.org/10.3852/12-011
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MY COTAXON
http://dx.doi.org/10.5248/130.27 Volume 130, pp. 27-31 January-March 2015
A new species of Terriera (Rhytismatales, Ascomycota) on Photinia villosa
QinG Lr, YUAN Wu’, DAN-DAN LU’, YA-FEI Xu’, & YING-REN LIN”
" School of Life Science &? School of Forestry & Landscape Architecture, Anhui Agricultural University, West Changjiang Road 130, Hefei, Anhui 230036, China
*CORRESPONDENCE TO: yingrenlin@yahoo.com
ABSTRACT — A new Terriera species, T: aequabilis on fallen leaves of Photinia villosa from the Mount Sangingshan National Park, Jiangxi Province, China, is described, illustrated, and discussed. This taxon is distinguished from T. cladophila by smaller ascomata that are not associated with conidiomata, black thin zone lines, a well-developed excipulum, and unbranched paraphyses. The type specimen is deposited in the Reference Collection of Forest Fungi of Anhui Agricultural University, China (AAUF).
KEY worps — taxonomy, morphology, Rhytismataceae, Rosaceae
Introduction
Eriksson (1970) established the genus Terriera based on T. cladophila (Lév.) B. Erikss. (© Hysterium cladophilum Lév.), which produces a different paraphysial type and opening mechanism from genera Lophodermium Cheval. and Sporomega Corda. Minter (1996) provided a detailed description for T. cladophila. Johnston (2001), who systematically studied monocotyledon- inhabiting Lophodermium, divided that heterogeneous genus into five groups, one of which was transferred to Terriera. IndexFungorum (2014) lists 25 specific and two varietal epithets in Terriera.
Here, Terriera aequabilis on fallen leaves of oriental photinia from Jiangxi Province, China, is described as a new species.
Materials & methods
Mature ascomata were selected from the collected specimens. Under the dissecting microscope with a 10—50x magnification, the macroscopic appearance of ascomata and zone lines was observed. After rehydration of reference materials in water for 15 min., vertical transverse 10—15um thick sections were sliced from ascomata using a freezing microtome and mounted in 0.5% (w/v) cotton blue in water for observing ascomatal
28 ... Li & al.
outlines. Gelatinous sheaths surrounding ascospores and paraphyses were examined in water or 0.1% (w/v) lactophenol-cotton blue. The color of internal structures and ascospore contents were observed in water. Squash mounts were prepared in 5% KOH solution for measurements of asci, ascospores, and paraphyses. Figures of the external shapes and internal structures of the fruitbodies were drawn using a microscopic painting tube. The type specimen is deposited in the Reference Collection of Forest Fungi of Anhui Agricultural University, China (AAUF).
Taxonomy
Terriera aequabilis Qing Li & Y.R. Lin, sp. nov. Fires 1-5 MycoBaAnk MB 808818
Differs from Terriera cladophila by smaller ascomata not associated with conidiomata, frequent black and thin zone lines, a much thinner covering stroma, unbranched paraphyses swollen conspicuously at the apex, and ascospores with a visible mucous sheath.
Type: China, Jiangxi, Mount Sanqingshan National Park, Bingyudong, alt. 1550 m, on fallen leaves of Photinia villosa (Thunb.) DC. (Rosaceae), 21 August 2012, Y.R. Lin, S.J. Wang & L. Zhang 2657 (Holotype, AAUF 68765).
ErymMo.oey: aequabilis (Latin = uniform), referring to the single ascospore, which has
an equal diameter throughout its length. Cotontgs on both sides of leaves, forming distinct subcircular or irregular, yellow-brown bleached areas each with an obvious edge, 6-10 mm diam., which tend to coalesce into larger irregular shapes.
ZONE LINES usually frequent, dark brown or black, thin, entirely or partly surrounding the paler areas.
CONIDIOMATA not observed.
AscomarTa developing on both sides of leaves, predominantly on the lower side, scattered over the pale areas, with two ascomata sometimes coalescent. In surface view, ascomata 320-720 x 250-430 um, elliptical to subcircular, straight or slightly curved to one side, ends rounded. Whole surface of ascoma black, except sometimes for a paler region at each end, with a clearly defined edge, slightly shiny, moderately raising above the substrate surface but somewhat sunken near the split, opening by a single longitudinal split extending most of the length of the ascoma. Immature ascomata appearing as a single dark brown protrusion, more or less rotund in outline, not forming a pale zone along the future line of opening. In median transverse vertical section, ascomata subepidermal with epidermal cells becoming filled with fungal tissue as ascoma develops, 165-190 um deep. COVERING STROMA 15-20 um thick near the opening, becoming thinner towards the edge and connecting to the basal stroma, composed mainly of textura angularis with blackish-brown to dark brown, thick-walled cells 3-5.5 um diam. Lip cells absent. Along the edge of the ascoma opening, there is a short extension, 10-18um thick, adjacent
Terriera aequabilis sp. nov. (China) ... 29
\\ ASS YTES SURE F) WISSSEY
ORDO US a St see OS SoS
Fics 1-5. Terriera aequabilis (holotype, AAUF 68765) on Photinia villosa 1. Habit on a leaf. 2. Detail of ascomata and a zone line. 3. Ascoma in transverse vertical section. 4. Portion of ascoma in transverse vertical section. 5. Paraphyses, asci, and ascospores.
30 ... Li&al.
to the top of the covering stroma, which is comprised of strongly carbonized tissue with no obvious cellular structure. ExcrpuULUM well-developed, 10-15 uum thick, arising from the inner layer of the basal stroma, septate, comprised of colorless textura porrecta-intricata with hyphae ca 0.8 um diam., sometimes a reticular structure formed by the interlaced hyphae at the upper part. BASAL STROMA 6-10 um thick, dark brown, consisting of 2(-3) layers of 3-5 um diam., angular, thick-walled cells. Colorless to grayish-brown textura prismatica with cell 3-8.5 um diam., 20-45 um thick, existing in the triangular area, visible in sections between the covering and basal stroma at each edge of the ascoma. SUBHYMENIUM 15-22 um thick, rather flat, consisting of hyaline textura- angularis and textura intricata. PARAPHYSES 1-1.3 um wide, filiform, sparsely septate, not branched, gradually or suddenly swollen to 2.8-5 um at the apex, extending ca 10 um beyond asci, immersed in a ca 0.5 um thick gelatinous matrix, hyphal bridges no observed at the base. Asc1 maturing sequentially, cylindric-clavate, 75-105 x 4.5-5.5 um, short-stalked, thin-walled, apex round or slightly obtuse, J-, 8-spored. Ascosporgs arranged fasciculately, with a uniform diameter throughout the single ascospore, 55-78 x 0.8-1 um, filiform, hyaline, aseptate, ends rounded, covered by a 0.3-0.5 um thick gelatinous sheath.
ADDITIONAL SPECIMENS EXAMINED — On dead leaves of Photinia villosa: CHINA,
J1ANGx1: Mount Sanqingshan National Park, Huaiyushan, alt. ca 600 m, 18 August
2012, S.J. Wang & Y.R. Lin 2632 (AAUF 68740); Sanmucun, alt. ca 850 m, 20 August
2012, F. Zhou & L. Zhang 2645 (AAUF 68753); Yulinfeng, alt. ca 810 m, 21 August 2012,
Y.R. Lin, S.J. Wang & L. Zhang 2704 (AAUF 68812). ComMMENTsS — Terriera aequabilis is somewhat similar to the type species T. cladophila in the shape of ascomata, asci, and ascospores. However, T. cladophila occurs on dead or living twigs and has larger (350-900 x 300-600 um) ascomata that are associated with conidiomata, a much thicker (<40 um) covering stroma composed of textura globulosa or angularis, paraphyses with frequently and irregularly branched and irregularly twisted swollen upper parts, sometimes with hyphal bridges at the base between adjacent paraphyses, ascospores without mucous sheath, and brown diffuse zone lines occasionally present (Minter 1996).
Terriera minor (Tehon) P.R. Johnst. [= Lophodermium minus (Tehon) P.R. Johnst.], the most widely distributed species, differs from the new species in oblong to oblong-elliptic, paraphyses branching 2-3 times in upper 30-40um, extend 20-30 um beyond asci and sometimes irregularly swollen at the apices, longer and wider (100-130 x 6-7 um) asci that are tapering abruptly to small, rounded apex, wider (1.5-2 um) ascospores slightly tapering towards both ends and 0-1-septate, and a poorly developed subhymenium (Johnston 1989). In addition, Tehon’s (1918) observation that a thin, hyaline epithecium was formed by apically coiled paraphyses differs from Johnston's description.
Terriera aequabilis sp. nov. (China) ... 31
The similar Terriera simplex Y.L. Lin et al. is distinguished from T. aequabilis by larger (650-1000 x 350-480 um), sometimes triangular ascomata with conidiomata, a very poorly developed excipulum, wider (1.2-1.5 um) paraphyses occasionally swollen to 2.5-3 um near the apex, synchronously maturing asci with truncate to obtuse apices, and ascospores tapering to the rounded base (Gao et al. 2012).
Acknowledgements
The authors are grateful for the pre-submission comments and suggestions provided by Dr Z. Wang (Yale University, USA) and Dr M. Ye (Hefei University of Technology, China) and to F Zhou and L. Zhang for the field investigations. This study was supported by the National Natural Science Foundation of China (No. 31270065, 31170019).
Literature cited
Eriksson B. 1970. On Ascomycetes on Diapensiales and Ericales in Fennoscandia. I. Discomycetes. Symbolae Botanicae Upsalienses 19(4): 1-71.
Gao XM, Zheng CT, Lin YR. 2012. Terriera simplex, a new species of Rhytismatales from China. Mycotaxon 120: 209-213. http://dx.doi.org/10.5248/120.209
IndexFungorum. 2014. [www.indexfungorum.org (viewed online on 1 May 2014)].
Johnston PR. 1989. Lophodermium (Rhytismataceae) on Clusia. Sydowia 41: 170-179.
Johnston PR. 2001. Monograph of the monocotyledon-inhabiting species of Lophodermium. Mycological Papers 176: 1-239.
Minter DW. 1996. Terriera cladophila. IMI Descriptions of Fungi & Bacteria,. no. 1296.
Tehon LR. 1918. Systematic relationship of Clithris. Botanical Gazette 65: 552-555. http://dx.doi.org/10.1086/332288
SpeciesFungorum. 2014. [www.speciesfungorum.org (viewed online on 1 May 2014)].
ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
MYCOTAXON
http://dx.doi.org/10.5248/130.33 Volume 130, pp. 33-40 January-March 2015
New records of one Amygdalaria and three Porpidia taxa (Lecideaceae) from China
Lu-Lu ZHANG, XIN ZHAO, & LING Hu’
Key Laboratory of Plant Stress Research, College of Life Sciences, Shandong Normal University, Jinan, 250014, P. R. China
* CORRESPONDENCE TO: lichenhuling@gmail.com
AxBstRAcT —Four lichen taxa of Lecideaceae, Amygdalaria consentiens var. consentiens, Porpidia carlottiana, P. lowiana, and P. tuberculosa, are reported for the first time from China.
Key worps —Asia, Lecideales, lichens
Introduction
The family Lecideaceae Chevall. contains about 23 genera and 547 species, of which the largest genus is Lecidea Ach., containing about 427 species (Kirk et al. 2008, Fryday & Hertel 2014). In China, twenty-three species of the other genera in Lecideaceae have been reported, including two each of Amygdalaria Norman, Bellemerea Hafellner & Cl. Roux, and Immersaria Rambold & Pietschm.; one each of Lecidoma Gotth. Schneid. & Hertel, Paraporpidia Rambold & Pietschm., and Stenhammarella Hertel; and 16 species of Porpidia Korb. (Hertel 1977, Wei 1991; Aptroot & Seaward 1999; Aptroot 2002; Aptroot & Sparrius 2003; Obermayer 2004; Guo 2005; Zhang et al. 2010, 2012; Wang et al. 2012; Ismayi & Abbas 2013; Hu et al. 2014).
Amygdalaria and Porpidia are obviously very closely related. Both have large halonate ascospores, a high hymenium, Porpidia-type asci, and a dark pigmented hypothecium and are (with a few exceptions) restricted to lime-free, silicate rocks. However Amygdalaria can be best distinguished from Porpidia by the presence of cephalodia, the higher hymenium (over 130 um), the larger ascospores (generally 20-35 x 10-16 um) with conspicuous, rather compact epispores, and a tendency toward a brownish or yellowish pink thallus (Inoue 1984, Brodo & Hertel 1987, Gowan 1989, Smith et al. 2009).
34 ... Zhang, Zhao, & Hu
During our research on the lecideoid taxa of Lecideaceae in China, we have identified four taxa new to the country: Amygdalaria consentiens vat. consentiens, Porpidia carlottiana, P. lowiana, and P. tuberculosa.
Materials & methods
The studied specimens are preserved in the Lichen Section of Botanical Herbarium, Shandong Normal University, Jinan, China (SDNU); Herbarium Mycologicum Academiae Sinicae - Lichenes, Beijing, China (HMAS-L); and the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China (KUN). The morphological and anatomical characters were examined under a stereo- microscope (COIC XTL7045B2) and a polarizing microscope (OLyMpus CX41). The thalli and medullae were tested for identification with K (10% aqueous KOH solution), C (saturated aqueous NaClO solution), I (a 10% aqueous KI solution), P (a saturated p-phenylenediamine solution in 95% ethyl alcohol). The lichen substances were identified using standardized thin layer chromatography techniques (TLC) with solvent system C (Orange et al. 2010). Photos of these lichens were taken under Olympus SZX16 and BX61 with DP72.
Taxonomic descriptions
Amygdalaria consentiens (Nyl.) Hertel, Brodo & Mas. Inoue, J. Hattori Bot. Lab. 56: 322 (1984) var. consentiens Pret
MorPHoLocy — THALLUS continuous, cracked-areolate, usually thick, light orange to brownish gray, often becoming oxidated orange; medulla I-; prothallus black, thin and conspicuous; cephalodia mostly flush with thallus surface, almost the same colour as the thallus, but occasionally pinker or gray, containing Stigonema; soredia absent. APOTHECIA entirely sunken in thallus, (0.35-)0.45-0.9 mm diam., usually one or two per areole; disc dark brown to black, margin slightly to moderately rusty orange pruinose. Excipie black- brown, 50-70 um wide, without crystals; epihymenium brownish, with orange crystals; hymenium hyaline, 150-200 um tall; subhymenium colorless, 45-60 um tall; hypothecium blackish brown, usually thin, bowl-shaped; paraphyses strongly anastomosed and branched, almost moniliform at the apical part, with +swollen apices. Asci clavate, Porpidia-type; ascospores hyaline, simple, ellipsoid, 28-32(-35) x 14-17(-19) um, halonate. Pycnip1A immersed, conidia bacilliform, 6-9 x c. 1 um.
CHEMISTRY — Thallus and medulla K-, C-, KC-, P-. No lichen substances were detected by TLC.
DISTRIBUTION — Amygdalaria consentiens has been reported from Asia, Europe, and North America (Inoue 1984, Brodo & Hertel 1987, Smith et al. 2009). New to China.
SPECIMEN EXAMINED: CHINA. YUNNAN, Luquan, Mt. Jiaozixueshan, alt. 3800 m, on rock, 26 Oct. 2008, Z.J. Ren 20108488 (SDNU).
Amygdalaria & Porpidia species new to China... 35
Fic. 1 Amygdalaria consentiens var. consentiens (Ren 20108488, SDNU). A: thallus; B: prothallus; C: apothecium section; D: epihymenium with crystals; E: amyloid reaction of ascus; F: ascus and ascospores; G: ascospores; H: paraphyses.
COMMENTS —Amygdalaria consentiens var. consentiens is morphologically similar to A. consentiens var. japonica and A. continua, but A. consentiens var. japonica contains stictic and constictic acids, while A. continua has a smooth to finely rimose thallus which is rarely cracked-areolate.
Porpidia carlottiana Gowan, Bryologist 92: 39 (1989) Fia. 2 MorPHoLocy — THALLUS crustose, finely cracked to subrimose, 0.15-0.25 mm thick, whitish gray; medulla I-; prothallus continuous between thallus
36 ... Zhang, Zhao, & Hu
20 pm 20 pm
Eb ad
Fic. 2 Porpidia carlottiana (Wang 20127123, SDNU). A: thallus; B: apothecium section; C: amyloid reaction of ascus; D: ascospores; E: exciple and epihymenium without crystals.
patches, black, thin; soredia absent. APOTHECIA scattered or contiguous, sunken in thallus when young, becoming sessile on a broad base when mature, 0.5-1.1(-1.5) mm diam.; disc black, plane to weakly convex, usually with heavy whitish pruina; margin bare, distinct, regular to flexuose. ExcrpLe dark brown to black at margin, brown within, 90-105 um wide, without crystals; epihymenium yellowish brown or olive brown, without crystals; hymenium hyaline, 90-105(-130) um tall; subhymenium 20-25 um, hypothecium blackish brown; paraphyses strongly anastomosed and apically branched. Asci clavate, Porpidia-type; ascospores hyaline, simple, ellipsoid, 17-20 x 7-10 um, halonate. Pycnip1a not observed.
CHEMISTRY — ‘Thallus and medulla K-, C-, KC-, P-. 2’-O-methyl- superphyllinic acid was detected by TLC.
DISTRIBUTION — Porpidia carlottiana has been reported from North America (Gowan 1989). New to China.
SPECIMEN EXAMINED: CHINA. YUNNAN, Lijiang, Mt. Laojunshan, alt. 3800 m, on rock,
5 Nov. 2009, H.Y. Wang 20127123 (SDNU). ComMENts — Porpidia carlottiana is closely related to P. rugosa, which also produces pruinose apothecia and 2’-O-methylsuperphyllinic acid. However, P. rugosa always has soredia, and the apothecia rarely seen.
Amygdalaria & Porpidia species new to China... 37
Porpidia lowiana Gowan, Bryologist 92: 49 (1989) Fic. 3
MorPHOoLocy — THALLUS crustose, continuous, finely cracked to rimose- areolate, 0.2-0.5 mm thick, yellowish gray to whitish, sometimes dark gray or greenish gray; medulla I-; prothallus absent; soredia absent. APOTHECIA scattered to contiguous, soon becoming broadly sessile, 0.5-1.3 mm diam.; disc black, non-pruinose to moderately pruinose; margin bare, distinct, even to weakly crenulate, regular to flexuose. ExcrpLe mostly black and carbonaceous at exciple margin, hyaline within, 55-90 um wide, with crystals that are soluble in K and C; epihymenium olive brown, without crystals; hymenium hyaline, 70-110 um tall; subhymenium 18-26 um, hypothecium blackish brown; paraphyses strongly anastomosed and apically branched. Asci clavate, Porpidia-type; ascospores hyaline, simple, ellipsoid or tear-drop-shape, 14-16(-25) x (4.5-)6-8 um, halonate. Pycnip1A not observed.
CHEMISTRY — ‘Thallus and medulla K-, C-, KC-, P-. Confluentic, 2’-O-methylmicrophyllinic, and 2’-O-methylperlatolic acids were detected by TLE.
DISTRIBUTION — Porpidia lowiana has been reported from Europe and North America (Gowan 1989, Smith et al. 2009). New to China.
Fic. 3 Porpidia lowiana (Ren 20102097, SDNU). A: thallus; B: apothecium section; C: amyloid reaction of ascus; D: ascospores; E: exciple with crystals.
38 ... Zhang, Zhao, & Hu
SPECIMENS EXAMINED: CHINA. HELONGJIANG, Tahe, Mt. Mengkeshan, alt. 555 m, on rock, 14 Aug. 2009, Q. Ren 20102097 (SDNU). INNER Monco ia, Mangui, alt. 900 m, on rock, 13 Sep. 1977, J.C. Wei, 074581 (HMAS-L); SicHUAN, Dukou, Mt. Dabaoding, alt. 1950 m, on rock, 21 Jun. 1983, L.S. Wang 83-184 (KUN); YUNNAN, Xinping, Mt. Mopan, alt. 2000 m, on rock, 20 Dec. 2008, L.S. Wang 08-30003 (KUN); Chuxiong, Mt. Zixi, alt. 2060 m, on rock, 31 Aug. 2005, L.S. Wang 05-25263 (KUN); Lincang county, Matai village, alt. 1650 m, on rock, 20 Oct. 2003, L.S. Wang 03-22921, 03-22922 (KUN); Yimen, alt. 1600 m, on rock, 10 Dec. 1990, L.S. Wang 11707 (KUN). ComMENts — Porpidia lowiana is closely related to P cinereoatra, from which it differs mainly in having a thinner, yellower thallus, thicker proper margin (c. 0.05 mm in P. cinereoatra), and sessile apothecia (mainly sunken in P. cinereoatra).
Porpidia tuberculosa (Sm.) Hertel & Knoph, Beih. Nova Hedwigia 79: 438 (1984) Fic. 4
MorPHOLOGY — THALLUS crustose, continuous, subrimose to distinctly rimose-areolate, 0.2-0.3(-0.5) mm thick, medium gray or sometimes rusty orange; medulla I+ violet; prothallus usually present when abutting another lichen, black; soralia present, 0.3-0.7 mm diam., round to irregular, scattered to crowded, shallowly erose or abraded, usually with slightly raised rim; soredia farinose to granular, white, pale gray or flecked blue-gray or blackish, I+ violet. APOTHECIA occasional, scattered, soon becoming sessile, 0.9-1.5 (-1.7) mm diam.; disc black, plane to subconvex, weakly to moderately pruinose; margin bare, distinct, regular to flexuose. ExcIPLE greenish to brownish black at exciple margin, dark brown within, 90-140 um wide, without crystals; epihymenium olive brown, without crystals; hymenium hyaline, 90-105 um tall; subhymenium 35-50 um, hypothecium blackish brown; paraphyses strongly anastomosed and apically branched. Ascr clavate, Porpidia-type; ascospores hyaline, simple, ellipsoid, 17.5-22 x 7-8 um, halonate. PycNnip1A immersed, conidia bacilliform, 7.5-10 x c. 1 um.
CHEMISTRY — ‘Thallus and medulla K-, C-, KC-, P-. Confluentic, 2’-O-methylmicrophyllinic, and 2’-O-methylperlatolic acids were detected by TES
DISTRIBUTION — Porpidia tuberculosa has been reported from Asia, Macaronesia, Europe, and North & South America (Hertel 1984, Smith et al. 2009). New to China.
SPECIMENS EXAMINED: CHINA. HusBEI, Shennongjia, Mt. Dashennongijia, alt. 3000 m, on rock, 27 Jun. 1984, J.C. Wei, 067994, 068005, 074292 (HMAS-L). YUNNAN, Luquan, Mt. Jiaozixueshan, alt. 3800 m, on rock, 27 Oct. 2008, H.Y. Wang 20081180, 20082302 (SDNU). ComMENtTs — Porpidia tuberculosa, P. rugosa and P. soredizodes all have a thallus with soralia. However, P rugosa has a more verrucose thallus,
Amygdalaria & Porpidia species new to China... 39
100 um
Fic. 4 Porpidia tuberculosa (Wei 067994, HMAS-L). A: thallus; B: apothecium section; C: amyloid reaction of ascus; D: conidia; E: exciple and epihymenium without crystals.
I- medulla, and contains 2’-O-methylsuperphyllinic and glaucophaeic acids. Porpidia soredizodes has an I- medulla as well as an overall smaller thinner (<0.2 mm) darker thallus that contains stictic acid.
Acknowledgements
The authors thank Dr. A. Aptroot (ABL Herbarium, Soest, the Netherlands) and Dr. Shou-Yu Guo (State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for presubmission reviews. This study was supported by Program for Scientific Research Innovation Team in Colleges and Universities of Shandong Province, the National Natural Science Foundation of China (31170187), Foundation of Key Laboratory, CAS (KLBB-201306), and the Scientific Research Foundation of Graduate School of Shandong Normal University (BCX1406).
Literature cited
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Aptroot A, Seaward MRD. 1999. Annotated checklist of Hong Kong lichens. Tropical Bryology 17: 57-101.
Aptroot A, Sparrius LB. 2003. New microlichens from Taiwan. Fungal Diversity 14: 1-50.
AO ... Zhang, Zhao, & Hu
Brodo IM, Hertel H. 1987. The lichen genus Amygdalaria (Porpidiaceae) in North America. Herzogia 7: 493-521
Fryday AM, Hertel H. 2014. A contribution to the family Lecideaceae s. lat. (Lecanoromycetidae inc. sed., lichenized Ascomycota) in the southern subpolar region; including eight new species and some revised generic circumscriptions. Lichenologist 46(3): 389-412. http://dx.doi.org/10.1017/S0024282913000704
Gowan SP. 1989. The lichen genus Porpidia (Porpidiaceae) in North America. Bryologist 92: 25-59.
Guo SY. 2005. Lichens. 31-82, in: WY Zhuang (ed.). Fungi of northwestern China. Mycotaxon Ltd., Ithaca, New York.
Hertel H. 1977. Gesteinsbewohnende Arten der Sammelgattung Lecidea (Lichenes) aus Zentral-, Ost- und Siidasien. Khumbu Himal, Ergebnisse des Forschungsunternehmens Nepal Himalaya. 6: 145-378.
Hertel H. 1984. Uber saxicole, lecideoide Flechten der Subantarktis. Beihefte zur Nova Hedwigia. 79: 399-499.
Hu L, Zhao X, Sun LY, Zhao ZT, Zhang LL. 2014. Four lecideoid lichens new to China. Mycotaxon 128: 83-91. http://dx.doi.org/10.5248/128
Inoue M. 1984. Japanese crustose lichen genera formerly reported under Lecidea sensu lato. 1. Amygdalaria Norman. Journal of the Hattori Botanical Laboratory 56: 321-330.
Ismayi G, Abbas A. 2013. Bellemerea alpina — new lichen species record from China. Plant Science Journal 31(4): 333-335.
Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi. 10th edition. CABI Bioscience: CAB International. 771 p.
Obermayer W. 2004. Additions to the lichen flora of the Tibetan region. Bibliotheca Lichenologica 88: 479-526.
Orange A, James PW, White FJ. 2010. Microchemical methods for the identification of lichens. 2nd edition. London: British Lichen Society.
Smith CW, Aptroot A, Coppins BJ, Fletcher A, Gilbert OL, James PW, Wolseley PA (eds). 2009. The lichens of Great Britain and Ireland. Natural History Museum Publications, in association with The British Lichen Society.
Wang XY, Zhang LL, Joshi Y, Wang HY, Hur JS. 2012. New species and new records of the lichen genus Porpidia (Lecideaceae) from western China. Lichenologist 44(5): 619-624. http://dx.doi.org/10.1017/S0024282912000242
Wei JC. 1991. An enumeration of lichens in China. International Academic Publishers, Beijing.
Zhang LL, Wang HY, Sun LY, Zhao ZT. 2010. Four lichens of the genus Lecidea from China. Mycotaxon 112: 445-450. http://dx.doi.org/10.5248/112.445
Zhang LL, Wang LS, Wang HY, Zhao ZT. 2012. Four new records of lecideoid lichens from China. Mycotaxon 119: 445-451. http://dx.doi.org/10.5248/119.445
ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
MYCOTAXON
http://dx.doi.org/10.5248/130.41 Volume 130, pp. 41-46 January-March 2015
Anungitea guangxiensis and Ellisembia longchiensis, two new species from southern China
JI-WEN XIA, YING-RuI Ma, & XIU-GUO ZHANG
Department of Plant Pathology, Shandong Agricultural University, Taian, 271018, China
*CORRESPONDENCE TO: zhxg@sdau.edu.cn, sdau613@163.com
ABSTRACT — Two new hyphomycete species, Anungitea guangxiensis and Ellisembia longchiensis, are described and illustrated from specimens collected on dead branches in southern China. Anungitea guangxiensis is characterized by conidiogenous cells that are polyblastic, integrated, terminal, smooth, and denticulate (with cylindrical denticles) and conidia produced in acropetal chains. Ellisembia longchiensis is characterized by distinct, single, unbranched, erect conidiophores, monoblastic conidiogenous cells, and distoseptate conidia that have a small, globose, mucilaginous apical sheath.
Key worps — conidial fungi, taxonomy
Introduction
Anungitea was established by Sutton (1973) as a monotypic genus with A. fragilis B. Sutton as the type species. The genus is characterized by conidia borne on conspicuous denticulate loci of the terminal or intercalary conidiogenous cells and which form acropetal, unbranched chains. A further 19 names have been proposed in Anungitea (Castafeda 1986; Castafieda & Kendrick 1990, 1991; Castaneda et al. 1997; Crous et al. 1995, 2014; Kirk 1983; Matsushima 1975; Rambelli et al. 2008, 2009, 2010; Sutton & Hodges 1978), but subsequently A. globosa B. Sutton & Hodges was synonymised with A. fragilis (Kirk 1982; Matsushima 1987; Castaneda Ruiz et al. 1996), and A. triseptata R.F. Castafieda & W.B. Kendr. was transferred to Anungitopsis (Castaheda & Kendrick 1991).
The genus Ellisembia was introduced by Subramanian (1992) to accommodate Sporidesmium-like species with determinate or irregularly percurrently extending conidiogenous cells that produce distoseptate conidia. Wu & Zhuang (2005) merged Imicles Shoemaker & Hambl. (Shoemaker & Hambleton 2001) into Ellisembia and expanded the generic concept to include species that produce typically lageniform, ovoid, or doliiform percurrently extending conidiogenous cells.
42 ... Xia, Ma, & Zhang
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Fic. 1. Anungitea guangxiensis (holotype, HSAUP H6351). A,C. Conidiophores with conidia. B. Apex of conidiophore. D. Conidia. E,F. Apex of conidiophores with conidia.
Many taxa of hyphomycetes have been already described from China (Wu & Zhuang 2005, Zhang et al. 2009a,b, 2011, 2012, Ma et al. 2010, 2012a,b,c, Ren
Anungitea & Ellisembia spp. nov. (China) ... 43
et al. 2012). In our studies on hyphomycetes from deciduous stems and rotten wood in southern China, we collected two hyphomycetes that possess the morphological characteristics of Anungitea and Ellisembia. Both are proposed herein as new species.
Anungitea guangxiensis J.W. Xia & X.G. Zhang, sp. nov. FIG. 1 MycoBank MB 811157 Differs from Anungitea heterospora by its much larger conidia; and from A. longicatenata by its longer, cylindrical conidia. Type: China, Guangxi Province: Mount Dayao, on dead stems of unidentified broad- leaved tree, 26 Oct 2012, J.W. Xia (Holotype, HSAUP H6351; isotype, HMAS 243458).
EryMo_oey: in reference to the province where the type was found.
Colonies on natural substrate effuse, pale brown to brown, hairy. Mycelium partly superficial, partly immersed in the substratum, composed of septate, pale brown, smooth, 1-2 um wide hyphae. Conidiophores distinct, single, unbranched, erect, straight or slightly flexuous, cylindrical, smooth, thick- walled, pale brown to brown, 6-11-septate, 70-160 x 2.5-4.5 um. Conidiogenous cells polyblastic, integrated, terminal, sympodial, hyaline to pale brown, 10-18 x 2.5-4.5 um, with 1-6 short denticles. Conidia acrogenous, cylindrical, in unbranched acropetal chains, dry, subhyaline to hyaline, 1-septate, truncate at the base and rounded at the apex, 16-22 um long, 3-4 um wide.
ComMENTs - Among the known species, Anungitea guangxiensis bears some resemblance to A. heterospora P.M. Kirk and A. longicatenata Matsush. in conidial shape. However, the primary conidia of A. heterospora are smaller (8-9.5 x 3-4 um, l-septate) than those of A. guangxiensis and occasionally slightly constricted at the septum (Kirk 1983). The conidia of A. longicatenata differ by being shorter (11-18 x 2.5-4 um, 0-2-septate), narrowly obovoid to cylindrical, and tapering at both ends (Matsushima 1975).
Ellisembia longchiensis J.W. Xia & X.G. Zhang, sp. nov. Fic. 2 MycoBank MB 811159 Differs from Ellisembia mucicola by its longer, narrower, obclavate conidia with more
numerous distosepta; and from E. suttonii by its longer, narrower conidia and its conidiogenous cells lacking percurrent proliferations.
Type: China, Sichuan Province: national forest park of Longchi, on dead stems of unidentified broad-leaved tree, 17 Apr 2012, J.W. Xia (Holotype, HSAUP H6246-1; isotype, HMAS 243459).
EryMo_oey: in reference to the type locality. Colonies on natural substrate effuse, pale brown to brown, hairy. Mycelium partly superficial, partly immersed in the substratum, composed of septate, pale brown, smooth, 2-5 um wide hyphae. Conidiophores distinct, single, erect,
44 ... Xia, Ma, & Zhang
A : B 20um
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ns
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Fic. 2. Ellisembia longchiensis (holotype, HSAUP H6246-1). A. Conidiophores with conidia. B. Conidia.
straight or slightly flexuous, cylindrical, smooth, thick-walled, pale brown to brown, 2-4-septate, 55-75 x 3.5-5 um. Conidiogenous cells monoblastic, integrated, terminal, pale brown to brown, 13.5-15 x 3.5-5 um. Conidial
Anungitea & Ellisembia spp. nov. (China) ... 45
secession schizolytic. Conidia holoblastic, solitary, acrogenous, straight or slightly curved, obclavate, smooth-walled, pale brown to brown, paler towards the apex where it is hyaline, 9-12-distoseptate, 55-86 um long, 6-7 um thick in the broadest part, 3-4.5 um wide at the truncate base, with a small, globose, mucilaginous apical sheath.
Comments - Ellisembia longchiensis is closely related to E. mucicola W.P. Wu and E. suttonii W.P. Wu. Ellisembia mucicola differs by its shorter, wider, ellipsoid to fusiform conidia (42-52 x 2.5-4 um, 8-9-septate); and E. suttonii differs by its shorter, wider conidia (45-52 x 9-12 um, 10-12-septate) and its percurrently proliferating conidiogenous cells (Wu & Zhuang 2005).
Acknowledgments
The authors express gratitude to Dr. Eric H.C. McKenzie and Dr. Bryce Kendrick for serving as pre-submission reviewers and for their valuable comments and suggestions. This project was supported by the National Natural Science Foundation of China (Nos. 31093440, 31230001) and the Ministry of Science and Technology of the People’s Republic of China (Nos. 2006FY120100).
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Castaneda Ruiz RE, Kendrick WB. 1990. Conidial fungi from Cuba: II. University of Waterloo Biology Series 33: 1-61.
Castafieda Ruiz RE, Kendrick WB. 1991. Ninety-nine conidial fungi from Cuba and three from Canada. University of Waterloo Biology Series 35: 1-132.
Castafieda Ruiz RF, Gené J, Guarro J. 1996. Litter hyphomycetes from La Gomera (Canaries). Mycotaxon 59: 203-215.
Castaneda Ruiz RF, Kendrick WB, Guarro J. 1997. Notes on conidial fungi. XIV. New hyphomycetes from Cuba. Mycotaxon 65: 93-105.
Crous PW, Wingfield MJ, Kendrick WB. 1995. Foliicolous dematiaceous hyphomycetes from Syzygium cordatum. Can. J. Bot. 73: 224-234.
Crous PW, Groenewald JZ, Shivas RG. 2014. Fungal Planet 222 - 10 June 2014. Anungitea eucalyptorum Crous & R.G. Shivas, sp. nov. Persoonia 32: 198-199.
Kirk PM. 1982. New or interesting microfungi. IV. Dematiaceous hyphomycetes from Devon. Transactions of the British Mycological Society 78: 55-74.
Kirk PM. 1983. New or interesting microfungi. IX. Dematiaceous hyphomycetes from Esher Common. Transactions of the British Mycological Society 80: 449-467.
Ma LG, Ma J, Zhang YD, Zhang XG. 2010. A new species of Spadicoides from Yunnan, China. Mycotaxon 113: 255-258. http://dx.doi.org/10.5248/113.255
Ma LG, Ma J, Zhang YD, Zhang XG. 2012a. Spadicoides camelliae and Diplococcium livistonae, two new hyphomycetes on dead branches from Fujian Province, China. Mycoscience 53: 25-30. http://dx.doi.org/10.1007/s10267-011-0138-z
Ma J, Zhang YD, Ma LG, Ren SC, Castafieda Ruiz RE, Zhang XG. 2012b. Three new species of Solicorynespora from Hainan, China. Mycological Progress 11: 639-645. http://dx.doi.org/10.1007/s11557-011-0775-9
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MaJ, Zhang YD, Ma LG, Castaneda Ruiz RE, Zhang XG. 2012c. Three new species of Sporidesmiella from southern China. Mycoscience 53: 187-193. http://dx.doi.org/10.1007/s10267-011-0152-1
Matsushima T. 1975. Icones microfungorum a Matsushima lectorum. Matsushima, Kobe. 209 p.
Matsushima T. 1987. Matsushima Mycological Memoirs 5: 1-100.
Rambelli A, Venturella G, Ciccarone C. 2008. Dematiaceous hyphomycetes from Pantelleria Mediterranean maquis litter. Flora Mediterranea 18: 441-467.
Rambelli A, Venturella G, Ciccarone C. 2009. More dematiaceous hyphomycetes from Pantelleria Mediterranean maquis litter. Flora Mediterranea 19: 81-113.
Rambelli A, Tempesta S, Venturella G, Ciccarone C. 2010. Dematiaceous hyphomycetes from Pantelleria Mediterranean maquis litter. Flora Mediterranea 20: 211-233.
Ren SC, Ma J, Ma LG, Zhang YD, Zhang XG. 2012. Sativumoides and Cladosporiopsis, two new genera of hyphomycetes from China. Mycological Progress 11: 443-448. http://dx.doi.org/10.1007/s11557-011-0759-9
Shoemaker RA, Hambleton S. 2001. “Helminthosporium” asterinum, Polydesmus elegans, Imimyces, and allies. Canadian Journal of Botany 79: 592-599. http://dx.doi.org/10.1139/cjb-79-5-592.
Subramanian CV. 1992. A reassessment of Sporidesmium (hyphomycetes) and some related taxa. Proceeding of the Indian National Science Academy, B 58: 179-190.
Sutton BC. 1973. Hyphomycetes from Manitoba and Saskatchewan, Canada. Mycological Papers 132: 1-143.
Sutton BC, Hodges CS. 1978. Eucalyptus microfungi. Chaetendophragmiopsis gen. nov. and other hyphomycetes. Nova Hedwigia 29: 593-607.
Wu WP, Zhuang WY. 2005. Sporidesmium, Endophragmiella and related genera from China. Fungal Diversity Research Series 15: 1-351.
Zhang K, Ma J, Wang Y, Zhang XG. 2009a. Three new species of Piricaudiopsis from southern China. Mycologia 101: 417-422. http://dx.doi.org/10.3852/08-147
Zhang K, Ma LG, Zhang XG. 2009b. New species and records of Shrungabeeja from southern China. Mycologia 101: 573-578. http://dx.doi.org/10.3852/09-006
Zhang YD, Ma J, Ma LG, Castafieda Ruiz RF, Zhang XG. 2011. New species of Phaeodactylium and Neosporidesmium from China. Sydowia 63: 125-130.
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MY COTAXON
http://dx.doi.org/10.5248/130.47 Volume 130, pp. 47-56 January-March 2015
Fistulina subhepatica sp. nov. from China inferred from morphological and sequence analyses
Jz Sonc, MEI-LING HAN, & BAo-Kai Cur
Institute of Microbiology, Beijing Forestry University, Beijing 100083, China Beijing Key Laboratory for Forest Pest Control, Beijing Forestry University, Beijing 100083, China
* CORRESPONDENCE TO: cuibaokai@yahoo.com
ABSTRACT — Fistulina subhepatica sp. nov. is described from Yunnan Province, southwestern China. The main characters of F subhepatica are its pileate basidiomata with rose to reddish brown pileal surface and white pore surface, individual and easily separable pores (6-9 per mm), a monomitic hyphal structure with clamped generative hyphae, and ellipsoid basidiospores (4-6 x 3-4 um). Phylogenetic analysis inferred from the combined ITS (internal transcribed spacer region) and nLSU (the nuclear large subunit ribosomal RNA) dataset support F. subhepatica as a distinct new species in Fistulina.
Key worps — Agaricales, Agaricomycetes, Fistulinaceae, phylogeny, rDNA, taxonomy
Introduction
Fistulina Bull. was established by Bulliard (1790: t.464) and typified by F. buglossoides Bull. [= E hepatica (Schaeff.) With.]. It is a small but cosmopolitan genus and causes a brown rot of hardwoods. Morphologically, the genus is characterized by an annual growth habit, reddish to brown pileus surface, separate but closely packed tubes, a monomitic hyphal system, and ovoid basidiospores (Gilbertson & Ryvarden 1986, Nunez & Ryvarden 2001, Ryvarden & Gilbertson 1993).
Previous multi-gene phylogenetic studies showed that Fistulina fell into the euagarics clade and was closely related to Schizophyllum Fr., Auriculariopsis Maire, and Porodisculus Murrill (Binder et al. 2005, Bodensteiner et al. 2004).
Taxonomic and phylogenetic studies focusing on brown-rot fungi in China have been carried out recently, and some new species have been described (Cui 2013, Cui & Dai 2013, Cui & Li 2012, Cui et al. 2011, Li & Cui 2013a, Li et al. 2013, Shen et al. 2014, Zhou & Dai 2012, Zhou & Wei 2012). As a continuation of these surveys, one undescribed species of Fistulina was discovered based on
48 ... Song, Han, & Cui
morphological characters and phylogenetic analysis of the ITS and nLSU rRNA gene regions.
TABLE 1. List of species, vouchers, and GenBank accession numbers for sequences used in phylogenetic analysis. New sequences are indicated by bold font.
GENBANK ACCESSION NO.
SPECIES VOUCHER nLsU ITS Boletus satanas Lenz Bs 2 AF336242 DQ534567 TDB 1000c DQ533973 Coniophora olivacea (Fr.) P. Karst. 402 AF098376 Favolaschia andina Singer KG 0025 HM246679 HM246678 F. calocera R. Heim PDD 70689 AY572006 PDD 71528 AY572007 SR.KEN. 346 AF261417 E cf. calocera JM 98/372 AF261419 EF, peziziformis (Berk. & M.A. Curtis) Kuntze PDD 67440 AY572008 Favolaschia sp. KG 0027 HM246682 HM246681 Fistulina antarctica REG 550 AY571002 CBS 701.85 AY293181 DQ486702 CIEFAP 115 AY571003 EF. hepatica DSH 93-183 AY293182 REG 593 AY571004 AY571038 EF. pallida CBS 508.63 AY571005 AY571039 E. subhepatica Dai 11714 KJ925057 Dai 12416 KJ925053 KJ925058 Cui 11130 KJ925054 KJ925059 Dai 13216 KJ925055 KJ925060 Dai 13244 (HT) KJ925056 KJ925061 Jaapia argillacea CBS 252.74 GU187581 GU187524 Mycena epipterygia (Scop.) Gray GLM 45969 AY207249 M. galericulata (Scop.) Gray GLM 45970 AY207251 TENN 7495 AF261412 RV 87/14.01 AF042636 M. pura (Pers.) P. Kumm. CBH 371 FN394630 KF913023 IS 10/11/2000 FN394634 FN394611 CBH 367 FN394631 KF913022 CBH 358 FN394629 FN394608 M. rosea Gramberg CBH 097 FN394628 FN394556 TL 12393 FN394641 FN394555 UP 2 FN394632 FN394550 M. sanguinolenta (Alb. & Schwein.) P. Kumm. 430360 AB512401 AB512311 Panellus edulis Y.C. Dai et al. HMJAU 7066 GQ219739 GQ219731 HMJAU 7214 GQ219738 GQ219730
Fistulina subhepatica sp. nov. (China) ... 49
Paxillus involutus (Batsch) Fr. UBC F16285 EU486436
Poromycena manipularis (Berk.) R. Heim JM 98/217 AF261423
Porodisculus pendulus HHB-15376-sp AY293204 DAOM 198417 AF261593
Poromycena sp. JM 98/128 AF261429
Suillus cavipes (Opat.) A.H. Sm. & Thiers TDB-646 AF071535
Schizophyllum amplum (Lév.) Nakasone FCUG 1803 AF141873
S. commune Fr. NBRC 4928 AB733339 AB733163 NBRC 6502 AB733340 AB733164 NBRC 30496 AB733341 AB733165 NBRC 30749 AB733342 AB733166
S. fasciatum Pat. CBS 267.60 AF261589 L43385
S. umbrinum Berk. FL 02.1 AF261590 AF249391
HT = holotype
Materials & methods
Morphological studies
The studied specimens are deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Microscopic examinations followed Dai et al. (2010). Sections were studied at magnifications up to 1000x using a Nikon E 80i microscope and phase contrast illumination (Nikon, Tokyo, Japan). Drawings were made with the aid of a drawing tube. Microscopic features, measurements, and drawings were made from slide preparations stained with cotton blue and Melzer’s reagent. Basidiospores were measured from sections cut from the tubes. In presenting the variation in the basidiospore size, 5% of measurements that were excluded from each end of the range are given in parentheses. In the text, the following abbreviations were used: IKI = Melzer’s reagent, IKI- = negative in Melzer’s reagent, KOH = 5% potassium hydroxide, CB = cotton blue, CB- = acyanophilous, CB+ = cyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios among the specimens studied, n = number of spores measured from given number of specimens. Special color terms follow Petersen (1996).
Molecular procedures and phylogenetic analysis
The Phire Plant Direct PCR Kit (Finnzymes, Vantaa, Finland) was used to obtain PCR products from dried specimens, according to the manufacturer's instructions. A small piece of dried fungal specimen was lysed in 30 ul dilution buffer for DNA extraction. After incubating for 3 min at room temperature, 0.75 ul of the supernatant was used as template for a 30 ul PCR reaction. The internal transcribed spacer (ITS) regions were amplified with the primers ITS4 and ITS5 (White et al. 1990), and the nuclear large subunit (nLSU) ribosomal RNA gene regions with the primers LROR and LR7 (http://www. biology.duke.edu/fungi/mycolab/primers.htm). The PCR procedure for ITS was as follows: initial denaturation at 95°C for 3 min, followed by 34 cycles at 94°C for 40 s, 54°C for 45 s, and 72°C for 1 min, and a final extension of 72°C for 10 min. The
50 ... Song, Han, & Cui
PCR procedure for nLSU was as follows: initial denaturation at 94°C for 1 min, followed by 34 cycles at 94°C for 30 s, 50°C for 1 min and 72°C for 1.5 min, and a final extension of 72°C for 10 min. The PCR products were purified and directly sequenced in Beijing Genomics Institute, China, with the same primers. All newly generated sequences were submitted to GenBank (TABLE 1).
Sequences generated for this study were aligned with additional sequences downloaded from GenBank (TaBLE 1) using BioEdit (Hall 1999) and ClustalX (Thompson et al. 1997). Prior to phylogenetic analysis, ambiguous positions at the start and the end were deleted and gaps were manually adjusted to optimize alignment. Sequence alignment was deposited at TreeBASE (http://purl.org/phylo/treebase; submission ID 15918).
Phylogenetic analysis was done as in Li & Cui (2013b). Maximum parsimony analysis and Bayesian inference (BI) was applied to the combined ITS and nLSU dataset. Jaapia argillacea Bres. was used as outgroup to root trees (Bodensteiner et al. 2004). The maximum parsimony tree was constructed in PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates (Felsenstein 1985). Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimonious tree (MPT) generated.
MrMODELTEST2.3 (Nylander 2004, Posada & Crandall 1998) was used to determine the best-fit evolution model for BI. BI was calculated with MrBayes3.1.2 (Ronquist & Huelsenbeck 2003) according to the calculated model. Four Markov chains were run for 2 runs from random starting trees for 1.5 million generations, and trees were sampled every 100 generations. The first one-fourth generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for maximum parsimony (MP) 275% and Bayesian posterior probabilities (BPP) 20.95 were considered as significantly supported.
Results
The combined ITS and nLSU dataset include sequences from 49 fungal collections. The dataset has an aligned length of 1388 characters including gaps, of which 812 characters are constant, 135 are parsimony-uninformative, and 441 are parsimony-informative. Maximum parsimony analysis yielded 9 equally parsimonious trees (Length = 1373, CI = 0.650, RI = 0.867, RC = 0.564, HI = 0.350), and a strict consensus tree of these trees is shown in Fic. 1. The best model for the combined ITS and nLSU partition is a GTR+I+G model. BI produced a topology similar to that from MP analysis with an average standard deviation of split frequencies = 0.007406.
In the phylogenetic tree (Fic. 1), the unknown Chinese Fistulina collections grouped in a well-supported lineage (MP = 88%, BPP = 1.00), which had a
Fistulina subhepatica sp. nov. (China) ... 51
Favolaschia calocera SR.KEN. 346 a Soins Favolaschia calocera PDD 70689 97/1.00 deleiay Favolaschia calocera PDD 71528 -/0.98| © Favolaschia cf. calocera JM 98/372 IN Favolaschia sp. KG 0027 78/1.00] Favolaschia andina KG 0025 Favolaschia peziziformis PDD 67440 Poromycena manipularis JM 98/217 59/- ||; Mycena galericulata TENN 7495 Mycena galericulata GLM 45970 Mycena epipterygia GLM 45969 Mycena rosea CBH 097 98/0.97 ae Mycena rosea UP 2 Mycena rosea TL 12393 LK) Mycena pura CBH 371 100/1.00 Mycena pura CBH 367 Mycena pura CBH 358 Mycena pura I§ 10/11/2000 89/1.00 Mycena sanguinolenta 430360 Poromycena sp. JM 98/128 Mycena galericulata RV 87/14.01 100/1.00 Panellus edulis HMJAU 7066 Panellus edulis HMJAU 7214 Fistulina subhepatica Dai 11714 Fistulina subhepatica Dai 13244
100/-
99/1.00 88/1.00) Fistulina subhepatica Dai 13216 87/0.99 100/1.00 Fistulina subhepatica Cut 11130 Fistulina subhepatica Dai 12416 Fi stu lin a 100/1.00 99/199 Fistulina hepatica REG 593 Fistulina hepatica DSH 93-183 Fistulina antarctica REG 550 100/1.00 Fistulina antarctica CBS 701.85 98/0.99 100/1.00 64/- Fistulina antarctica CIEFAP 115 Porodisculus pendulus HHB-15376-sp Porodisculus pendulus DAOM 198417 93/0.99 L_. Fistulina pallida CBS 508.63 Schizophyllum commune NBRC 30749 90/- Schizophyllum commune NBRC 30496 58/- 89/0.99 pelzepioiiuin commune NBRC 4928 Schizophyllum commune NBRC 6502 Schizophyllum fasciatum CBS 267.60 100/1.00
Schizophyllumumbrinum FL 02.1 79/- © Schizophyllumamplum FCUG 1803 66/0.99 — Boletus satanas Bs 2 91/0.98]L Boletus satanas TDB 1000c 89/1.00 Paxillus involutus UBC F16285 Suillus cavipes TDB-646
Coniophora olivacea 402
Jaapia argillacea CBS 252.74
100/-
30
Figure 1. The phylogenetic position of Fistulina subhepatica based on ITS+nLSU sequence data. Topology is from maximum parsimony analysis. Bootstrap values >50% and Bayesian posterior probabilities 20.95 are indicated at the nodes.
sister relationship with E hepatica [= F buglossoides, the generic type]. The new species is described on the next page.
52... Song, Hany-& Cui
FiGurRE 2. Fistulina subhepatica (holotype), basidiomata in situ. Scale bar = 2 cm.
Taxonomy
Fistulina subhepatica B.K. Cui & J. Song, sp. nov. FIGs. 2-3 MycoBank MB 809111 Differs from Fistulina hepatica by its thin- to slightly thick-walled tramal generative
hyphae with clamp connections and larger basidiospores.
Type: China, Yunnan Province, Jingdong County, Ailaoshan Nature Reserve, on dead tree of Lithocarpus (Fagaceae), 12.V1I.2013, Dai 13244 (holotype, BJFC 014732; GenBank KJ925056, KJ925061).
EryMoLoecy: subhepatica (Lat.): refers to the morphological similarity to Fistulina
hepatica. FruiTBopy — Basidiomata annual, pileate, fleshy, and readily exuding a reddish blood-like sap when squeezed or bruised when fresh, leathery when dry. Pileus sessile to laterally substipitate, dimidiate to reniform or subcircular, projecting up to 25 cm, 30 cm wide, and 6 cm thick at base. Pileal surface rose to reddish brown when fresh, becoming fuscous to black upon drying, faintly radially furrowed when fresh; margin acute, concolorous. Pore surface white when fresh, turning darker when bruised and becoming cinnamon-buff to reddish brown when dry; pores 6-9 per mm, consisting of individual, crowed but easily separable tubes. Context clay-buff to black, leathery when dry, up to 5 cm thick. Tubes concolorous with pore surface, leathery, up to 1 cm long.
Fistulina subhepatica sp. nov. (China) ... 53
iabaateiae aes 10 pm 10 ym
Cc
SSS 10 ym
FiGure 3. Fistulina subhepatica (holotype). a: Basidiospores. b: Basidia and basidioles. c: Cystidioles. d: Cystidial elements from dissepiment edges. e: Hyphae from trama. f: Hyphae from context. Scale bars = 10 um.
54 ... Song, Han, & Cui
HYPHAL STRUCTURE — Hyphal system monomitic; generative hyphae with clamp connections, IKI-, CB-, more or less dissolving in KOH.
CONTEXT — Generative hyphae hyaline, thin-walled, rarely branched, interwoven, 5-12 um in diam, but with inflated portions up to 18 um, gloeoplerous hyphae present.
TuBES — Generative hyphae hyaline, thin- to slightly thick-walled, rarely branched, parallel along the tubes and densely in sections from dried specimens, 3.5-8 um in diam. Basidia clavate with four sterigmata and a basal clamp connection, 15-32 x 5-7 um; basidioles in shape similar to basidia, but slightly smaller. Cystidial elements present in the dissepimental edges, hyaline, thin-walled, 75-90 x 5-7 um.
Spores — Basidiospores ellipsoid, hyaline, thick-walled, smooth, IKI-, CB+, 4-6 x 3-4(-4.5) um, L = 4.82 um, W = 3.37 um, Q = 1.36-1.52 (n = 150/5).
ADDITIONAL SPECIMENS EXAMINED — CHINA. YUNNAN PROVINCE, LINCANG, Xiaodaohe Forest Farm, on dead tree of Castanopsis (Fagaceae), 10.V1I.2013, Dai 13216 (BJFC 014706; GenBank KJ925055, KJ925060); NANHUA County, Dazhongshan Nature Reserve, on fallen angiosperm trunk, 15.VII.2013, Cui 11130 (BJFC 015245; GenBank KJ925054, KJ925059); CHuxIoNG, Zixishan Forest Park, on dead tree of Lithocarpus,
28.VUI.2010, Dai 11714 (BJFC 008827; GenBank KJ925057); 11.VI.2011, Dai 12416 (BJFC 010696; GenBank KJ925053, KJ925058).
OTHER SPECIMEN EXAMINED — Fistulina hepatica. FINLAND. VANTAA, Tammosto Nature Reserve, on dead tree of Quercus (Fagaceae), 8.1X.2012, Dai 12874 (BJFC 013153).
Discussion
The previous reports of Fistulina hepatica in China (Dai 2012, Dai et al. 2011, Wang et al. 2011, Yuan & Dai 2008) were from identifications based only on morphological characters. According to the current phylogenetic analysis and more careful morphological examinations, these Chinese samples are newly described as F. subhepatica. Macroscopically, FE hepatica is quite similar to FE. subhepatica in its pinkish brown to more reddish or purplish brown pileal surface, white pore surface when fresh, and individual pores; microscopically, however, F. hepatica differs from F. subhepatica by having smaller basidiospores, simple septate and thin-walled trama generative hyphae (Gilbertson & Ryvarden 1986, Nunez & Ryvarden 2001, Ryvarden & Gilbertson 1993). Moreover, F. hepatica forms a distinct lineage separate from F. subhepatica (Fic. 1). In addition, E hepatica is widely distributed in temperate Europe and grows mainly on Quercus and Castanea, whereas F. subhepatica is found in subtropical China, occurring mostly on Castanopsis and Lithocarpus. We did not find FE hepatica on Quercus in northern China, although the big and old oak trees are suitable for the fungus.
Fistulina subhepatica sp. nov. (China) ... 55
Fistulina antarctica Speg., closely related to F. subhepatica in the phylogenetic analysis, differs morphologically from FE subhepatica by its longer basidiospores (5-7 x 3-4 um, Spegazzini 1887).
Fistulina pallida Berk. & Ravenel resembles F. subhepatica in its brownish pore surface and individual pores; however, it produces smaller basidiomata and wood-brown pileus surface (Gilbertson & Ryvarden 1986). Phylogenetically, E pallida falls outside the “core Fistulina clade” and groups with Porodisculus pendulus (Fr.) Murrill.
Acknowledgments
The authors are grateful to Prof. Yu-Cheng Dai (BJFC, China) for collecting specimens and improving the text. We express our gratitude to Drs. Li-Wei Zhou (China) and Tatiana B. Gibertoni (Brazil), who reviewed the manuscript. The research was financed by Beijing Higher Education Young Elite Teacher Project (YETP0774) and the National Natural Science Foundation of China (Project No. 31170018).
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Bodensteiner P, Binder M, Moncalvo JM, Agerer R, Hibbett DS. 2004. Phylogenetic relationships of cyphelloid Homobasidiomycetes. Molecular Phylogenetics and Evolution 2: 501-515. http://dx.doi.org/10.1016/j.ympev.2004.06.007
Bulliard JBE. 1790. Herbier de la France 10: t.433-480.
Cui BK. 2013. Antrodia tropica sp. nov. from southern China inferred from morphological characters and molecular data. Mycological Progress 12: 223-230. http://dx.doi.org/10.1007/s11557-012-0829-7
Cui BK, Dai YC. 2013. Molecular phylogeny and morphology reveal a new species of Amyloporia (Basidiomycota) from China. Antonie van Leeuwenhoek 104: 817-827. http://dx.doi.org/10.1007/s10482-013-9994-1
Cui BK, Li HJ. 2012. A new species of Postia (Basidiomycota) from Northeast China. Mycotaxon 120: 231-237. http://dx.doi.org/10.5248/120.231
Cui BK, Li HJ, Dai YC. 2011. Wood-rotting fungi in eastern China 6. Two new species of Antrodia (Basidiomycota) from Mt. Huangshan, Anhui Province. Mycotaxon 116: 13-20. http://dx.doi.org/10.5248/116.13
Dai YC. 2012. Polypore diversity in China with an annotated checklist of Chinese polypores. Mycoscience 53: 49-80. http://dx.doi.org/10.1007/s10267-011-0134-3
Dai YC, Cui BK, Liu XY. 2010. Bondarzewia podocarpi, a new and remarkable polypore from tropical China. Mycologia 102: 881-886. http://dx.doi.org/10.3852/09-050
Dai YC, Cui BK, Yuan HS, He SH, Wei YL, Qin WM, Zhou LW, Li HJ. 2011. Wood-inhabiting fungi in southern China 4. Polypores from Hainan Province. Annales Botanici Fennici 48: 219-231. http://dx.doi.org/10.5735/085.048.0302
Felsenstein J. 1985. Confidence intervals on phylogenetics: an approach using bootstrap. Evolution 39:783-791,
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Li HJ, Cui BK. 2013a. Two new Daedalea species (Polyporales, Basidiomycota) from South China. Mycoscience 54: 62-68. http://dx.doi.org/10.1016/j.myc.2012.07.005
Li HJ, Cui BK. 2013b. Taxonomy and phylogeny of the genus Megasporoporia and its related genera. Mycologia 105: 368-383. http://dx.doi.org/10.3852/12-114
Li HJ, Han ML, Cui BK. 2013. Two new Fomitopsis species from southern China based on morphological and molecular characters. Mycological Progress 12: 709-718. http://dx.doi.org/10.1007/s11557-012-0882-2
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ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
MYCOTAXON
http://dx.doi.org/10.5248/130.57 Volume 130, pp. 57-59 January-March 2015
Antherospora sukhomlyniae, a new species of smut fungi on Hyacinthella in Crimea (Ukraine)
KYRYLO G. SAVCHENKO*
Department of Plant Pathology, Washington State University, Pullman, WA 99164, United States
* CORRESPONDENCE TO: kyryll.savchenko@wsu.edu
AsBstTRACT — Antherospora sukhomlyniae, a new species of smut fungus parasitic on Hyacinthella sp. in Crimea (Ukraine), is described and illustrated.
Key worps — anther smut, biodiversity, mycobiota, Ustilaginomycetes
Introduction
During a collecting trip for smut fungi in Crimea (Ukraine), carried out by the author in 2009, several specimens of smut fungi were collected. Based on the study of the specimen from anthers of Hyacinthella sp. (Asparagaceae), I propose here a new smut fungus, Antherospora sukhomlyniae, that was collected in Karadag reserve in the southeastern part of the Crimean peninsula.
Materials & methods
Sorus and spore characteristics were studied using dried herbarium specimens. Spores were dispersed in a droplet of lactic acid on a microscope slide, covered with a cover glass, gently heated to boiling point to rehydrate the spores, cooled, and then examined using a Carl Zeiss Axiostar light microscope (LM) at 1000x magnification. For scanning electron microscopy (SEM), spores were attached to specimen holders by double-sided adhesive tape and coated with gold. The surface structure of spores was observed at 15 kV and photographed with a scanning electron microscope JEOL JSM-6700EF.
The holotype specimen is conserved in the Herbarium, Department of Biodiversity and Biotechnology of Fungi, Institute of Evolution, University of Haifa, Israel (HAI).
Taxonomy
Antherospora sukhomlyniae K.G. Savchenko, sp. nov. FIG. 1 MycoBAnk 808616
58 ... Savchenko
FiGurE 1. Antherospora sukhomlyniae (holotype) in flowers of Hyacinthella sp.: spores. a = LM; b-d = SEM. Bars: a, b= 10 um, c = 2 um, d= 1 um.
Differs from all other Antherospora species by its host specialization on Hyacinthella and from A. albucae and A. urgineae by its smaller spores, from A. eucomis by its irregularly shaped spores, and from A. tourneuxii by its thicker spore walls.
Type: Ukraine, Autonomous Republic of Crimea, Karadag reserve, in flowers of Hyacinthella sp., 5.V.2009, leg. K.G. Savchenko (Holotype HAI 6521).
EryMo_oey: named in honor of the Ukrainian mycologist Maryna M. Sukhomlyn.
Parasitic on Hyacinthella. Sori in all inner floral organs of deformed and swollen flowers, producing blackish brown, powdery mass of spores, for a long time enclosed by the outermost floral envelopes. Infection systemic, with all flowers of the plant infected. Spores ovoid, ellipsoidal, subglobose to irregular, (6.5-)7-10 x 7.5-13(-14) um, yellowish brown. Spore wall even, ca. 0.8 um thick, finely densely punctate-verrucose, with finely wavy spore profile.
Antherospora sukhomlyniae sp. nov. (Ukraine) ... 59
DISTRIBUTION - Known only from the type locality in Crimea, on Hyacinthella sp.
COMMENTS -— Previous studies have demonstrated that while all species of Antherospora attack hosts in the Asparagaceae, they display a high level of specificity, with each species restricted to host plants from a single genus, or in some cases, from a single species (Bauer et al. 2008, Piatek et al. 2011, 2013). As no Antherospora species has been reported on Hyacinthella, 1 propose a new species here.
Antherospora sukhomlyniae belongs to the group of Antherospora species with sori destroying all inner floral organs of infected flowers (Vanky 2012), which includes A. albucae (Syd. & P. Syd.) R. Bauer et al., A. eucomis Vanky, A. tourneuxii (A.A. Fisch. Waldh.) R. Bauer et al., and A. urgineae (Maire) R. Bauer et al. However, these species can be distinguished from A. sukhomlyniae: A. albucae by its larger spores (7-14.5 x 9.5-22.5 um; Vanky 2012); A. urgineae by its larger spores (7-12 x 9.5-17.5 um; Vanky 2012); A. eucomis by its regularly shaped spores with thinner spore walls (0.5 um; Vanky 2009); and A. tourneuxii by its thinner spore walls (0.5 um; Vanky 2012).
Acknowledgments
The author thanks Lori M. Carris and Vasyl P. Heluta for peer-reviewing the manuscript, Shaun Pennycook for useful corrections, and Vitalii Sapsai for help with the SEM microscopy.
Literature cited
Bauer R, Lutz M, Begerow D, Piatek M, Vanky K, Bacigalova K, Oberwinkler F. 2008. Anther smut fungi on monocots. Mycological Research 112: 1297-1306. http://dx.doi.org/10.1016/j.mycres.2008.06.002
Piatek M., Lutz M., Chater A.O. 2013. Cryptic diversity of the Antherospora vaillantii complex on the Muscari species. IMA Fungus 4: 5-19.
Piatek M., Lutz M., Smith P.A., Chater A.O. 2011. A new species of Antherospora supports the systematic placement of its host plant. IMA Fungus 2: 135-142.
Vanky K. 2009. Taxonomic studies on Ustilaginomycetes - 29. Mycotaxon 110: 289-324. http://dx.doi.org/10.5248/110.289
Vanky K. 2012. Smut fungi of the world. APS Press, USA.
ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/130.61 Volume 130, pp. 61-68 January-March 2015
Tuber xanthomonosporum, a new Paradoxa-like species from China
YUAN QING", SHU-HONG LI’, CHENG-YI Liv?, LIN Li’, MEI YANG}, XIAO-LEI ZHANG’, XIAO-LIN L14, LIN- YONG ZHENG?* 4, & YUN WANG®®
' Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, School of Life Science, Sichuan University, 24 (South part) First Ring Road, Chengdu, Sichuan, China
? Biotechnology & Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, 9 Xueyun Road, Kunming, Yunnan, China
Sichuan Panzhihua Academia of Agriculture and Forestry, 1719 Panzhihua Road, Panzhihua, Sichuan, China
‘Sichuan Academy of Agricultural Sciences, 20 Jingjusi Road, Chengdu, Sichuan, China
‘Department of Light Chemical Engineering, Xichang College, Mapingba Road, Xichang, Sichuan, China
°Yunnan Institute for Tropical Crop Research, 99 Xuanwei Road, Jinghong, Yunnan, China
* CORRESPONDENCE TO: “zly6559@126.com "wangynz@yahoo.com
ABSTRACT — A new species, Tuber xanthomonosporum, is described based on specimens collected under Pinus yunnanensis in Panzhihua, Sichuan Province, China. Tuber xanthomonosporum invariably has only one spore per ascus. It can be distinguished from the three other Chinese Paradoxa-like species — T: gigantosporum, T: sinomonosporum, and T. glabrum — by its whitish to yellow-brownish gleba, two-layered peridium, and spiky cystidia. Molecular analysis also supports T: xanthomonosporum as a unique species.
KEY worps — taxonomy, Pezizales, Yunnan
Introduction
The genus Paradoxa was erected by Mattirolo in 1935 in the Tuberaceae to accommodate Paradoxa monospora Mattir., a species which, invariably, has one-spored asci (Montecchi & Sarasini 2000; Leessoe & Hansen 2007). For the following 50 years the genus remained monotypic until Wang & Hu (2008) transferred Tuber gigantosporum into Paradoxa. Wang & Li (1991) originally described T’ gigantosporum based on a single specimen collected from Huidong County, Sichuan, China, in 1988. The species was characterized by its extremely large elliptical ascospores and one-spored asci, a feature that was eventually recognized as being important when a second T: gigantosporum
62 ... Qing & al.
collection was made in Yongren County, Yunnan, China, in 2002. Since then three other Paradoxa-like species have been reported: Tuber sp. 1 (Kinoshita et al. 2011), Paradoxa sinensis (Fan et al. 2012), and Tuber glabrum (Fan et al. 2014). Recent molecular phylogenetic analysis of truffle species supported Paradoxa within Tuber (Kinoshita et al. 2011, Fan et al. 2013), and subsequently Fan et al. (2014) transferred P sinensis into Tuber with the replacement name
T. sinomonosporum.
In 2012 during a survey in Panzhihua in the southwestern corner of Sichuan, China, we found truffles that invariably had one-spored asci. Here we describe this Paradoxa-like truffle as a new species of Tuber and briefly review the
molecular relationships of truffle species with single-spored asci.
TABLE 1: Origin of the fungal ITS sequences. (New sequences are indicated
in bold font.)
TAXON
Choiromyces meandriformis
Gymnohydnotrya australiana
Tuber sp. 1 Tuber sp. 2 T. borchii
T. canaliculatum
T. excavatum
T. foetidum
T. fulgens
T. glabrum
T. indicum
T. latisporum
VOUCHER OR CODE
isolate RH691 isolate GB285
isolate OSC130601
isolate K201 isolate K215
clone Bo6
strain Z43 Unknown JT12670
JT23942
TE-M
isolate zb3281_215 TE-WD
isolate B-2452 isolate B-2489 isolate y466
isolate zb3386_202 isolate M2435
strain T39 strain T47 strain T49 HKAS 44315 HKAS 42380
GEOGRAPHICAL ORIGIN
USA USA
USA
Japan Japan Italy Italy France USA USA Poland Germany Poland Hungary Hungary Germany Germany USA China China China China China China China
GENBANK NO.
HM485330 HM485331
JQ925629
AB553344 AB553356 AF106890 AF250291 AF132505 GQ221455 GQ221456 KC330228 HM152020 KC330227 AJ557543 AJ557544 HM152014 HM152024 HM485358 KFO002731 KF002732 JQ638997 JQ639005 JQ639007 DQ898183 DQ898184
Tuber xanthomonosporum sp. nov. (China) ... 63
T. liui HKAS 48269 China DQ898182 T. macrosporum isolate JT 19458 USA HM485372 isolate $7510 Germany JF926121 T. maculatum clone Macl Italy AF106889 1967 New Zealand EU753269 T. melanosporum strainl015 Israel AF167096 LHB-mic-Tmel-Qc-09-1 Spain GU810153 LHB-mic-Tmel-Qr-09-1 Spain GU810152 T. panzhihuanense strain DXJ260 China JQ978644 strain DXJ276 China JQ978650 T. pseudoexcavatum isolate CJ408 USA HM485381 strain E China JQ638958 strain Q China JQ638982 strain T48 China JQ639006 T. rufum 1798 Italy EF362473 1447 Italy EF362477 T. sinomonosporum BJTCFAN150 China KF002729 T. umbilicatum HKAS48267 China GU979032 HKAS44316 China GU979031 isolate T104 China FJ797879 isolate T117 China FJ797880 T. xanthomonosporum YAAS 13185 China KJ162154 YAAS L3186 China KJ162155 YAAS L3187 China KJ162156 T. zhongdianense HKAS 45388B China DQ898186
wang0299 China DQ898187
Materials & methods
Morphological studies
Specimens of a truffle with asci containing a single spore were collected from Panzhihua, Sichuan, China, in 2012. Specimens are deposited in the Herbarium of Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan, China (YAAS).
The samples were examined at the Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming, China and Sichuan Panzhihua Academy of Agriculture and Forestry, Panzhihua, Sichuan, China. Macroscopic characters were described from fresh specimens and microscopic features described from dried specimens following the methods of Yang & Zhang (2003). Photographs were made under a Nikon E400 microscope. For scanning electron microscopy (SEM), spores were scraped from the dried gleba onto double-sided tape and mounted directly on a SEM stub. They were then coated with gold-palladium and examined and photographed with a JEOL, JMS-5600LV SEM.
64 ... Qing & al.
DNA amplification & sequence analysis
DNA was extracted using CTAB (Doyle 1987) modified by adding 200 wL 5M potassium acetate after 4 x CTAB treatments. The primers ITSIF (Gardes & Bruns 1993) and ITS4 (White et al. 1990) were used to amplify the ITS-rDNA region. PCR reaction solution and cycling parameters used by Chen & Liu (2007) were adopted. The amplification products were electrophoresed on a 1% agarose gel and purified with Sangons purification kit. Sequencing was performed with a BigDye® Terminator v3.1 Cycle Sequencing Kit on an ABI 3730XL automatic sequencer.
In order to determine whether the specimens represented a new species, we compared our Tuber xanthomonosporum ITS-rDNA sequences with 48 ITS-rDNA sequences downloaded from NCBI. An ITS-rDNA sequence of Gymnohydnotrya australiana B.C. Zhang & Minter was also included as outgroup. Sequence alignment and phylogenetic analysis protocols followed Chen & Liu (2007).
Results
Phylogenetic analysis
A total of 52 ITS sequences were included in a phylogenetic analysis (TABLE 1, PLATE 2). Of the 712 total characters analyzed, 248 are constant, 51 parsimony-uninformative, and 413 parsimony-informative (L = 1979, CI = 0.481, RI = 0.812, RC = 0.390). ITS sequence analysis revealed that the three Paradoxa-like species (Tuber sp. 1, T: sinomonosporum, T. glabrum) form a well-supported subclade (bootstrap support = 98%) within Clade I, which also included T: macrosporum, T. canaliculatum, and Tuber sp. 2. In contrast, T. xanthomonosporum does not cluster with the other Paradoxa-like species but forms a new Subclade I in Clade II (with low bootstrap support) alongside Subclade II (/melanosporum) and Subclade III (/rufum).
Taxonomy
Tuber xanthomonosporum Qing & Y. Wang, sp. nov. PLATE 1 MycoBAnk MB 807668
Differs from other Tuber species with single-spored asci by its whitish to yellow- brownish gleba and two-layered peridium with spiky cystidia.
TyPE: China, Sichuan Province, Panzhihua, 26°29’N 102°01’E, under Pinus yunnanensis Franch., 15 Nov. 2012: Y. Qing Pan1201 (Holotype, YAAS L3185; GenBank KJ162154).
EryMoLoecy: xanthomonosporum from Latin refers to the yellow brownish gleba and single spored asci.
PiaTE 1. Tuber xanthomonosporum (YAAS L3185, holotype): 1, 2. Fresh ascomata showing surface features and cut sections. 3. Cross section of peridium showing two layers of pseudo- parenchymatous and interwoven tissue with some cystidia. 4. Cross section of outer layer of peridium showing two kinds of cystidia: talland tapered (arrows), and short and obtuse (arrowhead). 5-7. Ascospores and asci (invariably with only one spore). 8. Ascospores showing ornamentations. Scale bars: 1, 2 = 1 cm; 3, 8 = 20 um; 4, 7 = 50 um; 5 = 100 um; 6 = 40 um.
Sh 14 (20°
66 ... Qing & al.
Ascomata subglobose or irregular and much lobed with deep furrows, firm and a little rubbery, whitish to yellow-brownish, up to 3.0 cm broad, rough with hairs. Opor slightly garlicky when mature. PERIDIuM 160-200 um thick, composed of two layers; the outer layer complex, 50-70 um thick, pseudoparenchymatous, composed of irregular globose to rectangular cells, 19-32 x 13-15 um diam and thin-walled; the outmost cells giving rise at the surface to cystidia of two types; one tall, thin, tapered, reaching 50-90 x 2-4 um and sometimes with a small teat on the wall, hyaline; the other short, obtuse, 1-2-septate, 15-20 x 4-5 um; the inner layer 110-130 um thick, composed of mainly interwoven, colourless hyphae of 2-4 um diam with some big cells of 2-4 um diam, that gradually merge into the gleba tissue. GLEBA solid, whitish when young, becoming yellow-brownish at maturity, marbled with distinct, white and meandering veins, merging at many points with the peridium. Asc1 60 x 70 um, globose to subglobose, sessile, slightly thick walled 2-3 um thick, invariably 1-spored and randomly dispersed in glebal tissue. AscospoREs globose, 41-43 x 43-46 um; spore walls 2.5 um thick, brown at maturity, ornamented with a regular alveolate-reticulum, 2-3 um deep, constituted of mostly hexagonal meshes 8-15 x (5-)7-13 um, 4-5 across the spore width.
ECOLOGY & DISTRIBUTION — Hypogeous, solitary or in groups in calcareous soils with pH 6.8-7.6 under Pinus yunnanensis and forming ectomycorrhizae with its roots, alt. 1500-2000 m, fruiting from autumn to winter. Known only from Sichuan, China.
ADDITIONAL SPECIMENS EXAMINED: CHINA, SICHUAN PROVINCE, Panzhihua,
26°29’N 102°01’E, under Pinus yunnanensis, 15 Nov. 2012, C.Y. Liu Pan-No-10 (YAAS L3186; GenBank KJ162155); C.Y. Liu Pan-No-40 (YAAS L3187; GenBank KJ162156).
Discussion
The new Tuber species, T: xanthomonosporum from Sichuan, produces only one spore per ascus, the diagnostic character of the now defunct genus, Paradoxa (Mattirolo 1935; Fan et al. 2014). Recent phylogenetic studies of Paradoxa-like species (Kinoshita et al. 2011; Fan et al. 2014) found that they group with Tuber species. Our phylogenetic analysis groups T. xanthomonosporum with Tuber species but not with other Paradoxa-like species, despite sharing the common character of having only one spore per ascus. Our work therefore supports the conclusion by Fan et al. (2014) that Paradoxa-like truffle species do not form a monophyletic group. While Tuber sinomonosporum [= P. sinensis], T. glabrum, and T. sp. 1 (the Japanese Paradoxa-like new species) do group together, they also group with T! macrosporum and T. canaliculatum in Clade I (/macrosporum). These species in Clade I share the same morphological feature of having red-brown gleba. Tuber xanthomonosporum groups in Clade II with the /melanosporum and /rufum subclades but with very low bootstrap support (<50%). Morphologically, T. xanthomonosporum differs greatly from
Tuber xanthomonosporum sp. nov. (China) ... 67
Gymnohydnotrya australiana JQ925629 98 Tuber sp. 1 AB553344 T. sinomonosporum KF002729 100» 7. glabrum KF002731 100 T. glabrum KF002727 Clade I Tuber sp. 2 AB553356 macrosporum 100) 7. canaliculatum GQ221455 T. canaliculatum GQ221456 100-- 7. macrosporum HM485372 T. macrosporum JF926121 T. xanthomonosporum KJ162154 T. xanthomonosporum KJ162155 Subclade I T. xanthomonosporum KJ162156 T. indicum JQ638997 T. indicum JQ639005 100 T. indicum JQ639007 T. melanosporum AF167096
100
100
08 ye T. melanosporum GU810153 Subelade II T. melanosporum GU810152 melanosporum 100r 7. pseudoexcavatum HM485381 Clade II 100 T. pseudoexcavatum JQ638958
& 100 7. pseudoexcavatum JQ638982
T. pseudoexcavatum JQ639006 T. rufum EF362473 100 T. rufum EF362477 T. rufum JF926123 T. umbilicatum GU979032_ | Subclade TIT T. umbilicatum GU979031 rufum go) 2. umbilicatum FJ797879 95! 7. umbilicatum FJ797880 T. borchii AF106890 T. borchii AF250291 83 T. borchii AF132505 100 T. liui DQ898182 T. zhongdianense DQ898186 1001 7, zhongdianense DQ898187 100) 7. latisporum DQ898183 100 T. latisporum DQ898184 109 T. panzhihuanense JQ978644 100! 7. panzhihuanense JQ978650 100) 7. foetidum AJ557543 100 T. foetidum AJ557544 100 7. maculatum AF106889 T. maculatum EU753269 T. excavatum KC330228 loop 7. excavatum HM152020 T. excavatum KC330227 T. fulgens HM152014 100 T. fulgens HM152024 T. fulgens HM485358 100 Choiromyces meandriformis HM485330 Choiromyces meandriformis HM485331
100 100 100
67 100
100
_10
PLATE 2. One of eight most parsimonious trees constructed with ITS sequences of Tuber xanthomonosporum and related species. MP Bootstrap values greater than 50% are indicated at nodes.
the species in these two subclades in its whitish to yellow-brownish gleba and reticulate spores. Given the few species and low support for the species now placed in Clade I, the phylogenetic relationships need further study with more samples and gene loci.
Acknowledgements
We are grateful to Dr. Ian Hall and Dr. A. Zambonelli for reviewing the paper. We also thank Prof. Fan Li and Mr. H.K. Xiong for providing Paradoxa-like specimens for our study. This study was supported financially by the National Science Foundation of China (No.31160010) and the Science and technology support program of Sichuan Province (No.2012NZ0003 and No.2013NZ0029).
68 ... Qing & al.
Literature cited
Chen J, Liu PG. 2007. Tuber latisporum sp. nov. and related taxa, based on morphology and DNA sequence data. Mycologia 99: 475-481. http://dx.doi.org/10.3852/mycologia.99.3.475
Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure from small quantities of fresh leaf tissues. Phytochemical Bulletin 19: 11-15.
Fan L, Cao JZ, Li Y. 2012. Tuber microsphaerosporum and Paradoxa sinensis spp. nov. Mycotaxon 120: 471-475. http://dx.doi.org/10.5248/120.471
Fan L, Feng S, Cao JZ. 2014. The phylogenetic position of Tuber glabrum sp. nov. and T. sino- monosporum nom. nov., two Paradoxa-like truffle species from China. Mycological Progress 13(2): 241-246. http://dx.doi.org/10.1007/s11557-013-0908-4
Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes- application to the identification of mycorrhizae and rusts. Molecular Ecology 2(2): 113-118. http://dx.doi.org/10.1111/j.1365-294X.1993.tb00005.x
Kinoshita A, Sasaki H, Nara K. 2011. Phylogeny and diversity of Japanese truffles (Tuber spp.) inferred from sequences of four nuclear loci. Mycologia 103(4): 779-794. http://dx.doi.org/10.3852/10-138
Lzessoe T, Hansen K. 2007. Truffle trouble: what happened to the Tuberales? Mycological Research 111(9): 1075-1099. http://dx.doi.org/10.1016/j.mycres.2007.08.004
Mattirolo O. 1935. Catalogo ragionato dei funghi ipogei raccolti nel Canton Ticino e nelle provincie Italiane confinanti. Beitrage zur Kryptogamenflora der Schweiz 8: 1-53.
Montecchi A, Sarasini M. 2000. Fungi ipogei d’Europa. A.M.B. Fondazione Centro Studi Micologici. Vicenza.
Wang Y, Hu HT. 2008. New species of Paradoxa gigantospora comb. nov. from China. Mycotaxon 106: 199-202.
Wang Y, Li ZP. 1991. A new species of Tuber from China. Acta Mycologica Sinica 10(4): 263-265.
White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315-322, in: MA Innis et al. (eds). PCR protocols: a guide to methods and applications. Academic, New York.
Yang ZL, Zhang LE. 2003. Type studies on Clitocybe macrospora and Xerula furfuracea var. bispora. Mycotaxon 88: 447-454.
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MY COTAXON
http://dx.doi.org/10.5248/130.69 Volume 130, pp. 69-72 January-March 2015
Distance1D - a protein profile analytical program designed for fungal taxonomy
DuSAN MATERIC??’, BILJANA KUKAVICA}, & JELENA VUKOJEVIC4
'Faculty of Agriculture & Teachers Training Faculty, University of East Sarajevo, Semberskih ratara bb, 76300 Bijeljina, Bosnia and Herzegovina *Faculty of Science, The Open University, Walton Hall, MK7 6AA Milton Keynes, UK *Faculty of Natural Sciences and Mathematics, University of Banja Luka, Mladena Stojanovica 2, 78000 Banja Luka, Bosnia and Herzegovina ‘Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia
* CORRESPONDENCE TO: dusan.materic@gmail.com
ABSTRACT —Taxonomic analysis of macromycete fruiting bodies is a challenging task that utilizes morphological, biochemical, and molecular methods. Many biochemical and molecular methods have been developed to test or confirm identifications or phylogenetic positions independently of morphological data. SDS electrophoresis has been shown to be a good biochemical method for protein separation. Although protein profiles can be analyzed by commercially available software, there is no software designed specifically for fungal taxonomic research. We have developed an open source portable program that uses protein profiles of fungal fruiting bodies to calculate relative differences between species for use in generating to generate more accurate phylogenetic trees.
Key worps — biochemistry, fungi, Perl, proteome
Introduction
The challenging task of macromycete taxonomy uses morphological, biochemical, ecological, and molecular data (Guarro et al. 1999, Blackwell et al. 2006, Korabecna 2007, Lutzoni & Vilgalys 1995). Sodium dodecyl sulfate (SDS) electrophoresis is a good biochemical method for protein separation and biomarker discovery, which when applied to the proteomes of fungal fruiting bodies gives a good number of separated proteins, revealing small (or sometimes large) differences in protein expression (Materi¢ 2012). The method, which is robust and reliable, could serve as an independent taxonomic tool for fungi (Guarro et al. 1999, Tyrrell 1969).
70 ... Materi¢, Kukavica, & Vukojevi¢é
The aim of this work was to develop a user-friendly open source program using electrophoresis data for fungal taxonomy. Such a program would compare and calculate relative differences between taxa based on results from SDS gel electrophoresis and similar techniques (e.g., western blot, zymography).
Materials & methods
We sampled and identified 21 fruiting bodies representing differently related species. Proteins were separated according to the method described in Materic (2012), and protein profiles were obtained for each species. The quality of protein profiles depends upon many factors during sample homogenization, protein extraction and electrophoresis, and an optimized protocol can be found in Materic¢ et al. (2012). In this work we have chosen to show the results of only seven species: however, more detailed dendrograms are available in Materi¢ (2012).
Protein profile data are stored in .csv format and examples of files can be found along with the source-code in Materi¢ (2013). Protein profile files include two sets of data: (1) molecular weights (MW) of proteins; and (2) relative abundance. MW and relative abundances can be obtained using specialized software such as TotalLab (Phoretix, Newcastle, United Kingdom). However, abundances should be recalculated as relative abundances with range of 1 (least abundant) to 5 (most abundant). In order to create a distance matrix table, protein profiles are compared by distancelD. The distance matrix tables are transformed into dendrograms using the program package PHYLIP (Felsenstein 2002).
Results & discussion
Protein electrophoresis, which has been widely used in solving taxonomic and systematic problems, produces results that are independent of morphological and molecular data (Guarro et al. 1999, Tyrrell 1969). Thus, it is crucial to have appropriate software that deals with taxonomic issues rather than with pure biochemical properties. Apart from distance1D, there is no such software specifically designed to interpret fungal taxonomy from protein profile data.
Distance1D is a program designed to calculate relative differences between two protein profiles, which could then be used to generate dendrograms (Fic. 1). The program is designed and written specifically for use in fungal taxonomy (mainly for analyzing protein profiles of fungal fruiting bodies), but its usage could be wider. The program is meant to be open source, user-friendly, and portable. Portability results from the program's being written in Perl, which can be run on any operating system. As the program uses the Tk library for generating widgets (Windows-like interface), Perl/Tk should be installed along with Perl.
Our previous work produced good protein profiles of fungal fruiting bodies from SDS gel electrophoresis (Materi¢ 2012) that are stored in separate files (Fie. 1):
Distance 1D for analyzing fungal profiles ... 71
A)
wW [ SDS Electrophoresis
Fruiting bodies (Boletus edulis)
distancelD 0.1
Image analysis and storing
Species 1: | /GELS2-3-2Be.lin protein profiles in a .csv files Species 2: /GELS/2-3-3Sg.lin
TOTAL DIF: 150.7
(mwa): (abd):
Za > 2 ; pa Boletus edulis
Boletus luridus
Distance matrix yee D)
Distances obtained using distance1D
Boletusedulis _|000.0[093.9]217.5] 134.6] 170.0]188.1]150.7
Boletus luridus | 093.9]000.0|182.8]151.0/169.6]1903]161.8) Neighbour joining Lactarius piperatus — |217.5|182.8/000.0/ 113.0] 133.3]131.0]166.1
Suillus granulatus
Russula cyanoxantha
i Tree drawing using i
Lactarius piperatus
Russula foetens
Russula emetica
FIGURE 1. Diagram showing stages of an experiment used in order to gain biochemically based phylogenetic trees. A: Fruiting bodies. B: Result of SDS electrophoresis (channels: M = molecular mass marker; 1 = Boletus luridus; 2 = B. edulis; 3 = Suillus granulatus; 4 = Russula foetens; 5 = Lactarius piperatus; 6 = R. cyanoxantha; 7 = R. emetica; Materi¢ et al 2012). C: Interface of the program distance1D. D: Distance matrix table generated by the program. E: Phylogenetic tree drawn by PHYLIP program package (similar results were obtained by analyzing ITS II regions of rDNA as control; Materi¢ 2012).
The program as an input takes the paths of two files (two file names) where protein data are stored in .csv format. An algorithm searches for optimal pairing of proteins by looping through the parameters, such as MW sensitivity. After alignment and optimization, the following differences are scored: (1) presence of new protein lines (new protein), (2) molecular weight difference of protein pairs, and (3) abundance difference of protein pairs. The scoring system, which could be adapted according to user requirements, is set up as follows: “10” scored for each new protein line, “1” scored for each 1kDa MW shift, and “2” scored for abundance difference. If DNA/RNA gel data are going to be used, the abundance difference for each fragment should be set the same.
There are several important advantages to using this program: the program generates distances that give accurate phylogenetic trees (more accurate than a single DNA sequence such as rDNA ITS1; Materi¢ 2012); the parameters can be changed to suit particular research; the program can be easily adapted
72 ... Materié, Kukavica, & Vukojevi¢
for use with any protein/DNA/RNA electrophoresis data; and the program is suitable for all operating systems and is open source and free.
Acknowledgments The authors thank Milan Matavulj (University of Novi Sad, Serbia) and Mirjana Stajic (University of Belgrade, Serbia) for presubmission review.
Literature cited
Blackwell M, Hibbett DS, Taylor JW, Spatafora JW. 2006. Research Coordination Networks: a phylogeny for kingdom Fungi (Deep Hypha). Mycologia 98: 829-837. http://dx.doi.org/10.3852/mycologia.98.6.829
Felsenstein, J. 2002. PHYLIP (Phylogeny Inference Package) version 3.6 a3.
Guarro J, Gené J, Stchigel AM. 1999. Developments in fungal taxonomy. Clinical Microbiology Reviews 12: 454-500.
Korabecna M. 2007. The variability in the fungal ribosomal DNA (ITS1, ITS2, and 5.8S rRNA gene): its biological meaning and application in medical mycology. 783-787 in Communicating current research and educational topics and trends in applied microbiology (A Méndez- Vilas, ed.). Formatex, Spain.
Lutzoni F, Vilgalys R. 1995. Integration of morphological and molecular data sets in estimating fungal phylogenies. Canadian Journal of Botany 73: 649-659. http://dx.doi.org/10.1139/b95-307
Materi¢ D. 2012. Biohemijska, molekularna i bioinformaticka analiza taksona gljiva podcarstva Dikarya. Master's thesis, Bosnia and Herzegovina, University of Banja Luka.
Materi¢ D. 2013. Distance1D - source code. GitHub. https://github.com/dusanac/distance1D
Materi¢ D, Kukavica B, Boroja M, Vukojevic¢ J. 2012. Optimizacija protokola za ekstrakciju proteina iz plodonosnih tijela gljiva (vrste rodova: Boletus, Russula, Lactarius i Agaricus) za SDS- elektroforezu. Skup 4(1):36-41.
Tyrrell D. 1969. Biochemical systematics and fungi. Botanical Review 35: 305-316. http://dx.doi.org/10.1007/BF02858875
ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
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http://dx.doi.org/10.5248/130.73 Volume 130, pp. 73-77 January-March 2015
Coccomyces prominens sp. nov. (Rhytismataceae) on Rhododendron coeloneurum in China
YA-FEI Xu’, YUAN Wu’, YAN-QIONG MENG’, SHI-JUAN WANG?, & YING-REN LIN”
' School of Life Science &? School of Forestry & Landscape Architecture, Anhui Agricultural University, West Changjiang Road 130, Hefei, Anhui 230036, China
*CORRESPONDENCE TO: yingrenlin@yahoo.com
ABSTRACT — A fungus found on leaves of Rhododendron coeloneurum from Garze prefecture (Sichuan Province), China, is described as Coccomyces prominens. The new species is similar to C. urceoloides but differs by its much larger intraepidermal ascomata and the presence of conidiomata. The type specimen is deposited in the Reference Collection of Forest Fungi of Anhui Agricultural University, China (AAUF).
Key worps — foliicolous fungus, morphological character, taxonomy, Ericaceae
Introduction
Coccomyces De Not. is the second-largest genus of Rhytismataceae (Rhytismatales, Leotiomycetes, Ascomycota), represented by 116 species recorded by Kirk et al. (2008) plus a few recently added. Members of this genus are widely distributed and occur on leaves, twigs, bark, or wood of vascular plants, especially Ericaceae, Fagaceae, and Lauraceae (Sherwood 1980). Twenty-eight Coccomyces species have been reported from China (Korf & Zhuang 1985; Lin 1998; Hou et al. 2006, 2007; Jia et al. 2012; Zheng et al. 2012; Wang et al. 2013; Yang et al. 2013) since Teng (1934) first recorded C. dentatus (J.C. Schmidt) Sacc. on Quercus and Castanea and C. delta (Kunze) Sacc. on Lauraceae.
Here, Coccomyces prominens on leaves of Rhododendron coeloneurum from Luding county, Garze prefecture, Sichuan Province, China, is described as a new species.
Materials & methods Mature fruiting bodies were selected from the collected specimens. External shapes, size, color, ascomatal and conidiomatal openings, and zone lines were observed under
74 ... Xu & al.
a dissecting microscope at 10—-50x magnification. After rehydration in water, 10-20 tum thick vertical transverse sections of fruiting bodies were made using a freezing microtome and mounted in 0.7% (w/v) cotton blue in water. Gelatinous sheaths surrounding ascospores and paraphyses were observed in water or 0.1% (w/v) cotton blue in phenol glycerin. The colors of internal structures and paraphysis contents were observed in water. Measurements were made using material in 5% KOH and from ca 30 paraphyses, asci, ascospores, conidiogenous cells, and conidia for each specimen. Figures were drawn of the external and internal structures of the fruiting bodies using a microscopic drawing tube. The type collection is deposited in the Reference Collection of Forest Fungi of Anhui Agricultural University, Hefei, China (AAUF).
Taxonomy
Coccomyces prominens Y.F. Xu & Y.R. Lin, sp. nov. Fics 1-6 MycoBank MB 808960 Differs from Coccomyces urceoloides by its much larger, intraepidermal ascomata with
an obvious pre-formed dehiscence mechanism, its wider ascospores enveloped in a gelatinous sheath, and its production of conidiomata.
Type: China, Sichuan, Garze prefecture, Luding, Hailuogou, alt. ca 3400m, on fallen leaves of Rhododendron coeloneurum Diels (Ericaceae), 22 June 2009, Y.G. Liu & Y.R. Lin 2768 (Holotype, AAUF 68876).
ETYMOLOGY: prominens (Latin = prominent), referring to the ascomata, which are noticeable on the substratum. ZONE LINES usually frequent, brown or dark brown, thin to wide, somewhat diffused, nearly always partly surrounding the bleached spots.
ConipiomMara in similar positions to ascomata on the substratum, scattered to clustered, sometimes several coalescent. In surface view, conidiomata 150-220 um diam., rounded or subrounded, with a clearly marked outline, black-brown, moderately raising the leaf surface, discharging spores through an apical ostiole. In vertical transverse section, conidiomata intraepidermal, lenticular to semicircular. UPPER WALL 6.5—12 um thick, almost not attenuating towards the base, composed of disparately sized angular cells. BASAL WALL 35-50 um thick in the middle, becoming 13-20 um thick at periphery, consisting of black-brown, angular and elongate, thick-walled cells 3.5-8 um diam. SUBCONIDIOGENOUS LAYER 5-8 um thick, composed of colorless, thin- walled angular cells 1.2-2 um diam. CONIDIOGENOUS CELLS 10-14 x 1.5-2.2 um, cylindrical but tapered towards the apex, hyaline, proliferating sympodially and percurrently. Conrp1a 4.5-7 x 0.5-0.8 um, cylindrical, straight or slightly curved to one side, ends rounded or bluntly pointed, hyaline, aseptate, smooth- walled. TRICHOGYNES 35-45 x 2-3 um, cylindrical, tapering towards the apex, 1—2-septate near the base.
Ascomarta epiphyllous, scattered or occasionally coalescent, in subcircular or irregular pale areas with obvious edges. In surface view, ascomata 800-1400
Fs)
Coccomyces prominens sp. nov. (China) ...
Pee ory
6. Coccomyces prominens (ex holotype, AAUF 68876) on Rhododendron coeloneurum.
Fics 1
and a zone line observed
ascomata, under a dissecting microscope. 3. Ascoma in vertical transverse section. 4. Portion of ascoma in
1. A leaf bearing fruiting bodies and zone lines. 2. Conidiomata,
asci, and ascospores. 6. Conidioma in vertical transverse
vertical transverse section. 5. Paraphyses,
section.
76 ... Xu &al.
um diam., quadrilateral to octagonal, black-brown to black, not shiny or slightly shiny, edge defined, moderately raising the substrate surface, with an obvious pre-formed dehiscence mechanism, opening by 4-8 radial splits nearly extend to the edge of ascoma. Lips absent. In median vertical transverse section, ascomata intraepidermal. COVERING sTROMA 55-65 um thick near the opening, decreasing to 25-30 um thick adjacent to the base, composed of black-brown textura angularis-epidermoidea with thick-walled cells 4—6.5 um diam., connecting to the basal stroma. Adjacent to the top of the covering stroma, a flimsy structure develops consisting of angular, thin-walled, almost colorless cells staining with cotton blue. BasaL stroma 18-22 um thick, comprised of 2—3(—4) layers of dark brown, angular, thick-walled cells 4-6 um diam. ExcrpuLUM well-developed, 45-60 um thick, arising from the marginal paraphyses and the internal matrix of stroma, consisting of closely agglutinated septate hyphae 2—5 um diam., mostly short-celled, with swollen greyish-brown apices. INTERNAL MATRIX OF STROMA moderately developed, 20-50 um thick, comprising hyaline, gelatinised textura intricata. SUBHYMENIUM colorless, 10-15 um thick, composed of textura angularis. PARAPHYSES 160-190 x 1.8—2.2 um, filiform, 0-1-septate, occasionally branched, yellowish-brown in the upper quarter, surrounded by a 0.6—0.8 um thick gelatinous matrix, gradually irregularly swollen to 3-5 um and containing disparately-sized oil drops near the apex, forming a 35-50 um thick epithecium inserted with a number of hyaline, refractive, sub-oblong solid gels 12-32 x 8-14 um. AscI ripening sequentially, 110-170 x 12—15 um, cylindrical-clavate, short-stalked, thin-walled, apex bluntly pointed to subtruncate-conical, J-, 8-spored. ASCOSPORES arranged fasciculately, 80-120 x 2-2.4 um, filiform, hyaline, aseptate, enveloped in a 0.6—0.9 um thick gelatinous sheath.
HOST SPECIES, HABITAT AND DISTRIBUTION: Producing ascomata and conidiomata on fallen leaves of Rhododendron coeloneurum. Known only from the type locality, Sichuan Province, China.
COMMENTS — Coccomyces prominens resembles C. urceoloides Spooner in the well-developed excipulum with more or less brown hyphal tips, gradually swollen paraphyses at the apex, and refractive solid gels in the epithecium. However, C. urceoloides has smaller, hypophyllous, subepidermal ascomata (350-550 um diam.) not associated with conidiomata; much smaller, narrowly cylindrical asci (120-135 x 5.5—-6 um); and smaller ascospores (80-98 x ca 1 um) with a mucilaginous cap at the proximal end (Spooner 1990).
Coccomyces sinensis Y.R. Lin & Z.Z. Li is easily distinguished from C. prominens by its triangular to pentagonal ascomata, smaller asci (108-130 x 5.3—-6.0 um), smaller ascospores (54—90 x 1—1.2 um), subhymenium consisting of textura porrecta-intricata, and distinct paraphyses that are abruptly swollen
Coccomyces prominens sp. nov. (China) ... 77
to subfusoid-ventricose with a subcylindrical mucro near the apex (Lin et al. 2001).
The similar C. monticola Sherwood differs in the smaller and orbicular ascomata (500-800 um diam.), a thinner covering stroma (20 um thick), slightly swollen paraphysial apices (2.5 um wide), and much narrower asci (8-9 um wide; Sherwood 1980).
Acknowledgements
We are grateful to Dr J.E. Taylor and Dr M. Ye for serving as pre-submission reviewers, and to Dr Y.G. Liu for the field investigations. This study was supported by the National Natural Science Foundation of China (No. 31270065, 31170019).
Literature cited
Hou CL, Piepenbring M. 2007. Two new species of Rhytismataceae on twigs of conifers from Yunnan Province, China. Mycotaxon 102: 165-170.
Hou CL, Kirschner R, Chen CJ. 2006. A new species and new records of Rhytismatales from Taiwan. Mycotaxon 95: 71-79.
Jia GJ, Lin YR, Hou CL. 2012 [“2011”]. A new species of Coccomyces (Rhytismatales, Ascomycota) from Mt. Huangshan, China. Mycotaxon 118: 231-235. http://dx.doi.org/10.5248/118.231
Kirk PM, Cannon PF, Minter DW, Stalpers JA (eds). 2008. Dictionary of the fungi, 10th ed. CAB International. Wallingford. 771 p.
Korf RP, Zhuang WY. 1985. Some new species and new records of Discomycetes in China. Mycotaxon 22: 483-514.
Lin YR. 1998. Studies on Coccomyces de Notaris and Neococcomyces gen. nov. in China. [Ph.D. thesis; in Chinese]. Northeast Forestry University. Harbin. 98 p.
Lin YR, Li ZZ, Xu ZS, Wang JR, Yu SM. 2001. Studies on the genus Coccomyces from China IV [in Chinese]. Mycosystema 20: 1-7.
Sherwood MA. 1980. Taxonomic studies in the Phacidiales: The genus Coccomyces (Rhytismataceae). Occasional Papers of the Farlow Herbarium of Cryptogamic Botany 15: 1-120.
Spooner BM. 1990. Coccomyces and Propolis (Rhytismatales) from Mt Kinabalu, Borneo. Kew Bulletin 45: 451-484. http://dx.doi.org/10.2307/4110513
Teng SC. 1934. Notes on Discomycetes from China. Sinensia 5: 431—465.
Wang SJ, Lin YR, Tang YP, Li K. 2013 [“2012”]. A new species of Coccomyces (Rhytismatales, Ascomycota) on Ilex elmerrilliana. Mycotaxon 122: 287-291. http://dx.doi.org/10.5248/122.287
Yang MS, Lin YR, Zhang L, Wang XY. 2013 [“2012”]. Coccomyces hubeiensis, a new fungus of Rhytismatales from China. Mycotaxon 122: 249-253. http://dx.doi.org/10.5248/122.249
Zheng Q, Lin YR, Yu SM, Chen L. 2012 [“2011”]. Species of Rhytismataceae on Lithocarpus spp. from Mt Huangshan, China. Mycotaxon 118: 311-323. http://dx.doi.org/10.5248/118.311
ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/130.79 Volume 130, pp. 79-89 January-March 2015
Type studies on Amauroderma species described by J.D. Zhao et al. and the phylogeny of species in China
MENG-JIE Li”? & Ha1l-SHENG YUAN”
"State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110164, P. R. China ? University of Chinese Academy of Sciences, Beijing 100049, P. R. China
* CORRESPONDENCE TO: hsyuan@iae.ac.cn
ABSTRACT — Holotypes of eight Amauroderma species described by Ji-Ding Zhao and co-workers, together with 93 additional specimens collected from China, were examined. Four names are demoted to synonymy (A. amoiense and A. wuzhishanense = A. rugosum; A. dayaoshanense = Pyrrhoderma sendaiense; A. fujianense = Ganoderma fornicatum), two represent accepted species (A. austrosinense, A. yunnanense), and two are of uncertain application (A. longgangense, A. jiangxiense) because of the poor or sterile condition of the holotype material. Illustrated descriptions of A. austrosinense and A. yunnanense are given based on the holotypes. Four other species reported from China (A. perplexum, A. rugosum, A. subresinosum, A. yunnanense) were sequenced for molecular analysis, and a phylogenetic tree was constructed by maximum parsimony and Bayesian analyses of the nuclear internal transcribed spacer of ribosomal DNA (ITS rDNA) sequences. A key to accepted Amauroderma species in China is provided.
KEY worDs — Ganodermataceae, morphology, phylogenetics, taxonomy
Introduction
Amauroderma Murrill was established by Murrill and typified by A. regulicolor (Berk. ex Cooke) Murrill (Murrill 1905). This genus is mainly characterized by sessile or stipitate (mesopodal, pleuropodal) basidiocarps with a laccate or dull pileus; a trimitic hyphal system; and ellipsoid, subglobose to globose bitunicate basidiospores with an asperulate inner wall (Furtado 1981, Ryvarden 2004). Macroscopically, Amauroderma shares with Ganoderma P. Karst. a similar basidiocarp shape of central or lateral stipe and a laccate or dull surface. Ganoderma can be separated from Amauroderma by its distinctly truncate basidiospores, and most Ganoderma species grow on dead wood, while most Amauroderma species grow in the ground from buried roots (Ryvarden 2004).
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Amauroderma is widely distributed in the tropics and subtropics south of 25°N in China within Guangdong, Guangxi, Yunnan, and Hainan Provinces. Amauroderma species are regarded as economically valuable because of their famous medicinal properties and pathogenicity (Dai et al. 2007, 2009; Jiao et al. 2013; Chan et al. 2013). Index Fungorum (www.indexfungorum.org) has listed 126 names under this genus. In China, taxonomic research on Amauroderma began with Teng, who recorded 10 species (Teng 1936, 1939, 1963). Later Zhao et al. (1979, 1983, 1984) and Zhao & Zhang (1986, 1987a,b, 2000) reported 20 species, nine of which were new. The types of these new species [except Amauroderma guangxiense J.D. Zhao & X.Q. Zhang (not traced)] are preserved in the Herbarium Mycologicum Academiae Sinicae (HMAS), the Institute of Microbiology, Chinese Academy of Sciences. Tai (1979) and Fan & Liu (1990) have also contributed to Amauroderma records in China.
In this study we investigated the type specimens of Amauroderma described by Ji-Ding Zhao and his colleagues and 93 other recently collected specimens from southern China based on morphological characters and rDNA sequence analyses. The taxa are presented below in alphabetical order according to the original names.
Materials and Methods
Morphology
The studied specimens were deposited in the Herbaria of the Institute of Applied Ecology, Chinese Academy of Sciences (IFP); the Institute of Microbiology, Chinese Academy of Sciences (HMAS); and the Institute of Microbiology, Beijing Forestry University (BJFC). Microscopic procedures followed He & Dai (2012). Sections were studied at magnifications up to 1000x using a Nikon Eclipse 80i microscope with phase contrast illumination. Measurements were made from sections stained with Cotton Blue. In presenting basidiospore size variation, 5% of measurements are excluded from each end of the range and given in parentheses. The following abbreviations are used: CB = Cotton Blue, CB+ = cyanophilous, CB- = acyanophilous, IKI = Melzer’s reagent, IKI- = negative in Melzer’s reagent, KOH = 5% potassium hydroxide, L = mean basidiospore length (arithmetic average of all basidiospores), W = mean basidiospore width (arithmetic average of all basidiospores), Q = variation in the L/W ratios between the specimens studied, and n = number of basidiospores measured from given number of specimens. Special color terms follow Petersen (1996).
Molecular phylogeny
Amplification of ITS sequences from herbarium specimens was conducted using Phire® Plant Direct PCR Kit (Finnzymes, Finland) with ITS5 and ITS4 primers (White et al. 1990). PCR procedure was as follows: initial denaturation at 98 °C for 5 min, followed by 39 cycles at 98 °C for 5 s, 59 °C for 10 s and 72 °C for 5 s, and a final extension at 72 °C for 2 min. PCR amplification was confirmed on 1% agarose electrophoresis gels
Amauroderma type studies (China) ... 81
TABLE 1. ITS sequences of Amauroderma and Tomophagus species used in the molecular analyses.
SPECIES VOUCHER LOCALITY GENBANK No. A. perplexum Cui 6496 Hainan, China KJ531650 Dai 10811 Hainan, China KJ531651 Wei 5562 Hainan, China KJ531652 A. rugosum Cui 6285 Hainan, China KJ531656 Zhou 153 Guangxi, China KJ531657 Dai 7862 Hainan, China KJ531658 Zhou 136 Guangxi, China KJ531659 Zhou 347 Guangxi, China KJ531660 Zhou 66 Guangxi, China KJ531661 Yuan 4554 Hainan, China KJ531662 Dai 9904 Hainan, China KJ531663 Cui 9011 Guangdong, China KJ531664 Cui 9012 Guangdong, China KJ531665 Wei 5234 Hainan, China KJ531666 Cui 8882 Guangdong, China KJ531667 Dai 9553 Hainan, China KJ531668 Dai 9566 Hainan, China KJ531669 Dai 4345 Hainan, China KJ531670 Dai 10040 Hainan, China KJ531671 Cui 4078 Fujian, China KJ531672 Dai 10307 Hainan, China KJ531673 Zhou 523 Guangxi, China KJ531674 Zhou 547 Guangxi, China KJ531675 Dai 12324 Yunnan, China KJ531676 Dai 12390 Yunnan, China KJ531677 Cui 9006 Guangdong, China KJ531678 THP 30 Unknown HM 480835 A. subresinosum Wei 5569 Hainan, China KJ531649 ML 288 Malaysia JQ409358 A. yunnanense Cui 7974 Yunnan, China KJ531653 Dai 13021 Yunnan, China KJ531654 Yuan 2253 Yunnan, China KJ531655 T. colossus CGMCC 5.763 Philippines JQ081068
stained with ethidium bromide (Stéger et al. 2006). The PCR products were purified and directly sequenced in Beijing Genomics Institute, China, with the same primers. The most similar sequences (TABLE 1) were retrieved from GenBank (http://www.ncbi.nlm. gov) using the BLAST option. Sequences were aligned using ClustalX (Thompson et al.
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1997). Alignment was manually adjusted to allow maximum alignment and minimize gaps, and deposited in TreeBASE (http://treebase.org/treebase-web/; submission ID: 17050). Identity/similarity between two sequences was calculated using the “pairwise alignment, calculation of the similarity/identity” option of BioEdit v. 7.0.5 (Hall 2005). Maximum parsimony and Bayesian analysis were applied to the ITS dataset. All characters were weighted and gaps were treated as missing data. Maximum parsimony analysis (PAUP* version 4.0b10) was used (Swofford 2002). Trees were inferred using the heuristic search option with TBR branch swapping and 1,000 random sequence additions. Max-trees were set to auto-increase, branches of zero length were collapsed, and all parsimonious trees were saved. Clade stability was assessed using a bootstrap (BT) analysis with 1,000 replicates (Felsenstein 1985). Descriptive tree statistics, including tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for all trees generated under different optimality criteria.
JModelTest (Posada 2008) was used to determine the best-fit evolution model for each data set for Bayesian inference (BY). Bayesian inference was calculated with MrBayes3.1.2 with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist & Huelsenbeck 2003). Four simultaneous Markov chains were run with 3 million generations, and trees were sampled every 100 generations. Trees generated for the first one-fourth generations were discarded as burn-in. A 50% majority rule consensus tree of all remaining trees was calculated. Branches that received maximum parsimony bootstrap support (BSS) and Bayesian posterior probabilities (BPP) respectively greater than or equal to 75% and 0.95 were considered as significantly supported.
Taxonomy
We studied eight species of Amauroderma (Ganodermataceae) described from China by Ji-Ding Zhao et al. Four of the eight names are synonymized, two are accepted, and two are of uncertain application.
Amauroderma amoiense J.D. Zhao & L.W. Hsu, Acta Mycol. Sinica 2: 164. 1983. Ho.otype: China. Fujian Prov., Xiamen, sandy land, 13.V1.1976, Li Hui-zhong & Zhang Xiao-qing 28 (HMAS 42784).
= Amauroderma rugosum (Blume & T. Nees) Torrend, Brotéria, Sér. Bot. 18: 127. 1920. The type material is sterile, but all other characters fit A. rugosum well. For a detailed description of A. rugosum, see Ryvarden & Johansen (1980).
Amauroderma austrosinense J.D. Zhao & L.W. Hsu, Acta Mycol. Sinica
3: 20. 1984. Fig 1 Hototype: China. Hainan Prov., Bawangling, ground, 20.1V.1977, Han Shu-jin 902 (HMAS 42695).
BASIDIOCARPS annual, stipitate, umbelliform. PILEus single, suborbicular to circular, glabrous, dull, clay-buff to fulvous brown, densely concentrically zoned
Amauroderma type studies (China) ... 83
Fic. 1. Amauroderma austrosinense (holotype): basidiospores.
and slightly irregularly radially furrowed; MARGIN obtuse and deflexed when dry; STIPE sub-central or eccentric, concolorous with pileus, cavitate, up to 12 cm long and 1 cm thick. CONTEXT cinnamon, dense and hard, homogenous, up to 4mm thick. PORE SURFACE cream white when fresh, turning brown with age; PORES circular, entire, 6-8 per mm. TUBES woody, up to 3 mm long.
HYPHAL SYSTEM trimitic; GENERATIVE HYPHAE with clamp connections, hyaline, thin-walled, 2.8-4 um in diam; SKELETAL HYPHAE pale yellow to almost colorless, sparsely branched, 3.7-5.2 um in diam; BINDING HYPHAE hyaline, branched, tortuous, 1.5-2.2 um in diam; all the hyphae IKI-, CB+; tissues darkening in KOH. Basipiospores subglobose to globose, faintly yellowish, IKI-, CB+, doubled-walled, exospore smooth, endospore with conspicuous echinule, (6.5-)6.8-7.6(-8) x (5.9-)6.2-7(-7.3) um, L = 7.34 um, W = 6.58 um, Q = 1.12 (n = 30/1).
ADDITIONAL SPECIMEN EXAMINED: CHINA, HAINAN PRov., Changjiang County,
Bawangling Nat. Res., ground, 20.IV.1977 (HMAS 42695). REMARKS: Amauroderma austrosinense is an accepted species, remarkable for its umbelliform basidiocarps with distinct concentric rings, small pores, and basidiospores. A. camerarium (Berk.) J.S. Furtado is similar to A. austrosinense by sharing a clay-colored, densely concentrically zoned, and radially furrowed pileus. However, A. camerarium differs in its larger basidiospores (12-15 x 10-13 um; Ryvarden 2004).
Amauroderma leucosporum Corner may be confused with A. austrosinense by having similar-sized subglobose to globose basidiospores and also not turning blood red on bruising, but A. leucosporum can be distinguished by its finely to distinctly villous pileus and stipe (Corner 1983).
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Amauroderma dayaoshanense J.D. Zhao & X.Q. Zhang, Acta Mycol. Sinica 6551987 Ho .ortyPe: China. Guangxi Auto. Reg., Dayaoshan, dead wood, 1982, Wei Bing-gang 70 (HMAS 48282). = Pyrrhoderma sendaiense (Yasuda) Imazeki, Trans. Mycol. Soc. Japan 7: 4. 1966.
The type is sterile but has the characteristics of P sendaiense (see Nunez & Ryvarden (2000) for a detailed description).
Amauroderma fujianense J.D. Zhao, L.W. Hsu & X.Q. Zhang, Acta Microbiol. Sinica T9279. 1979:
Ho.otype: China. Fujian Prov., Sanming County, rotten wood, 28.VI.1976, Zhang Xiao-qing 68 (HMAS 37919). = Ganoderma fornicatum (Fr.) Pat., Bull. Soc. Mycol. Fr. 5: 71. 1889. The holotype is sterile but represents a juvenile specimen of G. fornicatum (see Patouillard (1889) for a detailed description).
Amauroderma jiangxiense J.D. Zhao & X.Q. Zhang, Acta Mycol. Sinica 6: 206. 1987. Ho .ortyPe: China. Jiangxi Prov., Jian County, rotten wood, 15.11.1983, Yang Ren-gen 152 (HMAS 50418). The basidiocarp is umbelliform and the pileus is black, laccate, and shiny, but the specimen is completely sterile. It probably represents a Ganoderma species, but the application of the name is uncertain.
Amauroderma longgangense J.D. Zhao & X.Q. Zhang, Acta Mycol. Sinica Sr227 L986,
Ho.otype: China. Guangxi Auto. Reg., Nonggang, on rotten wood, 1979, Wei Bing- gang 790111-G8 (HMAS 47621). Basidiospores subglobose, 6-8.5 x 5-8 um, with spinules 0.8-1.2 um on sporoderm. However, the single-walled basidiospore obviously differs from the double-walled spore of other Amauroderma species. The application of the name is uncertain.
Amauroderma wuzhishanense J.D. Zhao & X.Q. Zhang, Acta Mycol. Sinica 6: 208. 1987.
Ho.otype: China. Hainan Prov., Wuzhishan, on rotten wood, 22.VII.1956, Jiang Guang-zheng (HMAS 19311).
= Amauroderma rugosum (Blume & T. Nees) Torrend, Brotéria, Sér. Bot. 18: 127. 1920.
The holotype is in accordance with Amauroderma rugosum, with its pileus obscurely zonate, radially quite rugose, and warted. For a detailed description of A. rugosum, see Ryvarden & Johansen (1980).
Amauroderma type studies (China) ... 85
Fic. 2. Amauroderma yunnanense (holotype): basidiospores.
Amauroderma yunnanense J.D. Zhao & X.Q. Zhang, Acta Mycol. Sinica, Suppl. 1: 268. 1987 [“1986”]. FIG 2
Ho.otype: China. Yunnan Prov., Xichou County, ground, Xiaogiaogou, 14.V.1959,
Wang Qing-zhi 89 (HMAS 48231). BASIDIOCARPS annual, centrally to laterally stipitate, coriaceous to corky, brittle when dry. PiLeus single, flabelliform, suborbicular, umbilicate, PILEAL SURFACE pale yellowish brown to dark brown, adpressed velutinate, with distinct concentric zones at the margin and fine furrows in the middle; MARGIN inflexed, acute or subacute, entire or slightly lacerated and thin; stipe concolorous with the pileus, up to 7 cm long, inflated at base. ConTExT homogeneous, white to pale yellow. PoRE SURFACE white when fresh, pale straw yellow when dry, bruising unchanged; porgs angular, 2-3 per mm; TuBEs concolorous with pore surface, up to 5 mm thick.
HYPHAL SYSTEM trimitic; GENERATIVE HYPHAE with clamps, hyaline, thin-walled, 2-4.3 um in diam; SKELETAL HYPHAE thick-walled, pale yellow, arboriform, 3.5-5.5 um in diam; BINDING HYPHAE hyaline, branched, tortuous, 1.8-2.5 um in diam; all the hyphae IKI-, CB+; tissues darkening in KOH. Basipiospores broadly ellipsoid to subglobose, pale yellow brown, IKI-, CB+, doubled-walled, exospore smooth, hyaline, endospore with conspicuous echinule, (8.3—)8.9-10.3(-10.7) x (6.3-)6.7-8(-8.8) um, L = 9.35 um, W = 7.32 um, Q = 1.22-1.34 (n = 120/4).
ADDITIONAL SPECIMENS EXAMINED: CHINA, YUNNAN PRov., Chuxiong, Zixishan Forest Park, ground, 8.[X.2006, Yuan 2253 (IFP 013207); Ailaoshan, 5.[X.2007, Yuan 3367 (IFP 013182); Kunming, Qiongzhusi Park, 21.X.2009, Cui 7974 (BJFC 006463); Xiaoshao Village, 22.1X.2012, Dai 13021 (IFP 019123). REMARKS: Amauroderma yunnanense is an accepted species characterized by a brown tomentose pileus and stipe.
Amauroderma perplexum Corner and A. yunnanense both have a velutinate
pileus and stipe as well as stipes that dilate at the base; A. perplexum can be
86 ... Li & Yuan
separated by larger basidiospores, longer tubes, and a pore surface that changes color when bruised (Corner 1983).
Amauroderma partitum (Berk.) Wakef., which may be confused with A. yunnanense as they share similar angular pores that are 2-3 per mm, can be distinguished by its glabrous, slightly shiny pileus and larger basidiospores (12-15 x 8-10 um; Ryvarden 2004).
Molecular phylogeny
The nuclear ITS rDNA dataset contains 32 sequences representing A. perplexum, A. rugosum, A. subresinosum (Murrill) Corner, and A. yunnanense, with Tomophagus colossus (Fr.) Murrill as outgroup (TasBLe 1). After the ambiguous sites at both ends and the 5.8S region were trimmed off, the alignment comprised 561 characters, of which 462 were constant, 22 were variable but parsimony-uninformative, and 77 were parsimony-informative. Maximum parsimony analysis yielded 6 equally parsimonious trees (CI = 0.918, RI = 0.952, RC = 0.874, HI = 0.082). A strict consensus tree of these trees is presented in Figure 3. The 50% majority consensus tree generated by the Bayesian analysis showed a similar topology with the strict consensus MP tree, and only the topology from MP analysis is presented, while both bootstrap support value (BSS) and Bayesian posterior probabilities (BPP) are shown at the nodes (Fic. 3).
In the phylogenetic tree, each sampled Amauroderma species was resolved as a strongly supported clade (100% BSS and 1.00 BPP). Identity/similarity calculation result shows that identities of Amauroderma sequences are between 0.98 and 1. We failed to obtain sequences from the type specimens of Amauroderma described by Ji- Ding Zhao from China as the materials were too old to extract integrated DNA.
Key to species of Amauroderma in China
i. Pileal surface appressed welutinate:.o iiss talee ge hues Aol h eva heh Wale Thee 2 Te Pileal ‘surface: PlaDEOUS.§ 4p seven aybscton teh acbea dring.seg rhea son dhe soa dle puon toby baud he fobs deh fro 9 3 2. Pores 2-3 per mm, pore surface straw yellow, not reddening on bruising,
spores (8.3—)8.9-10.3(-10.7) x (6.3-)6.7-8(-8.8) um. .......... A. yunnanense 2. Pores 4-6 per mm, pore surface cream white, reddening on bruising,
spores (10.3-)10.8-14(-14.8) x (8.9-)9.2-12(-13.1) um......... A. perplexum 3. Pileus surface laccate, spores ovoid to broadly ellipsoid,
(13.7-)14.6-17.1(-17.5) x (8.3-)8.9-10.2(-11.3) um.......... A. subresinosum SaPileal Sut acevcUlle., £ reste dove. tdan ove len aortas Sahy eeselger estes ace aeearatenat hts gt mmaie ates -: 4, Pileal clay-buff to fulvous brown, pores 7-8 per mm,
spores (6.5—)6.8-7.6(-8) x (5.9-)6.2-7(-7.3) um .........0.00. A. austrosinense
4. Pileus taupe, dark brown to black, pores 4-6 per mm, spores (8.1—)8.4-11.7 (-12) x (7-)7.5-9.6(-10.3) um. ............. A. rugosum
Amauroderma type studies (China) ... 87
100/1.00 rAmauroderma subresinosum Wei 5569 Amauroderma subresinosum JQ 409358 Amauroderma yunnanense Cui 7974 ROUEOe Amauroderma yunnanense Dai 13021 100/1.00 Amauroderma yunnanense Yuan 2253 Amauroderma perplexum Cui 6496 100/08 Amauroderma perplexum Wei 5562 ‘Amauroderma perplexum Dai 10811 Amauroderma rugosum Cui 6285 52/0.52 Amauroderma rugosum Zhou 153 Amauroderma rugosum Dai 7862 Amauroderma rugosum Zhou 136 Amauroderma rugosum Zhou 347 Amauroderma rugosum Zhou 66 62/0.97 Amauroderma rugosum Yuan 4554 Amauroderma rugosum Dai 9904 Amauroderma rugosum HM 480835 Amauroderma rugosum Cui 9011 Amauroderma rugosum Cui 9012 Amauroderma rugosum Wei 5234 Amauroderma rugosum Cui 8882 100/1.00 Amauroderma rugosum Dat 9553 Amauroderma rugosum Dai 9566 Amauroderma rugosum Dai 4345 Amauroderma rugosum Dai 10040 Amauroderma rugosum Cui 4078 Amauroderma rugosum Dai 10307 Amauroderma rugosum Zhou 523 Amauroderma rugosum Zhou 547 Amauroderma rugosum Dai 12324 Amauroderma rugosum Dai 12390 Amauroderma rugosum Cui 9006 Tomophagus colossus JQ 081068 Liooorsiets 0.0 8.0
Fic. 3. Strict consensus tree obtained from Maximum Parsimony and Bayesian analyses of ITS sequences of Amauroderma. Tomophagus colossus was used as outgroup. Numbers at branches indicate parsimony bootstrap values and Bayesian posterior probabilities values higher than 50%.
88 ... Li & Yuan
Acknowledgements
The authors are grateful for presubmission review and helpful comments by Bao- Kai Cui (Institute of Microbiology, Beijing Forestry University) and Xiao-Yong Liu (Institute of Microbiology, Chinese Academy of Sciences). Special thanks are due to Dr. Katherine F. LoBuglio (Harvard University Herbaria, USA) for checking and improving the manuscript. This research was financed by the National Natural Science Foundation of China (Project Nos. 31170022 & 31470148).
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ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/130.91 Volume 130, pp. 91-103 January-March 2015
Cladonia corymbescens consists of two species
TEUVO AHTI, RAQUEL PINO-BODAS, & SOILI STENROOS
Botanical Museum, Finnish Museum of Natural History, University of Helsinki, PO. Box 7, FI-00014 Helsinki, Finland
* CORRESPONDENCE TO: teuvo.ahti@helsinki.fi
Asstract — The lichen Cladonia corymbescens (Cladoniaceae, Ascomycota) has been reported to have two main ranges, one in Melanesia and Australasia and another in the Himalayas and surrounding areas. When specimens from New Caledonia, Bhutan, and Thailand were subjected to molecular sequencing (ITS rDNA and RPB2), two distinct clades were detected. The clades were also supported by morphological and geographical differences supporting recognition of two distinct species. Cladonia corymbescens s. str. is present in the Philippines, Melanesia, and Australasia, while the South East Asian mainland material is referred to C. pseudofissa, a new combination at species level.
Key worps — Cladonia furcata group, lichens, molecular systematic, phylogeny, taxonomy
Introduction
One of the most common fruticose species of Cladonia in the mountains of western Melanesia is Cladonia corymbescens. The nomenclature of this species and its similarities with other species was thoroughly discussed by Stenroos (1988), who also reported it from China and Nepal. However, Abbayes (1974) was the first lichenologist to recognize this species from the Himalayas. His opinion was also accepted by Awasthi & Ahti (2007) and Rai et al. (2014), who stated that the species is widespread in the mountains in India and Nepal. In his world monograph, Vainio (1887) could not give a definite opinion on the type material of C. corymbescens from New Caledonia, and he referred the only Himalayan specimen representing that species that he apparently examined to C. erythrosperma var. thomsonii.
Several studies using molecular data have shown that many morphological Cladonia species are not monophyletic (Kotelko & Piercey-Normore 2010, Fontaine et al. 2011, Pino-Bodas et al. 2011, Steinova et al. 2013). The re- examination of the morphology based on molecular data in lichenized fungi has revealed that in several cases some phenotypical differences have been overlooked (Argitiello et al. 2007, Nufez-Zapata et al. 2010, Parnmen et al.
92 ... Ahti, Pino-Bodas, & Stenroos
2013, Muggia et al. 2014). The aim of this study was to use DNA sequence data to assess whether Cladonia corymbescens is a genetically homogeneous species and to revise its classification based on the phylogenetic results and morphological differences.
Materials & methods
Material for DNA analyses
Altogether 39 specimens of 29 species of Cladonia representing the supergroup Cladonia in the provisional classification of Stenroos et al. (2002) were selected for a phylogenetic analysis in this study. These specimens are preserved in the herbaria of University of Helsinki (H), University of Turku (TUR) or Complutense University, Biology Faculty (MACB); details of the collections are shown in TaBLE 1. Four specimens of Cladonia corymbescens, two from New Caledonia and two from the Himalayas, were available for DNA analyses, while 60 sequences had been generated in previous studies (Stenroos et al. 2002; Pino-Bodas et al. 2010a, 2010b, 2012a, 2012b, 2012c, 2013a, 2013b). Cladonia rangiformis Hoffm. was selected as outgroup based on previous results (Stenroos et al. 2002).
DNA extraction, PCR, and sequencing
Total DNA was extracted using DNeasy Plant Mini Kit (QUIAGEN, Germany) following the manufacturer's instructions. In this study two genetic regions were chosen: internal transcribed spacer of ribosomal DNA (ITS rDNA) and the second largest subunit of RNA polymerase IT (RPB2). Although the ITS rDNA region has been chosen as standard barcoding for fungi (Schoch et al. 2012), in Cladonia the ITS rDNA frequently fails in the identifications (Kelly et al. 2011; Pino-Bodas et al. 2013b) and RPB2 gives better results (Pino-Bodas et al. 2013b). The primers used to amplify the nuclear ITS nrDNA were ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990), and for RPB2 they were RPB2dRaq/RPB2rRaq (Pino-Bodas et al. 2010a) or CLRPB25F/CLRPB27R (Yahr et al. 2006). The amplification programs are listed in Pino-Bodas et al. (2013a). PCR was carried out with Ready-to-Go-PCR Beads (GE Healthcare Life Sciences, UK). Amplifications were prepared for a 25 uL final volume. PCR was performed using a Mastercycler ep Gradient S (Eppendorf, Westbury, NY). PCR products were purified with ExoSAP-IT (USB Corporation, USA) and sequenced in Macrogen Europe (Amsterdam, Netherlands).
Alignments and phylogenetic analyses
The forward and reverse strands were assembled and edited in Sequencher™ v. 4.1.4 (Gene Codes Corporation, Inc., Ann Arbor, Michigan, USA). BLAST searches were used to confirm that the obtained sequences belonged to Cladonia. The sequences were manually aligned in BIOEDIT (Hall 1999). Nine ambiguous positions were manually delimited and removed from the ITS rDNA alignment. The RPB2 alignment did not have ambiguous positions.
Every region was separately analyzed by Maximum Likelihood (ML), with 500 bootstrap replicates to assess the support of each node. The conflict between the loci was tested according Lutzoni et al. (2004). This method considers the existence of
Cladonia pseudofissa comb. nov. (Southeast Asia) ... 93
TABLE 1. List of Cladonia specimens used in the molecular study with voucher specimen information and GenBank accession numbers. (The new sequences and the new combination are in bold font.)
SPECIES COLLECTION CODE ITS rDNA RPB2 C. acuminata USA, Ahti 63278 (H) 1ACUMI JN621932 JN621965 C. apodocarpa USA, Harris 54250 (H) 1APODO KC526127 KC526068 C. borbonica New Caledonia, Dennetiére 109 (H) AT686 AF455214 KP732364 C. cariosa Spain, Burgaz (MACB 94208) 5CARI JN621909 JN621941 C. cartilaginea Brazil, Stenroos 4926 (TUR) LK45 AF455212 — C. ceratophylla Brazil, Stenroos 5081 (TUR) LK37 AF455171 — C. conista Spain, Burgaz (MACB 92796) 2HUMIL JF926613 JF926567 Russia, Zolotuchin 26a (H) 1CONIST JF926633 JF926568 C. corsicana Spain, Burgaz (MACB 100763) SP1 JE288797 JF288833 Spain, Burgaz (MACB 101074) SP2 JF288798 JF288834 C. corymbescens New Caledonia, Dennetiére 45 (TUR) AT680 AF455235 KP732366 New Caledonia, Christenhusz 6169d (H) CL166 KP732363 KP732367 C. cyathomorpha Spain, Burgaz (MACB) 1CYATH KC415941 KC525275 Norway, Ahti 68660 & Tonsberg (H) 3CYATH KC415943 = KC525276 C. firma Spain, Burgaz (MACB 91619) 1FIRM FM205907 FM207568 Spain, Burgaz (MACB 90655) 7FIRM FM205910 FM207576 C. foliacea Portugal, Burgaz (MACB 90506) 1FOL FM205894 FM207569 C. furcata USA, Ahti 58283 (TUR) AT638 AF455220 KP732369 C. humilis Spain, Burgaz (MACB 95931) 9HUMIL JF926615 JF926576 Croatia, Burgaz (MACB 101103) 20HUMIL JF926621 JF926580 C. multiformis Canada, Ahti 57065 (H) LK70 AF455213 KP732370 C. nana Brazil, Stenroos 4940 (TUR) LK34 AF455211 — C. neozelandica New Zealand, Wirth 28180 (TUR) AT590 AF455206 — C. petrophila USA, Ahti 56654 (H) LK68 AF455222. — C. peziziformis USA, Stenroos 5198 (TUR) AT631 AF455221 — USA, Ahti 56670 (H) LK38 AF455182 —_ C. pityrophylla Brazil, Stenroos 5096 (TUR) LK40 AF455238 — C. pseudofissa Thailand, Parnmen 285 (H) 1CORYMB KP732362 KP732365 Bhutan, Sgchting 9206 (H) LK35 AF455239 KP732368 C. pulvinata Spain, Burgaz (MACB 91646) 4PUL FM205911 =FM207579 C. pulvinella USA, Ahti 69191 (H) CLCAL4 KC415963 KC525287 C. pyxidata Greenland, Hansen (H) 17PYXI KC415983 = KC525269 C. rangiformis Netherlands, Van der Goes et al. (H) 5RANG JN811400 JN811429 Sweden, Skytén (H) 6RANG JN811401 JN811430 C. scabriuscula Canada, Ahti 56969 (H) LK11 AF455217 KP732371 C. subcariosa USA, Moore (H) 1SUBCARI JN621936 JN621969 C. subconistea Korea, Moon 7188 (H) 1SUBCONI KC415949 KC525296 C. subulata Spain, Burgaz (MACB 93151) 1SUBU FN86566 HM243210 C. turgida Canada, Lendemer (H) 1TURG JF288801 KC526089
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incongruity whenever a clade was supported with a bootstrap of more than 75% in a locus, while in other loci the individual sequences of this clade were part of another clade with bootstrap support =75%. The trees were checked manually. No conflicts were detected and the datasets were combined. The combined dataset was analyzed by Maximum Parsimony (MP), ML, and Bayesian analyses. MP analysis employed PAUP version 4.0.b.10 (Swofford 2002), using heuristic searches with 1000 random taxon- addition replicates with TBR branch swapping and MulTrees option in effect, and equally weighted characters and gaps treated as missing data. Bootstrap with 1000 replicates was used as the confidence analysis, using the heuristic option. The ML and Bayesian analyses were run considering four partitions (ITS rDNA and each codon position of RPB2). Maximum Likelihood analyses (ML) were done in RAXML 7.04 (Stamatakis 2006), assuming the model GTRGAMMA. A fast bootstrap with 500 replicates was implemented to assess the support of each node. Bayesian analysis was carried out using the program MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001). The models of evolution were selected with MrModeltest (Nylander 2004) under AIC criterion. The models selected were GTR+I+G for ITS rDNA and SYM+1+G for RPB2. Posterior probabilities (pp), approximated by sampling trees using Markov Chain Monte Carlo (MCMC), of each branch were calculated by counting the frequency of trees that were visited during the course of the MCMC analysis. Model parameters were estimated in each analysis for 5,000,000 generations sampled in four simultaneous chains, and every 1000th was saved into a file. When the analysis was finished we checked that standard deviation between the runs was <0.005. The program AWTY (Nylander et al. 2008) was used to determine when the chains reached the stationary stage. The first 1000 trees were deleted as the “burn in” and the 50% majority-rule consensus tree was calculated using the “sumt” command of MrBayes.
Morphology and chemistry
About 63 herbarium specimens of Cladonia corymbescens s. lat. deposited in Helsinki (H) were morphologically re-evaluated after the phylogenetic analyses. Authors Ahti and Stenroos had previously studied many additional specimens in various herbaria [e.g., Geneva (G), Leiden (L), Lucknow (LWG, LWG-LWU, LWG-AWAS)] as well as material borrowed from Edinburgh (E); see also the specimen lists in Stenroos (1988) and Rai et al. (2014).
The secondary metabolites were analyzed by thin layer chromatography (TLC) according to standardized procedures (Orange et al. 2001) using the solvents A (Toluene: dioxane: acetic acid, MERCK) and B (Hexane: diethylether: formic acid, MERCK).
Results
In this study we generated 10 new sequences (2 of ITS rDNA and 8 of RPB2). The combined dataset contained 1332 unambiguous characters (597 of ITS rDNA and 735 of RPB2), 267 of which were parsimony informative (152 of ITS rDNA and 115 of RPB2). The MP analysis generated two equally parsimonious trees of 886 steps. The ML analyses of the combined dataset generated a tree with likelihood value of -LnL = 6355.82, while the mean of likelihood of the Bayesian analysis was —LnL = 6589.51.
Cladonia pseudofissa comb. nov. (Southeast Asia) ... 95
77 --C. apodocarpa 1APODO C. petrophila LK68 C. borbonica AT686 100"— ©. corsicana SP 1 C. corsicana SP2 1por— C. cyathomorpha 1CYATH C. cyathomorpha 3CYATH C. conista 1CONIST C. conista 2HUMIL C. subconistea 1SUBCONI C. pulvinella CLCAL4 9gr— C. cartilaginea LK45 87 C. neozelandica AT590 85 C. nana LK34 100(— C. peziziformis AT631 C. peziziformis LK38 C. furcata AT638 C. multiformis LK70 100 C. humilis 2OHUMIL C. humilis 9HUMIL C. scabriuscula LK11 100[— ©. corymbescens AT680 (New Caledonia) C. corymbescens CL166 (New Caledonia) gi1-— C. ceratophylla LK37 A C. pityrophylla LK40 1por— C. pseudofissa comb. nov. 1CORYMB (Thailand) C. pseudofissa comb. nov. LK35 (Bhutan) 100/— ©. firma 1FIRM C. firma 7FIRM 78[—- C. acuminata 1ACUMI C. cariosa 5CARI C. foliacea 1FOL
100
100 g9f— C. pulvinata 4PUL 95 C. subcariosa 1SUBCARI le C. subulata 1SUBU
C. turgida 1TURG C. pyxidata 17PYXI| C. rangiformis 5RANG C. rangiformis BRANG
Fic. 1. Cladonia spp. phylogeny: consensus tree of the two most parsimonious trees based on the concatenated matrix of ITS rDNA and RPB2. The numbers on the branches represent bootstrap values (>75%).
In all the analyses the specimens from the Himalayan region and the specimens from New Caledonia formed two clades; however, the relationships
96 ... Ahti, Pino-Bodas, & Stenroos
om Lome C. porbonics RvO8S ¢. corsicana 74 re ose i fe CYATH 0.99/82] 1/100 . cyatnomorpna C. cyathomorpha 3CYATH_
1/100 C. cartilaginea LK45 -_C, neozelandica AT590 C. nana LK34 .
C. peziziformis AT631 C. peziziformis LK38 C. furcata AT638 C. multiformis LK70 1/100 - C. humilis ZOHUMIL C. humilis 9HUMIL 0.97/77 1/100 -C. conista 1CONIST 1/88 C. conista 2HUMIL C. subconistea_1SUBCONI
C. pulvinella CLCAL4
C. scabriuscula
1 1/88 C. ceratophylla LK37 ane eee aes eS pityrophylla LK40
C. pseudofissa comb. nov. 1CORYMB (Thailand) C. pseudofissa comb. nov. LK35 en 1/100; C. corymbescens AT680 (New Caledonia
C. corymbescens CL166 (New Caledonia) al Se eC apodocarpa 1APODO C. petrophila LK6
C. pulvinata 4PUL C. subcariosa 1SUBCARI
C. subulata 1SUBU - C. turgida 1TURG C. acuminata 1ACUMI C. cariosa 5CARI 14100 - C. rangiformis SRANG
C. rangiformis BRANG
Fic. 2. Cladonia spp. phylogeny: 50% consensus majority tree of Bayesian analysis based on the concatenated matrix of ITS rDNA and RPB2. Posterior probability of bayesian analysis (>0.95) and bootstrap values of ML analyses (>75%) are shown on the branches.
were not the same in all the analyses. In the MP analysis (Fie. 1) the specimens from the Himalayan region (in the tree C. pseudofissa) are closely related with C. pityrophylla Nyl. and C. ceratophylla (Sw.) Spreng. (Fic. 1, clade A), but this relationship was not well supported (bootstrap <70%). The specimens from New Caledonia cluster in a clade with specimens representing the C. furcata group, C. humilis group, C. corsicana (Rondon & Vézda) Pino-Bodas et al., C. borbonica Nyl., C. cartilaginea Mill. Arg., and C. neozelandica Vain. (Fie. 1, clade B). The ML and Bayesian analyses generated trees with the same topology, and only the 50% consensus majority-rule tree from Bayesian analyses is shown (Fic. 2). The specimens from New Caledonia (C. corymbescens) and the specimens from the Himalayan region (C. pseudofissa) formed two clades that are closely related, although the relationship was not supported in any of the analyses.
Other differences between the MP tree and the Bayesian tree are that C. pyxidata (L.) Hoffm. was shown as basal in the MP analysis, while the Bayesian analysis showed C. cariosa (Ach.) Spreng. and C. acuminata (Ach.) Norrl. in the basal clade. Additionally, the clade of C. conista (Nyl.) Robbins,
Cladonia pseudofissa comb. nov. (Southeast Asia) ... 97
C. subconistea Asahina, and C. pulvinella S. Hammer is closely related to a clade of C. cyathomorpha Stirt. ex Walt. Watson, C. corsicana, and C. borbonica in the MP analysis, but not in the Bayesian analysis.
Taxonomy
Cladonia corymbescens Ny]. ex Leight., Ann. Mag. Nat. Hist., ser. 3, 18: 407. 1866. Fig. 3A
TyPeE (Abbayes 1974: 112): New Caledonia, Mont de M’bée, 1855-1860 E. Vieillard 1785 (PC, lectotype, G, H-NYL 38414, PC-Lenormand, isolectotypes). [The lectotypification by Abbayes was overlooked by Ahti in Stenroos (1988: 126), Ahti (1993) and other recent authors, but the type collection cited is the same].
= Cladonia novoguineensis Zahlbr., Bot. Jahrb. Syst. 62: 455. 1929.
Type: Papua New Guinea [“Nordéstliches Neu-Guinea, Kaiser Wilhelmsland”], Morobe
Prov., Sarawaged Mts. [“Saruwaged-Gebirg”], 3600-4000 m, C. Keysser 66 [“68”] (W,
holotype). Primary thallus 0.5-1 x 1.5-2 mm, soon disappearing. Podetia (2—)3-7 cm tall, 0.7-1.5 mm thick, pale to dark brown, medulla hardly melanotic at base, erect, dichotomously, trichotomously or irregularly richly branched, axils open, at least some podetia stout, sterile podetia have thin, subulate tips, fertile podetia becoming thicker, forming characteristic lateral fissures and perforations, branches often flattened. Surface highly discontinuously corticated, especially towards the base, in part sparsely microsquamulose or with larger squamules, sometimes verruculose, apical parts hardly pruinose. Podetial wall 120-220 um thick, with indistinct cortex 10-20 um, medulla 150-200 um, and stereome 50-60 um, hard. Pycnidia terminal or sometimes lateral on podetia, rarely on basal squamules, containing hyaline slime, 1.5 mm in diam, conidia 6-7 x 1 um. Apothecia 0.8-1 mm wide, terminal, on every tip on fertile podetia, sometimes deformed, brown, spores not observed.
CHEMISTRY—K+ yellow, PD+ red, rarely PD-. CHEMOTYPE 1: Atranorin (major), fumarprotocetraric acid (major), protocetraric acid (minor), confumarprotocetraric acid (minor), rarely rangiformic and norrangiformic acids, occasionally 1-4 unknown minor substances and 1-3 terpenoids. CHEMOTYPE 2: Atranorin only, the fumarprotocetraric acid complex lacking (then PD-) (Stenroos 1988). If chemotype 2 proves to represent a distinct taxon, the name Cladonia novoguineensis is applicable for that (no fresh material for DNA analysis was available).
HasitatT— Terrestrial in bogs, grasslands, road banks, rarely on tree stumps, at 1500-4400 m in Papua New Guinea (Stenroos 1988: 126).
DISTRIBUTION—Australia (New South Wales, Queensland), Indonesia (Java), New Caledonia, New Zealand (North Island), Papua New Guinea, Philippines (Luzon).
98 ... Ahti, Pino-Bodas, & Stenroos
ADDITIONAL SPECIMENS EXAMINED (See also Stenroos 1988) — NEW CALEDONIA, between Yaté and Goro, 1966, Hill 11741 (BM, H). PAPUA NEW GUINEA, EAsTERN HIGHLANDS, Mt. Wilhelm, Pindaunde Lake, 3540 m, 1965, A. C. Jermy 5327 (BM, H; richly fertile!)}; MOROBE PRov., Mt. Sarawaket Southern Range, 4 km NEE of Lake Gwam, 2850 m, 1981, T. Koponen 31662 (H, LAE); SOUTHERN HIGHLANDS, Mt. Giluwe, 3500 m, 1982, H. Streimann 24151 (CANB, H, LAE; chemotype 2). PHILIPPINES, Luzon, Mountain Prov., Alab on road Baguio to Bontoc, 850 m, 1976, W. S. Gruezo 2030 (CAHP, H).
Cladonia pseudofissa (Asahina) Ahti, Pino-Bodas & S. Stenroos, stat. et comb. nov. Fic. 3B MycoBank MB 809612 = Cladonia rangiformis var. pseudofissa Asahina, Fl. E. Himalaya: 595. 1966. TyPE: India, Sikkim, Jongri, 4000 m, 1960, M. Togashi 157 (TNS, holotype). = Cladonia erythrosperma var. thomsonii Vain., Acta Soc. Fauna FI. Fenn. 4: 376. 1887, syn. nov. [non Cladonia thomsonii Ahti]. Type: India, "India orientalis’, [1847-51], J. D. Hooker & T. Thomson 2145 (PC, holotype). = Cladonia rangiformis var. incurva Mull. Arg., Flora 74: 72. 1891, syn. nov. [non Cladonia incurva Ahti]. Type: India, Himachal Pradesh (?), Damodar [“Damdar”] Valley, 4200 m, J. F Duthie (BM, holotype).
The earliest names for C. pseudofissa are C. erythrosperma var. thomsonii and C. rangiformis var. incurva, but they cannot be adopted at species level because these epithets are already occupied in Cladonia by C. thomsonii Ahti (1978) and C. incurva Ahti (1961).
Primary thallus rarely visible, soon disappearing, consisting of minute squamules. Podetia 1-4(-5) cm tall, to 1 mm thick, almost white, ashy grey or more rarely dark brown, medulla clearly melanotic at base; often very delicate, ascendant to erect, usually somewhat curly in appearance, without any stouter sterile podetia, repeatedly subcorymbosely dichotomously branched, apices subulate, ascyphose, axils usually perforated. Fertile podetia somewhat thicker. Surface very matt, minutely fibrous or pruinose, often very smooth and almost continuously corticate, with a few very small squamules, longitudinally somewhat fissured, esorediate. Podetial wall 150-160 um thick, with distinct cortex 10-15 um, medulla 75-100, stereome 25-50 um thick, softish rather than cartilaginous. Pycnidia always at tips of podetia, shortly stalked, pyriform, black, c. 2 x 1 mm; conidia or colour of slime not observed. Apothecia dark brown, 0.5-1 mm, subglobose, on tips of flattened branchlets, spores not observed.
CHEMISTRY—K+ yellow, PD+ orange red. Contains atranorin and the fumarprotocetraric acid complex.
HasitatT—On ground, often on thin soil over rocks.
Cladonia pseudofissa comb. nov. (Southeast Asia) ... 99
Fic. 3. A: Cladonia corymbescens (Papua New Guinea, T. Koponen 31662, H). B: Cladonia pseudofissa (Nepal, P. Ozenda 147, H). Scale bar = 1 cm.
DISTRIBUTION—Bhutan, China (Sichuan, Tibet, Yunnan), India (Arunachal Pradesh, Himachal Pradesh, Kerala, Sikkim, Tamil Nadu, Uttarkhand, West Bengal), Malaysia (Sabah), Nepal, Thailand.
ADDITIONAL SPECIMENS EXAMINED — BHUTAN, Paro District: below Jaley la Dzong, 2500-3000 m, 1998, U. Sochting 9206 (C, H; DNA voucher, Stenroos et al. (2002)
100 ... Ahti, Pino-Bodas, & Stenroos
and GenBank, as C. corymbescens). CHINA, SICHUAN: Miyi Co., 3200 m, 1983, L. S. Wang 83-834 (H, KUN). TrpeT: Kang Ding Co., 1976, Z. Mu 5304 (H, KUN); YUNNAN: Lijiang Co., 1985, L. S$. Wang 85-140 (H, KUN). INDIA, Srxxim: North Sikkim District, Phune-Yakche, 3000 m, 1996, G. P. Sinha 1103 (ASSAM, H). UTTARAKHAND: Pithoragarh District, Satgarh-Dhawj, 2700 m, 1989, D. K. Upreti L-18438 (H, LWG). [For details of further Indian collections, see Rai et al. 2014, as C. corymbescens.] NEPAL, FARWESTERN REGION: Seti, Dadachaur, Nhuna Khola, 2700 m, 1973, P. Ozenda 147C (Gy Eh).
Discussion
Cladonia pseudofissa was included by Abbayes (1974) in C. corymbescens. He also thought that C. perfossa Nuno, described from Taiwan, belongs here. However, as discussed by Ahti & Lai (1979) and Stenroos (1988: 126), C. perfossa is probably a distinct species (podetia short and thick; a topotype re-examined in H), although still very poorly known.
Asahina (1966) clearly included the present species in C. rangiformis because he found that it contains atranorin in addition to fumarprotocetraric acid; such a strain is known in C. rangiformis. In habit C. pseudofissa does resemble C. rangiformis to some extent, but C. rangiformis is not known to range east of northwestern Iran (Litterski & Ahti 2004: 233). They are distinguished by the longitudinally split podetia and melanotic part at the base of C. pseudofissa.
The two segregated species generally differ in habit and colour, C. corymbescens being taller, erect, brownish (much resembling C. furcata (Huds.) Schrad.) while C. pseudofissa is smaller, thinner, not readily erect, and with an ashy grey or whitish tint. In addition, C. corymbescens is hardly blackening (melanotic) at base, whereas C. pseudofissa is clearly blackening.
The phylogenetic analyses by Stenroos et al. (2002) demonstrated that C. corymbescens was not closely related to the species of the C. furcata group (C. farinacea (Vain.) A. Evans, C. furcata, C. multiformis G. Merr., and C. scabriuscula (Delise) Nyl.); it seemed to be related to C. petrophila R.C. Harris and C. apodocarpa Robbins instead. The phylogenetic study of Cladonia in Thailand by Parnmen et al. (2008) grouped one specimen corresponding to C. pseudofissa with C. rudis Ahti & Parnmen. Morphologically this species is closely related to the species of C. furcata (Ahti et al. 2008). Our present phylogenetic analyses do not allow conclusions on the phylogenetic relationships of C. corymbescens and C. pseudofissa. Until further Cladonia species are included in the phylogenetic analyses, the placement of these species will remain uncertain. The ML and Bayesian analyses suggest that they might be related, but this relationship is not supported, and the branches in both analyses are very long. The MP analysis groups Cladonia pseudofissa with C. ceratophylla and C. pityrophylla, but this relationship is not well supported either. A denser sampling of taxa is needed to clarify the phylogenetic relationships between the species.
Cladonia pseudofissa comb. nov. (Southeast Asia) ... 101
As far as is known, the geographic ranges of C. corymbescens and C. pseudofissa do not overlap; C. corymbescens is primarily Australasian to Melanesian and C. pseudofissa is Himalayan in distribution. However, the Cladonia flora of Indonesia is very poorly known.
Various data support C. pseudofissa asa species distinct from C. corymbescens. The phylogenetical analyses based on two independent loci separate Cladonia pseudofissa and C. corymbescens into two well-supported clades, clades that are also supported in the single gene analyses (data not shown). According to the genealogical concordance in phylogenetic species recognition (Taylor et al. 2000), these clades represent different species. In addition, morphological and distributional differences confirm that they are two different taxa.
Acknowledgments
We are grateful for fresh material of Cladonia collected by Dr. Maarten Christenhusz (London) in New Caledonia. We are also grateful to Dr. Philippe Clerc (Geneva), who sent our herbarium (H) a large collection of Cladonia from the Himalayas. Dr. Jaana Haapala helped us in photographing. Our sincere thanks to Dr. A. R. Burgaz and Dr. M. Piercey-Normore for their valuable comments to improve the manuscript.
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ISSN (print) 0093-4666 © 2015. Mycotaxon, Ltd. ISSN (online) 2154-8889
MY COTAXON
http://dx.doi.org/10.5248/130.105 Volume 130, pp. 105-130 January-March 2015
Morphology and phylogeny of four new Lactarius species from Himalayan India
KANAD Das", ANNEMIEKE VERBEKEN’, & JORINDE NUYTINCK”?
"Botanical Survey of India, Cryptogamy Unit, Central National Herbarium, Howrah 711103, W.B., India
?Ghent University, Department of Biology, Research Group Mycology, K.L. Ledeganckstraat 35, BE 9000, Gent, Belgium
*Naturalis Biodiversity Center, Section National Herbarium of the Netherlands, PO. Box 9517, 2300RA Leiden, The Netherlands
* CORRESPONDENCE TO: daskanadbsi@gmail.com
ABSTRACT —Four new species of Lactarius are described from Himalayan India. Lactarius olivaceoglutinus, L. pyriodorus, and L. yumthangensis belong to L. subg. Piperites and L. indochrysorrheus is closely related to some representatives of L. subg. Russularia. An ITS based phylogeny confirms the phylogenetic placement of the four new species, although the monophyly of neither Lactarius subg. Piperites nor L. subg. Russularia can be confirmed. The ITS data also suggest that the Indian species are closely related to some European and American species.
Key worps — ectomycorrhizal fungi, macrofungi, Russulaceae, Sikkim
Introduction
After segregation of the well-known ectomycorrhizal milkcaps into Lactarius Pers. and Lactifluus (Pers.) Roussel (Buyck et al. 2008, 2010), Lactarius sensu novo comprises three subgenera (Verbeken & Nuytinck 2013): L. subg. Piperites (Fr. ex J. Kickx f.) Kauffman, L. subg. Russularia (Fr. ex Burl.) Kauffman, and L. subg. Plinthogali (Burl.) Hesler & A.H. Sm. Lactarius subg. Piperites can be characterized as follows: pileus sticky to slimy/glutinous (more rarely dry and shiny), often hairy and/or zonate or with watery spots; stipe dry or sticky, often scrobiculate (Heilmann-Clausen et al. 1998, Basso 1999). In contrast, representatives of L. subg. Russularia typically have dry (rarely somewhat sticky) caps and stipes and colours that are predominantly orange or various tinges of brown (Heilmann-Clausen et al. 1998).
106 ... Das, Verbeken, & Nuytinck
Sikkim, a small (0.22% of the land surface) state in India, lies in Eastern Himalaya, which is part of the Himalaya Hotspot, one of the 34 Global Biodiversity Hotspots (www.biodiversityhotspots.org). Within a very small geographical area it is substantially diverse in flora, fauna, and mycobiota, the latter seriously underexplored. During mycological expeditions to different temperate to subalpine areas of the North District of Sikkim in 2009 (KD & AV), 2011 (KD), and 2012 (KD), we collected ectomycorrhizal (ECM) fungi, with Lactarius s.l. one of the dominant ECM genera in Dombang, Shingba Rhododendron Sanctuary, and Zema. Dombang is a subalpine coniferous to mixed (coniferous & broadleaf) forest dominated by Picea spinulosa, Abies densa, Tsuga dumosa, and Larix griffithii. Shingba Rhododendron Sanctuary contains subalpine coniferous and mixed forests distributed in Yumthang valley and its adjoining areas; apart from several Rhododendron species, this protected area is dominated by Abies densa, Picea spinulosa, Tsuga dumosa, Larix griffithii, Magnolia globosa, M. campbellii, Acer pectinatum, and Betula utilis. Zema is a subalpine coniferous forest dominated by Abies densa.
Although Sikkim harbours a large number of russulaceous taxa, only 32 species (out of ca 210 taxa recorded from India) have been reported (Berkeley 1852; Das 2009; Das et al. 2010, 2013; Das & Verbeken 2011, 2012; Van de Putte et al. 2012). We describe here three new species of Lactarius subg. Piperites (L. olivaceoglutinus, L. pyriodorus, and L. yumthangensis) and one new species of L. subg. Russularia (L. indochrysorrheus).
Materials & methods
Morphological study
Macromorphological characters were described from the fresh basidiomata in daylight. Colour codes and terms follow the COLOUR IDENTIFICATION CHART OF BRITISH FuNGus FLora (Henderson et al. 1969, here prefixed by “a:”) or the METHUEN HANDBOOK OF CoLourR (Kornerup & Wanscher 1981, here prefixed by “b:”). Spore print colour codes follow Kranzlin (2005, here prefixed by “c:”).
Micromorphological structures such as basidia, hymenial cystidia, pileipellis, stipitipellis, etc. were observed from free-hand sections of dry samples mounted in a mixture of 5% KOH, 30% Glycerol, Phloxine, and Cotton Blue using an Olympus CX41 compound microscope. Spores and spore ornamentation were studied in Melzer’s reagent. Drawings were made from SEM images obtained in different magnifications. Spore measurements were calculated based on 20 basidiospores per specimen; dimensions represent minimum-mean-maximum length x minimum-mean- maximum width, and Q = length/width ratio. Herbarium names are after Holmgren et
al. (1990).
DNA extraction, PCR amplification and sequencing DNA was extracted from dried fruiting bodies according to Nuytinck & Verbeken (2003) with slight modifications (Van de Putte et al. 2010). The internal transcribed
Lactarius spp. nov. (India) ... 107
TABLE 1. Collections of Lactarius and allied species used for molecular analyses.
SPECIES
Lactarius akahatsu
L. albocarneus
alboscrobiculatus aspideus atroviridis auriolla indochrysorrheus azonites
Reis ia fot Enea
brunneoviolaceus
caespitosus camphoratus chichuensis
Ea itech
chrysorrheus
L. aff. chrysorrheus
L. citriolens
L. controversus L. crassiusculus L. cyathuliformis L. flexuosus
L. formosus L. fuliginosus L. helvus
L. lilacinus L. luridus
L. montoyae
L. necator
L. olivaceoglutinus L. pallescens
L. peckii
L. pseudouvidus
VOUCHER
Verbeken 04-141 Verbeken 98-071 Verbeken 98-080
Le 175
Walleyn 3815 Verbeken 05-306 Walleyn 1601
KD 11-002 (Holotypus) Verbeken 00-124 Verbeken 04-220 Verbeken 04-249 Walleyn 1605 Eberhardt 24.08. 04-8
Oberwinkler 46773 Wang 1236
Nuytinck 01-089 D’Hooge 08-020 Eberhardt 04.10. 02-8 Verbeken 04-212 Verbeken 05-359 Eberhardt 20.09.04-3 Verbeken 00-117
Le 369
Eberhardt 04.09.04-3 Walleyn 2136 Walleyn 3178 Eberhardt 06.09.02-1 Le 382 (Holotypus) Basso 97-24 Eberhardt 08.09. 04-1 Walleyn 3774
Taylor 2003066 Eberhardt 17.09.04-3 Eberhardt 10.10. 04-4 Berteloot 11-011 Berteloot 11-012 Taylor 2004254 Walleyn 1455
KD 1065 (Holotypus) Verbeken 04-231
KD 11-103 (Holotypus)
Nuytinck 04-020 Eberhardt 24.08. 04-14 Eberhardt 24.08. 04-13
HERBARIUM GENT
GENT
GENT
CMU, SFSU, GENT GENT
GENT
GENT
BSHC, GENT GENT
GENT
GENT
GENT
UPS
TUB HKAS GENT GENT UPS GENT GENT UPS GENT
CMU, SFSU
UPS
GENT
GENT
URS
CMU, SFSU, GENT priv. herb.
UPS
GENT
UPS
UPS
UPS
GENT
GENT
UPS GENT BSD GENT
BSHC, GENT
GENT UPS UPS
ORIGIN
Thailand France
France Thailand
Czech Rep.
USA Sweden India Belgium France France Sweden Sweden
Germany China Belgium France Italy USA USA Sweden Italy Thailand Sweden Sweden
Czech Rep.
Sweden Thailand Sweden Sweden Belgium Sweden Sweden Sweden Belgium Belgium Sweden Belgium India France India
USA Sweden Sweden
GENBANK
KF133269 KJ742389 KF241545 EF141538 KJ742390 KF133270 KF133257 KJ742391 KF241540 KJ742392 KJ742393 KJ742394 KJ742395 FJ845421 AY606945 KF475766 KJ742396 KJ742397 KF133261 KJ742398 KJ742399 DQ422003 KF241544 EF560684 KF133266 KJ742400 KJ742401 DQ421992 EF141549 JQ446111 KF133263 KF133275 KJ742402 KJ742403 KJ742404 KF241547 KJ742405 KJ742406 KJ742407 EF560673 KF133276 KJ742408 DQ974747 KF133277 KJ742409 KJ742410
108 ... Das, Verbeken, & Nuytinck
L. pubescens Eberhardt 15.09.02-2 UPS Sweden DQ421996 L. pyriodorus KD 11-027 (Holotypus) | BSHC, GENT India KJ742411 L. quieticolor Eberhardt 10.09.04-1 UPS Sweden DQ422002 L. quietus Eberhardt 16.09.04-6 UPS Sweden KF133264 L. rufus Nuytinck 02-008 GENT Norway KF241543 L. sphagneti Walter 083 TUB Germany KJ742412 L. subdulcis Vervisch 06-024 GENT Belgium KF133279 L. subplinthogalus Verbeken 04-219 GENT USA KF241539 L. subsericatus Eberhardt 11.10.04-8 UPS Sweden DQ422011 L. thyinos Voitk 23-08-04 priv. herb. Canada KF133271 L. torminosus Walleyn 3183 GENT Czech Rep. KF133281 L. trivialis Van de Putte 10-011 GENT Russia KJ742413 Walleyn 3179 GENT Czech Rep. KJ742414 Eberhardt27.08. 02-17a UPS Sweden DQ421991 L. uvidus Van de Putte 10-027 GENT Russia KF241546 Nuytinck 01-033 GENT Finland KJ742415 Walleyn 1237 GENT France KJ742388 Walleyn 2119 GENT Sweden KJ742416 Eberhardt 28.08. 02-24 UPS Sweden KJ742417 L. vietus Eberhardt 11.10.04-1 UPS Sweden KF133267 L. vinaceorufescens Nuytinck 07-018 GENT Canada KF241542 L. yumthangensis KD 11-147 (Holotypus) | BSHC, GENT India KJ742418 Lactifluus volemus Eberhardt 09.08. 04-5 UPS Sweden DQ422008 Lactifluus vellereus Eberhardt 20.09.04-22 UPS Sweden DQ422034 Gace x Buyck 02-107 PC USA DQ421984 M. zonaria Desjardin 7442 SFSU, PC, BBH Thailand DQ421990 Russula cyanoxantha Eberhardt 29.09. 02-2 UPS France DQ422033 R. nigricans Eberhardt 20.09.04-7 UPS Sweden DQ422010
spacer region of the nuclear ribosomal DNA (ITS) was amplified and sequenced using primers ITS1-F and ITS4 (White et al. 1990, Gardes & Bruns 1993). PCR amplification protocols follow Le et al. (2007); sequencing was conducted with an ABI 3730XL or ABI 3700 by MacroGEN (Amsterdam, The Netherlands). Sequences were assembled and edited with Sequencher™ v4.9 (GeneCodes Corporation, Ann Arbor, Michigan, US.A.). Specimens and sequences used in the phylogenetic analysis are shown in TABLE l.
Alignment and phylogenetic analyses
Alignment was conducted with the on-line version of MAFFTv7 (Katoh & Standley 2013), applying the E-INS-I strategy, a very slow method recommended for fewer than 200 sequences with multiple conserved domains and long gaps. The alignment was manually refined in BioEdit v7.0.9.0 (Hall 1999). Ambiguously aligned positions were detected using Gblocks v0.91b (Castresana 2000), specifying less stringent conditions than default. The minimum number of sequences for a conserved position and for a flank position was set to half the number of sequences, the number of contiguous non- conserved positions was set to 10, and the minimum length of a block after gap cleaning to 5 and positions with gaps were not treated differently from other positions. The ITS
Lactarius spp. nov. (India) ... 109
L. pallescens DQ874747 USA L. brunneoviolaceus AV04,220 FRA L. brunneoviolaceus AV04,249 FRA L. brunneoviolaceus RW1605 SWE L. brunneoviolaceus UE24.08.04.8 SWE se L. luridus AT2003066 SWE L. luridus UE17.09.04.3 SWE SrA |e. luridus UE10.10.04.4 SWE L, luridus OB11.012 BEL 88 L. luridus OB11.011 BEL L. luridus AT2004254 SWE 98] [IL iuridus RW1455 BEL L. pyriodorus KD11.027 IND L. aspideus RW3815 CZE L. pseudouvidus UE24.08.04.14 SWE egbL. pseudouvidus UE24.08.04.13 SWE L. uvidus RW1237 FRA L. wvidus RW2119 SWE 7 L. uvidus JNO1.033 FIN 92) L. uvidus UE28,08,02.24 SWE L. wvidus KVP 10.027 RUS L formosus LTH382 THA L. olivaceoglutinus KD11.103 IND L. caespitosus FJ845421 L. albocameus AV98.071 FRA 964 L. albocarneus AV98.080 FRA L. alboscrobiculatus LTH175 THA 64 L, torminosus RW3183 CZE L. pubescens UE15.09.02.2 SWE L.controversus AV00.117 ITA Ey L.quieticolor UE10.09.04.1 SWE L. akahatsu JNO4.141 THA 84 L. thyinos AV23.08.04 USA L. auriolla RW1601 SWE 0b L. citriolens UE20.09.04.3 SWE
62
L quietus UE 16.09.04.6 SWE 58 L. helws UE08.09.04.1 SWE L. chichuensis XW1236 CHI 81 L. camphoratus FO46773 GER L. cyathuliformis UE04.09.04.3 SWE L, subdulcis JV06.024 BEL 3 L. subsericatus UE11.10.04.8 SWE
86 96
7 L. sphagneti LW083 GER 8 L. rufus JNO2,008 NOR 77 86) L. chrysonheus JNO1.089 BEL 100 fl. chrysorheus ED08.020 FRA 73 Lchrysortheus UE04.10.02.8 ITA = 100 L. aff. chrysormheus AVO4.212 USA L. aff. chrysomheus AVO5.359 USA 63|7—L. indochrysorrheus KD11.002 IND 64 L. vinaceorufescens JNO7.018 CAN L. peckii JNO4.020 USA L. yumthangensis KD11.147 IND L, fexuosus RW2136 SWE L flexuosus RW3178 CZE SIL. flexuosus UE0S.09.02.1 SWE L. trivalis UE27.08.02-17a SWE L. trivalis KVP10.010 RUS L. trivialis RW3179 CZE
83
100! L lilacinus RW3774 BEL L. atroviridis AVO5.306 USA L. necator AV04.231 FRA L. vetus UE11.10.04.1 SWE 100 ‘L. azonites AVO0.124 BEL 97) L. fuliginosus MTB97-24 SWE L. subplinthogalus AV04,219 USA
77] 76 L. montoyae typus IND L. crassiusculus LTH369 THA 100, 'M. ochricompacta BB02.107 USA M. zonaria DED7442 THA R. cyanoxantha UE29.09.02.2 SWE cy R nigricans UE20.09.04.7 SWE LE. volemus 09.08.04.5 SWE % Lt vellereus UE20.09.04-22 SWE
“1 0.05
PLATE 1. The obtained ML topology based on ITS sequences of Lactarius, Multifurca, Russula, and Lactifluus species. Bootstrap values >50% are indicated. Names in bold are the new Indian species described in this paper. The scale bar represents the number of nucleotide changes per site.
sequences were partitioned into 5 partitions: the ribosomal genes 18S, 5.88, and LSU and the spacer regions ITS1 and ITS2.
Maximum Likelihood (ML) analysis was performed in RAXML v7.0.3 (Stamatakis 2006), combining a ML search with the Rapid Bootstrapping algorithm for 1000 replicates. The model GTRGAMMA was estimated for each partition separately.
Results
Phylogeny
PLaTE 1 shows the obtained ML topology with bootstrap support (BS) values >50% displayed. The tree shows a well-supported genus Lactarius (97% BS). Within Lactarius, L. subg. Plinthogali receives a high support value (100% BS), but monophyly of neither Lactarius subg. Piperites nor L. subg. Russularia (sensu Heilmann-Clausen et al. 1998) is supported. Lactarius pyriodorus, L. olivaceoglutinus, and L. yumthangensis are closely related to species
110 ... Das, Verbeken, & Nuytinck
traditionally placed in L. subg. Piperites. Lactarius indochrysorrheus is closely related to the European L. chrysorrheus Fr. and the North American L. vinaceo- rufescens A.H. Sm. and L. aff. chrysorrheus, which are assigned to L. subg. Russularia in morphology-based taxonomy.
Species delimitation for this paper is mainly based on morphological features. We were not able to include enough specimens/sequences from the newly proposed species to test species delimitation in the phylogenetic tree.
Taxonomy
Lactarius indochrysorrheus K. Das & Verbeken, sp. nov. PLATES 2, 3, 6A,B,E MycoBank MB804887
Differs from Lactarius chrysorrheus by its more viscid pileus with its pileipellis an ixocutis covered by a thick gelatinous layer.
Type: India. Sikkim: North District, Dombang, 27°43’35.2”N 88°45’15.2”E, alt. 2920 m, 18.VIII.2011, K. Das, KD 11-002 (Holotype, BSHC; isotype, GENT).
EryMo_oey: an Indian look-alike of the European Lactarius chrysorrheus.
PiLEus 25-70 mm diam., convex with slightly pubescent and inrolled margin when young, gradually planoconvex to applanate with slightly depressed centre, rarely with a central papilla, sometimes becoming widely infundibuliform; margin decurved with maturity, becoming very irregularly wavy; surface smooth to greasy, viscid (sticky), shiny, brownish orange, pinkish buff to salmon (a: 45) or orange (b: 6A6) (cinnamon (a: 10) to somewhat rusty after maturity), gradually apricot cream to paler up to yellow (a: 5E), towards margin distinctly zonate with several zones over the whole diameter but most dense in the center; zones consisting of darker and watery spots; margin very faintly and shortly striate. LAMELLAE subdecurrent, crowded (18-22/cm at pileus margin), pale yellow (a: 4D) when young, gradually pink-spotted, finally becoming brown to reddish brown, with lamellulae in 9 series; edge entire, concolorous. STIPE 45-65 x 6-8.5 mm, slender, subcylindrical to cylindrical or slightly widened towards base; surface smooth, slightly greasy, strigose (hairy) at base, much paler than the pileus, especially in the upper part (very pale pinkish), very pale salmon to vinaceous, gradually darker up to rust to rusty tawny or pale brick red. CONTEXT hollow in stipe, pinkish yellow (a: 4D) to pale salmon, turning lemon yellow (a: 54) to greenish yellow (a: 57) after cut, changing to salmon (a: 45) with FeSO, and greenish with Guaiac. LaTex abundant, white, turning quickly to greenish yellow. Taste first mild, then becoming bitter and acrid. Opour not distinctive. SPORE PRINT pale cream (c: 10 Y).
BASIDIOSPORES 6.4—7.4-8.5 x 5.6-6.2-6.9 um, (Q = 1.08-1.18-1.33), subglobose to ellipsoid; ornamentation amyloid, <0.9 um high, composed of conical to spine-shaped warts, with short or medium, irregular to regular
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PLATE 2: Lactarius indochrysorrheus (Holotype KD 11-002). A. Fresh basidiomata showing lamellae and lamellulae. B. Basidiospores. C. Marginal cells. D. Basidia. Scale bars: B = 5 um; C, D = 10 um.
112 ... Das, Verbeken, & Nuytinck
ridges which are aligned or connected and forming a partial to incomplete reticulum; some isolated small warts present; plage sometimes distinct and amyloid. Basrp1a 30-44 x 9-13 um, 4-spored, subclavate to ventricose; sterigmata 2.5-4 x 1.5-2 um. PLEUROMACROCYSTIDIA 32-70 x 7.5-11 um, fairly abundant, emergent <10-30 um, narrowly clavate, cylindric with tapered apex or fusoid, often with mucronate to subcapitate apex, slightly thick-walled (wall <0.5 um); content refractive. PLEUROPSEUDOCYSTIDIA filamentous, 3-5 um wide. LAMELLAR EDGE fertile with basidia, cystidia and marginal cells. CHEILOMACROCYSTIDIA 35-55 x 8-9 um, moderately abundant, subclavate to narrowly clavate or ventricose to fusoid sometimes with mucronate apex; content refractive. CHEILOPSEUDOCYSTIDIA filamentous. MARGINAL CELLS 11-20 x 7-11 um, mostly clavate to subclavate, often multiseptate. HYMENOPHORAL TRAMA with lactifers. PILEIPELLIS an ixocutis, <170 um thick, with gelatinous layer mostly extended 10-15 um beyond the hyphal layer; hyphae repent to suberect, <3.5 um wide, branched, septate. STIPITIPELLIS an ixocutis, composed of repent hyphae mostly in parallel pattern; hyphae <3.5 um wide, branched, septate. STIPE TRAMA mostly with numerous nested sphaerocytes. CLAMP CONNECTIONS absent.
EcoLocy & DISTRIBUTION — Gregarious under Abies densa and Picea spinulosa in subalpine coniferous or mixed (coniferous and broadleaf) forest. August. Fairly common.
ADDITIONAL SPECIMENS EXAMINED: INDIA. SIKKIM: North District, Dombang, 27°43'35.2”N 88°45'15.2’E, alt. 2920 m, 14.VIII.2009, A. Verbeken, K. Das & K.V. Putte, AV-KD-KVP 032 (BSHC, GENT); North District, Shingba Rhododendron sanctuary, 27°43'42.1”N 88°44'58.8”E, alt. 2889 m, 24.VIII.2011, K. Das, KD 11-082 (BSHC); 27°44’03.5”N 88°44’23.3’E, alt. 3208 m, 26.VIII.2012, K. Das, KD 11-107 (BSHC). Notes — Zonate species with similar cap colours and milk that stains quickly sulphur to greenish yellow are European L. chrysorrheus, North American L. vinaceorufescens, and an undescribed American species [labeled L. aff. chrysorrheus in the phylogenetic tree and TABLE 1]. ITS sequence comparison suggests the new species described here is close to the three species but not conspecific.
Lactarius chrysorrheus is less sticky, which is reflected in the pileipellis structure, a loosely interwoven cutis without distinct slime layer (Heilmann- Clausen et al. 1998). Lactarius vinaceorufescens differs by a pileipellis with