Mycologia, 102(4), 2010, pp. 881–886. DOI: 10.3852/09-050 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897
Bondarzewia podocarpi, a new and remarkable polypore from tropical China Yu-Cheng Dai
confirm the affinity of the new taxon and infer evolutionary relationships among Bondarzewia species phylogenetic analyses were carried out based on ITS sequence data.
Institute of Microbiology, P.O. Box 61, Beijing Forestry University, Beijing 100083, China
Bao-Kai Cui1 Institute of Microbiology, P.O. Box 61, Beijing Forestry University, Beijing 100083, China
MATERIALS AND METHODS
Xiao-Yong Liu
Morphological studies.—Specimens are deposited at the Herbarium of the Institute of Applied Ecology, Chinese Academy of Sciences (IFP), and the Institute of Microbiology, Beijing Forestry University (BJFC). Sections were studied at magnification up to 10003 with a Nikon Eclipse E600 microscope and phase contrast illumination. Drawings were made with a drawing tube. Microscopic features, measurements and drawings were made from slide preparations stained with cotton blue and Melzer’s reagent. Spores were measured from sections cut from the tubes. In presenting variation in spore size 5% of measurements was excluded from each end of the range and given in parentheses. In the text we used these abbreviations: IKI 5 Melzer’s reagent, IKI2 5 negative in Melzer’s reagent, KOH 5 5% potassium hydroxide, CB 5 cotton blue, CB+ 5 cyanophilous, L 5 mean spore length (arithmetic average of all spores), W 5 mean spore width (arithmetic average of all spores), Q 5 variation in the L/W ratios between the specimens studied, n 5 number of spores measured from given number of specimens. Color terms are from Petersen (1996) and Anonymous (1969).
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
Abstract: Bondarzewia podocarpi is described as new and is distinguished from other species in the genus by regular pores, presence of cystidioles, thin generative hyphae and short basidia. It was found in tropical China and grows on living trees of Podocarpus. Parsimony analyses were applied to an ITS dataset. The results suggest that Bondarzewia is monophyletic with two well supported internal clades. One clade includes B. podocarpi and B. guaitecasensis, while the other includes B. berkeleyi and B. montana. Based on studied materials of Bondarzewia from the world, an identification key to the species of the genus is provided. Key words: Bondarzewia, ITS, phylogeny, polypore, Russulales, taxonomy
Molecular techniques.— DNA was extracted from dried herbarium materials following the methods of Gardes and Bruns (1993), Yao et al. (1999), Liu et al. (2001) and Cui et al. (2008a) with some modifications. About 10–30 mg dried specimens or 10 mg dried mycelia were ground in liquid nitrogen and mixed well with 600 mL prewarmed 2.5% CTAB extraction buffer containing 100 mM Tris (pH 8.0), 20 mM EDTA (pH 8.0), and 1.4 mol/L NaCl, followed by a water bath at 65 C for 50–80 min. A total of 600 mL chloroform-isoamyl alcohol (24 : 1 volume) was added to precipitate unwanted cell components and proteins. A total of 30 mL 3 M NaAc was added to the aqueous phase followed by 200 mL cold (220uC) isopropanol to precipitate DNA. Precipitated DNA was collected by centrifuge, washed with 500 mL ice-cold 70% ethanol, dried, then dissolved in 50 mL sterile distilled water and stored at 220 C. PCR amplifications follow the methods of Cui et al. (2008a) and Dai et al. (2008). Primers for PCR amplification were ITS4 and ITS5 described by White et al. (1990). A total of 50 mL reaction mixtures for PCR included genomic DNA in optimum concentration, 0.5 mM of each primer, 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM magnesium chloride (MgCl2), 200mM each of dATP, dCTP, dGTP and dTTP (Promega Co., USA), and 2 units Taq DNA polymerase (Sangon Ltd., Canada). PCR was performed in a Mastercycler (Eppendorf Inc., Germany) for 35 cycles,
INTRODUCTION
Although the taxonomy and diversity of poroid fungi in continental China have been studied recently (e.g. Cui and Dai 2006; Cui et al. 2007, 2008a, b; Dai and Cui 2005; Dai et al. 2007; Wei and Dai 2006; Zheng and Liu 2008), the polypore flora in tropical insular China, particularly of Hainan Island, is incomplete. This paper is a continuation of a series of studies on wood-decay fungi from southern China (Cui and Dai 2008; Dai et al. 2003, 2004a, b). Genus Bondarzewia Singer is characterized by strongly amyloid and ornamented basidiospores and is derived within the Russuales (Redhead 1993). Three species in the genus were recorded previously (Gilbertson and Ryvarden 1986; Ryvarden and Gilbertson 1993; Singer 1953, 1964). In studies of polypores from Hainan Island we found an undescribed species of Bondarzewia growing on Podocarpus trees. It is proposed here as Bondarzewia podocarpi. To Submitted 4 Feb 2009; accepted for publication 29 Dec 2009. 1 Corresponding author. E-mail:
[email protected]
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882 TABLE I.
MYCOLOGIA Species and sequences database accession numbers used in this study Species name
Sample number
Locality
Bondarzewia berkeleyi (Fr.) Bondartsev & Singer Bondarzewia guaitecasensis (Henn.) J.E. Wright Bondarzewia montana (Que´l.) Singer Bondarzewia podocarpi Y.C. Dai Bondarzewia podocarpi Y.C. Dai Fibroporia radiculosa (Peck.) Parmasto Fibroporia radiculosa (Peck.) Parmasto Heterobasidion australe Y.C. Dai & Korhonen Heterobasidion australe Y.C. Dai & Korhonen Heterobasidion australe Y.C. Dai & Korhonen Heterobasidion linzhiense Y.C. Dai & Korhonen Heterobasidion linzhiense Y.C. Dai & Korhonen Heterobasidion parviporum Niemela¨ & Korhonen Oxyporus subpopulinus B.K. Cui & Y.C. Dai Oxyporus subpopulinus B.K. Cui & Y.C. Dai Wrightoporia luteola B.K. Cui & Y.C. Dai
Li 1097 Mario 11898 — Dai 9261 Dai 10660 Dai 6473 Cui 2797 Dai 99002/1 Dai 02048/3 Dai 04171/1 Dai 04020/6 Dai 04021/4 Cui 2234 Cui 2313 Cui 2251 Dai 7221
China Argentina — China China China China China China China China China China China China China
a
GenBank number FJ644288 FJ644287 DQ200923a FJ644286 FJ644285 FJ644283 FJ644284 FJ627224 FJ627223 FJ627205 FJ627202 FJ627204 FJ627232 FJ644281 FJ644282 FJ644289
Sequence downloaded from GenBank.
including 94 C for 35 s, 55 C for 45 s and 72 C for 45 s. PCR products used for sequencing were cleaned with QIAquick PCR purification columns (QIAGEN Ltd., UK). DNA sequences were determined by direct sequencing with GeneCore Biotechnologies (Shanghai, China). Sequences were edited with Sequencher 3.1 (Gene Codes Corp., Ann Arbor, Michigan) and deposited in GenBank (TABLE I). Phylogenetic analyses.—We sequenced 15 taxa of Bondarzewia and related species. One sequence of Bondarzewia montana (Que´l.) Singer was downloaded from GenBank (Matheny et al. 2007). (Taxa used in the phylogenetic analyses are listed in TABLE I.) One dataset was prepared based on ITS sequences of 16 taxa (10 species), among which five represent four Bondarzewia species. DNA sequences were aligned with Clustal X using default settings (Thompson et al. 1997) and further adjusted manually in the data editor of PAUP* 4.0b10 (Swofford 2002). Parsimony analyses were performed with equally weighting of characters and transformations with gaps treated as missing data in PAUP*. Heuristic searches were performed with 1000 replicate searches, each with random taxon addition, MAXTREES set to auto-increase, and TBR branch swapping. Robustness of individual branches was estimated by maximum parsimony bootstrap proportions (BP) with 500 replicates. Each bootstrap replicate consisted of a single heuristic search with 50 random taxon addition replicates, MAXTREES set to auto-increase, and TBR branch swapping. Phylogeny reconstructions also were conducted with Bayesian inference with Metropolis-coupled Markov chain Monte Carlo techniques implemented by MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003). The best-fit model of nucleotide substitution (GTR+G), which was selected by hierarchical likelihood ratio tests (Huelsenbeck and Crandall 1997, Posada and Crandall 2001) implemented in MrModeltest 2.2 (Nylander 2004; Posada and Crandall 1998), was used in MrBayes 3.1.2 for phylogeny inferences. Two independent Bayesian
analyses were run simultaneously, each with one cold chain and three heated chains and with temperature set to 0.1, each initiated with a random tree, and flat prior. For each run one out of every 100 trees was sampled for 100 000 generations. The first 500 sampled trees from the two runs were discarded before summarizing the information, and the remaining 1500 sampled trees were used to calculate posterior probabilities which were considered statistically significant when P value was no less than 0.95. Convergence of the runs was diagnosed by the average standard deviation less than 0.01 and the potential scale reduction factor approaching 1 (Gelman and Rubin 1992). TAXONOMY
Bondarzewia podocarpi Y.C. Dai & B.K. Cui, sp. nov. FIGS. 1, 2 MycoBank MB515217 Carpophorum annuum, pileatum; facies pororum cremea; pori rotundi vel angulati 1–3 per mm. Systema hypharum dimiticum, hyphae generatoriae sine fibulis, hyphae skeletales contexti 2.5–5 mm diam. Sporae subglobosae, hyalinae, echinatae, amyloideae, CB+, 5.6–7.5 3 5.1– 6.5 mm.
Type. China. Hainan Province, Ledong County, Jianfengling Nature Reserve, on living tree of Podocarpus imbricatus, 16.XI.2007 Dai 9261 (holotype in IFP, isotype in BJFC and H). Etymology. podocarpi (Lat.): referring to the tree genus Podocarpus. Fruit body.—Basidiocarps annual, pileate, broadly attached to the substrate, solitary or a few imbricate, when fresh soft corky and watery, without odor or flavor, becoming hard corky to woody hard after drying. Pileus semicircular, projecting up to 7 cm,
DAI ET AL.: BONDARZEWIA PODOCARPI
FIG. 1. Microscopic structures of Bondarzewia podocarpi (drawn from the holotye). A. Basidiospores. B. Basidia and basidioles. C. Cystidioles. D. Hyphae from trama. E. Hyphae from context.
9 cm wide and 2 cm thick at base; margin acute. Pileal surface white to cream when juvenile, becoming rust brown from base to center on aging, azonate, glabrous; margin white when fresh, becoming brick when dry. Pore surface cream when fresh, becoming yellowish brown when dry; pores round to angular, 1– 3 per mm, mostly 2 per mm; dissepiments thin, entire to slightly lacerate. Context pale buff and woody hard when dry, up to 1 cm thick; tubes buff and hard corky when dry, up to 2 mm long. Hyphal structure.—Hyphal system dimitic to trimitic; generative hyphae simple septate; skeleto-binding hyphae IKI2, CB+; all hyphae unchanged in KOH. Context.—Contextual generative hyphae scanty, hyaline, thin-walled, 2–3 mm diam; skeletal hyphae dominant, hyaline, thick-walled with a narrow lumen to subsolid, frequently branched, flexuous, strongly interwoven, 3–7 mm diam (n 5 40/2); binding hyphae dominant, strongly flexuous, thick-walled, subsolid. Tubes.— Tramal generative hyphae hyaline, thinwalled, occasionally branched, interwoven, 2–3 mm
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FIG. 2. Basidiocarp of Bondarzewia podocarpi. A. Upper surface of B. podocarpi. B. Poroid surface of B. podocarpi. Bar: A 5 1 cm, B 5 1 cm.
diam; skeletal hyphae dominant, thick-walled with a narrow lumen to subsolid, occasionally branched, flexuous, interwoven, 2.5–3 mm diam (n 5 40/2). Cystidia absent; cystidioles present, subulate, hyaline, thin-walled, 20–23 3 6–6.5 mm. Basidia clavate, with a basal simple septum and four sterigmata, 20–30 3 7.2–9 mm; basidioles in shape similar to basidia but slightly smaller. Spores.—Basidiospores subglobose to broadly ellipsoid, hyaline, thick-walled, strongly echinulate, amyloid, CB+, (5.3–)5.6–7.5(–8) 3 (4.9–)5.1–6.5(–7) mm, L 5 6.55 mm, W 5 5.81 mm, Q 5 1.12–1.13 (n 5 90/3). Known distribution. Hainan Island, Hainan Province, China. Additional specimens examined (paratypes): CHINA. Hainan Province, Lingshui County, Diaoluoshan Nature Reserve, on base of living Podocarpus imbricatus, 8.XII.2008 Dai 10660 (BJFC). Ledong County, Jianfengling Nature Reserve, on living tree of Podocarpus imbricatus, 12.XII.2008 Dai 10662 & 10669 (BJFC).
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MYCOLOGIA
FIG. 3. Parsimony consensus tree illustrating the phylogeny of Bondarzewia podocarpi and related species based on ITS region sequence data. Parsimony bootstrap (before the slash markers) and Bayesian posterior probability (after the slash markers) less than 50% are not shown.
DISCUSSION
Bondarzewia podocarpi resembles B. berkeleyi (Fr.) Bondartsev & Singer and B. montana (Que´l.) Singer, but the latter two species occur in temperate areas (Gilbertson and Ryvarden 1986, Ryvarden and Gilbertson 1993). Bondarzewia berkeleyi grows mostly on trees of the Fagaceae and B. montana usually on the Pinaceae, whereas B. podocarpi occurs in Old World tropics on Podocarpaceae. Macroscopically B. podocarpi has a single pileus or few pilei while B. berkeleyi bears an imbricate cluster of basidiocarps. Bondarzewia montana can be readily distinguished from B. podocarpi by its purplish brown upper surface. Microscopically B. podocarpi differs from these other two species in the genus by having cystidioles, thinner generative hyphae (2–3 mm diam) and shorter basidia (20–30 mm). Bondarzewia berkeleyi and B. montana lack cystidioles, and their generative hyphae are slightly thicker (3–5 mm diam) and basidia longer (33–45 mm). In addition basidiospore ridges of B. berkeleyi are mostly longer than 1 mm while they are less than 1 mm in B. podocarpi. Bondarzewia guaitecasensis (Henn.) J.E. Wright was described from South America, growing on Nothofagus (Singer 1953, 1964). Bondarzewia podocarpi is similar to B. guaitecasensis by sharing short basidiospore ridges (less than 1 mm). However B. guaitecasensis differs in having larger pores, 1–2 per mm,
mostly 1 per mm, and its hyphae in mass are weakly amyloid. Its basidiospores are subglobose to broadly ellipsoid and larger, (7.8–)8–9.2(–10) 3 (7–)7.1– 8.2(–8.3) mm, L 5 8.69 mm, W 5 7.45 mm, Q 5 1.17 (n 5 30/1). In addition its basidia are longer, 35–40 um, and no cystidioles are present in its hymenia. Relationships among Bondarzewia species were investigated with ITS region from five taxa. The dataset had an aligned length of 854 bp with 286 parsimony informative positions. Two clades were recognized within Bondarzewia species (FIG. 3). The new species, B. podocarpi, forms a distinct and well supported clade with B. guaitecasensis (BP 5 93%). Another clade consists of B. berkeleyi and B. montana (BP 5 100%). The posterior probabilities of each clade constructed by Bayesian inferences were integrated into the consensus tree (FIG. 3) generated by parsimony analyses because Bayesian and parsimony analyses resulted in the same tree topology. KEY TO KNOWN SPECIES OF BONDARZEWIA IN THE WORLD
1.
Basidiocarps usually pileate or with a rudimentary stipe-like base, pores mostly 2 per mm, dissepiment edges even; cystidioles present, basidia , 35 mm long; known from tropical China on Podocarpaceae . . . . . . . . . . . B. podocarpi Y.C. Dai & B.K. Cui 19. Basidiocarps usually stipitate, pores mostly 1 per mm, dissepiment edges lacerate; cystidioles absent,
DAI ET AL.: BONDARZEWIA PODOCARPI
2.
29. 3.
39.
basidia . 35 um long; north or south temperate in distribution on Pinaceae or Fagaceae . . . . . . . . . . 2 Basidiospores with short and blunt spines, spines , 1 mm long; known only from southern South America on Nothofagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. guaitecasensis (Henn.) J.E. Wright Basidiospores with long and sharp spines, spines . 1 mm long; north temperate in distribution . . . . 3 Basidiocarps in large imbricate clusters at base of oaks, pileus ochraceous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. berkeleyi (Fr.) Bondartsev & Singer Basidiocarps mostly from a single stipe at base of conifer trees, pileus purplish brown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. montana (Que´l.) Singer
Other specimens studied: Bondarzewia berkeleyi. China. Henan Prov., Neixiang County, Baotianman Nat. Res., root of living Quercus, 24.VIII.2006 Li 1108 & 1097 (IFP). Xizang Auto. Reg., Linzhi County, base of living Quercus, 2.VIII.2004 Dai 5575 (IFP). B. montana. China. Xizang Auto. Reg., Lulang County, base of living Abies, 9.VIII.2004 Yu 56 (IFP). Yunnan Prov., Lijiang County, Yulongxueshan, Yunshanping, ground in forest of Picea, 3.IX.2006 Yuan 2187 (IFP). Czech Republic. Moravia, Area tuta ‘‘Velky´ Sˇpicˇa´k’’, on Abies, 8.VIII.1989 Vampola (H). Germany. Bayern, Bo¨ hmerwald, Zwiesel, Zwieslerwadhaus, Natuschutzgebiet, Mittelsteighu¨tte, on base of fallen Abies (H). USA. California, Calaveras, Calaveras Big Tree State Park, on root of dead Pinus (H). Washington, Olympic Peninsula, Sole duck River, fallen trunk of Tsuga, Lowe 7887 (H). B. guaitecasensis. Argentina. RioNegro, Parque Nacional Nahuel Huapi, Lago Frias, track to Puerto Blest, on base of living Nothofagus dombeyi, 7.V.1999 Mario 11898 (BJFC).
ACKNOWLEDGMENTS
We thank Dr Mario Rajchenberg (Centro Forestal CIEFAP, Argentina) for forwarding specimens for study. Yu-Cheng Dai thanks Dr Tuomo Niemela¨ (Helsinki, Finland) for support while working at the Botanical Museum of the University of Helsinki. The study is supported by the National Natural Science Foundation of China (project No. 30900006) and Beijing Forestry University (project No. BLYX200912).
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