Mycologia, 104(6), 2012, pp. 1433–1442. DOI: 10.3852/11-338 # 2012 by The Mycological Society of America, Lawrence, KS 66044-8897 A new species of Melanospora on truffles from China Li Fan the Melanospora complex, and the nonsporulating Cheng-lin Hou1 Papulaspora was demonstrated to represent sterile College of Life Science, Capital Normal University, Melanospora ascomata by Davey et al. (2008). Xisanhuanbeilu 105, Haidian, Beijing 100048, China Melanospora and its relatives were placed tradition- Paul F. Cannon1 ally within the Ceratostomataceae (Sordariales) based CABI and Royal Botanic Gardens, Kew, Richmond, primarily on morphological parallels with the Chaeto- Surrey TW9 3AB, United Kingdom miaceae (e.g. Hawksworth et al. 1995). The first molecular phylogenetic studies (Rehner and Samuels Yu Li 1995, Jones and Blackwell 1998) indicated that Institute of Mycology, Jilin Agricultural University, Melanospora belonged within the Hypocreales clade. Changchun 130118, China Zhang and Blackwell (2002) and Chaudhary et al. (2006) considered the Melanospora clade to be a sister group of the Hypocreales and found that sequences Abstract: The new species Melanospora subterranea is derived from other genera placed in the Ceratostoma- described from China, based on morphological and phylogenetic analyses. This is the first record of taceae (syn. Melanosporaceae) clustered within the main Melanospora species parasitizing Chinese black and Hypocreales clade. Zhang et al. (2006) concluded that white truffles (Tuber indicum and T. huidongense), the family belonged to the Hypocreomycetidae (i.e. the and its host range indicates that the new species clade containing the Coronophorales, Glomerellales, Halo- might be a disease threat to commercially exploited sphaeriales, Hypocreales, Microascales) but that it was a European truffles including Tuber melanosporum and sister group of the Coronophorales and merited recogni- Tuber magnatum. Apparent polyphyly within the tion as the new order Melanosporales.Theorderwas Ceratostomataceae can be explained at least in part published formally by Hibbett et al. (2007). A similar by inadvertent sequencing of the host fungus instead placement was proposed by Schoch et al. (2007) in a of the parasite. study of marine fungal lineages within the Hypocreomy- Key words: fungal parasitism, phylogeny, truffles cetidae. However, the apparent paraphyly of the order Melanosporales to date has not been addressed. During our studies on the genus Tuber (i.e. the true INTRODUCTION truffles) in China, an interesting microfungus be- The Melanospora complex, a distinctive group of longing to the genus Melanospora was found within microfungi with translucent ascomata, was revised by ascomata of T. indicum (the Chinese black truffle) Cannon and Hawksworth (1982) based primarily on when we cut the fruit body into slices, with the data obtained by SEM examination of the ascospores. parasitized ascoma showing densely crowded pin-like They adopted relatively narrow generic concepts, black dots that occupied nearly all the internal space accepting Sphaerodes as emended by von Arx (1981), of the tissues. The same fungus also was found in and erected new genera including Persiciospora with ascomata of T. huidongense, an endemic species of pitted ascospores. Cannon and Hawksworth (1982) Chinese white truffle. arranged the species with smooth-walled, lemon- shaped to ellipsoidal or ellipsoidal-citriform asco- MATERIALS AND METHODS spores and sunken germ pores in Melanospora, while those with reticulate ornamentation on the ascospore Morphological studies.—Macroscopic characters were de- surface and raised tubercle-like germ pores were scribed both from fresh and rehydrated dried specimens placed in Sphaerodes. Further species were added to and microscopic characters from razorblade sections the complex by Garcı´a et al. (2002, 2004), Hawks- mounted in 3% KOH (w/v), Melzer’s reagent and 0.1% (w/v) cotton blue in lactic acid. For scanning electron worth et al. (1999), Krug (1988), Nitzan et al. (2004), microscopy (SEM), spores were scraped from the dried host Stchigel et al. (1996, 1999) and Vujanovic and Goh gleba onto doubled-sided tape, which was mounted directly (2009). Chaudhary et al. (2006) published the new on an SEM stub and coated with gold/palladium. The genus Vittatispora from India, which also belongs to treated materials were examined and photographed with a Hitachi S-4800 SEM. Submitted 13 Oct 2011; accepted for publication 2 May 2012. 1 Corresponding authors. E-mail: [email protected] & houchenglincn Molecular methods.—Total genomic DNA was extracted @yahoo.com from ascomata with PeqLab E.Z.N.A._Fungal DNA kit 1433 1434 MYCOLOGIA following the manufacturer’s protocol after the samples support was found for clades containing Persiciospora were crushed by shaking for 3 min at 30 Hz (Mixer Mill MM and Fusarium (Gibberella) species, Syspastospora para- 301; Retsch, Haan, Germany) in a 1.5 mL tube together with sitica and Isaria farinosa, and Sphaerodes retispora and one 3 mm diam tungsten carbide ball. The large subunit Trichoderma harzianum. Scopinella solani clustered (LSU) region of the nuclear ribosomal DNA (rDNA) was weakly with species of Myrothecium, and a further well amplified with PCR with primers LROR/LR5 (White et al. supported clade (Clade 1 in FIG.1)contained 1990). PCR was performed in 50 mL reactions containing DNA template 4 mL, primer (10 mM) 2 mL each, 2 3 Master sequences derived from Melanospora species but with Mix (Tiangen Biotech Co. Ltd., Beijing) 25 mL. PCR only weak support for a sister group containing reactions were run as follows: initial denaturation at 95 C Cosmospora species. for 4 min, followed by 32 cycles at 94 C for 1 min, 58 C for 1 min, 72 C for 1 min and a final extension at 72 C for TAXONOMY 10 min. The PCR products were sent to Invitrogen Biotechnology Co. Ltd. (Beijing) for purifying, sequencing Melanospora subterranea L. Fan, C.L. Hou, P.F. and editing. The other sequence data of LSU rDNA Cannon & Y. Li, sp. nov. FIGS. 2–7 included in this study were downloaded from GenBank MycoBank MB563464 (TABLE I). Ascomata globosa, immersa, 200–250 mm diam, non- Phylogenetic analyses.—DNA sequences were aligned with ostiolata, leviter vel opace fulva, perlucida; peridium MUSCLE (Edgar 2004), within the MEGA5 phylogenetic membranosum, ex cellis fuscis, perlucidis (5–)7.5–15 mm software package (Tamura et al. 2011). The aligned dataset diam. Asci clavati, 45–60 3 15–20 mm, 2-spori. Ascosporae was analyzed with maximum parsimony (MP) and maxi- 15–22.5 3 10–15(–17.5) mm, ellipsoideae-citriformes, laeves. mum likelihood (ML) analyses, also within the MEGA5 Type: CHINA, YUNNAN Province, Kunming, in package, with heuristic searches using close neighbor tissue of Tuber indicum, 23 Oct 2007, Jin-Zhong Cao interchange (CNI) on 10 initial random trees. Gaps and 121, FAN1001 (BJTC holotype, K[M] 172128 isotype). missing data were taken into consideration where . 95% Other collections examined: CHINA, YUNNAN, Baoshan, in unambiguity was encountered. One thousand bootstrap tissue of Tuber huidongense, 15 Dec 2008, Li Fan, FAN1002 replications were used. A sequence derived from Chaeto- (BJTC). sphaerella phaeostroma (AY695264) was used as outgroup. Etymology: referring to the environment of the host species. RESULTS Stromata absent. Ascomata growing in the internal tissues of Tuber ascomata, 200–250 mm diam, solitary Phylogenetic analyses.—A total of 250 characters out or gregarious, immersed, globose, cleistothecial, of 864 were found to be parsimony informative. translucent, light brown to mid brown, appearing Maximum parsimony analysis resulted in one of 20 black when the ascospores are mature. Peridium most parsimonious trees with a length (TL) of 652 membranous, composed of hyaline or light brown, steps, consistency index (CI) of 0.619632, retention translucent, polygonal to irregular pseudoparenchy- index (RI) of 0.890845 and rescaled consistency matous cells (5–)7.5–15 mm diam. Interascal tissues index (RCI) of 0.551996 (for all sites). The nrLSU absent. Asci clavate, 45–60 3 15–20 mm, thin-walled, matric is available at www.treebase (http://purl.org/ short-stalked, without apical structures, evanescent, phylo/treebase/phylows/study/TB2:S12403) 2-spored. Ascospores 15–22.5 3 10–15(–17.5) mm, The phylogenetic analysis (FIG. 1) revealed two ellipsoidal-citriform, both ends slightly apiculate, strongly supported major clades representing the guttulate and dark brown to near black at maturity, Hypocreales and the Melanosporales. The latter clade smooth-walled, with two indistinct apical germ pores. was represented by sequences from 12 taxa, with Anamorph not seen. Vittatispora coorgii included as a basal lineage within the Melanosporales, and two strongly supported DISCUSSION subclades containing four and five sequences respec- tively. These subclades did not appear to correlate The new species described here, with its translucent exactly with taxa as defined with morphological ascocarps and smooth-walled ellipsoidal-limoniform methods. The new species, M. subterranea, clearly spores, clearly belongs to Melanospora, and the LSU could be demonstrated as belonging within the rDNA analysis (FIG. 1) confirms this placement. Two Melanosporales clade but did not definitely cluster similar species of that genus have cleistothecial within a particular subclade. ascomata (TABLE II). Microthecium geoporae (Oberm.) Sequences derived from taxa considered as belong- Ho¨hn. (basionym Guttularia geoporae Oberm.)