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392 Persoonia – Volume 45, 2020

Tolypocladium flavonigrum Fungal Planet description sheets 393

Fungal Planet 1175 – 19 December 2020 Tolypocladium flavonigrum Noisripoom, Tasanathai, Khonsanit & Luangsa-ard, sp. nov. Etymology. Named after the colour of fresh stromata, from the Latin ‘flavo’ The results of our phylogenetic study using LSU, tef1 and rpb1 meaning yellow, and ‘nigrum’ meaning black. sequences clearly separates T. flavonigrum from other species. Classification — Ophiocordycipitaceae, Hypocreales, Sorda Based on a megablast search of NCBIs GenBank nucleotide riomycetes. database, the LSU sequence of Tolypocladium flavonigrum had the highest similarity to T. japonicum (strain OSC 110991, Gen- Single or multiple stromata emerging directly from the ground Bank DQ51876.1; Identities = 850/908 (94 %), 33 gaps (3 %)), growing on unidentified Elaphomyces sp., clavate, 15–30 mm the closest hits using the tef1 sequence are T. japonicum (strain long, 1–2 mm wide, yellowish green when immature, black OSC 110991, GenBank DQ522330.1; Identities = 880/921 when mature. Terminal part of the stroma fertile, capitate, (96 %), no gaps), the closest hits using the rpb1 sequence yellow black to black, 2–5 mm diam. Perithecia crowded, or- are T. japonicum (strain OSC 110991, GenBank DQ522375.1; dinal in arrangement, completely immersed, elongate-ovoid, Identities = 534/566 (94 %), 3 gaps (0 %)). (560–)567–697(–750) × (200–)206–248(–250) µm. Asci Tolypocladium flavonigrum BCC 66576 cylindrical, 8-spored, (318–)330–416(–482) × 7–8 µm with 65/100/100 Tolypocladium flavonigrum BCC 66580 thickened ascus caps, 4.5–5 × 5 µm. Ascospores hyaline, -/65/99 Tolypocladium flavonigrum BCC 66578 filiform, (310–)330–375(–395) × 1.5–2 µm, breaking into 64 Tolypocladium japonicum OSC 110991 Tolypocladium fractum OSC 110990 cylindrical part-spores, 2–5 × 1.5–2 µm. Tolypocladium capitatum NBRC 100997 Culture characteristics — (Colonies developed from germi- Tolypocladium longisegmentum OSC 110992 Tolypocladium capitatum OSC 71233 nating ascospores. Ascospores germinated within 24 h on po- Tolypocladium fumosum WA 18945 tato dextrose agar (PDA). Colonies on PDA moderately growing, Tolypocladium inegoense SU 15 funiculose, c. 10 mm diam after 3 w at 25 °C. Colonies white to Tolypocladium paradoxum Tolypocladium paradoxum NBRC 106958 smoke grey with age. Colonies reverse cream. Conidiophores 90/94/100 Tolypocladium ophioglossoides OSC 106405 erect, arising from vegetative hyphae. Conidia single-celled, Tolypocladium ophioglossoides NBRC 8992 Tolypocladium cylindrosporum ARSEF 2920 hyaline, smooth, globose, 3–5 µm diam, produced in slimy Tolypocladium inflatum OSC 71235 Tolypocladium heads. 59/81/100 Tolypocladium inflatum CBS 824.70 Tolypocladium cylindrosporum NRRL 28025

Typus. Thailand, Kalasin Province, Khok Pa Si Community Forest, on 52/57/88 Tolypocladium album GB 5502 Elaphomyces sp., underground, 14 Aug. 2013, K. Tasanathai, W. Noisri Tolypocladium album CBS 393.89 Tolypocladium endophyticum MX 575 poom & A. Khonsanit (holotype BBH37600, culture ex-type BCC66576 = 100/99/100 Tolypocladium tropicale IQ 214 MY08887, ITS, LSU and tef1 sequences GenBank MN338090, MN337287 87/99/100 Tolypocladium tropicale MX 338 and MN338495, MycoBank MB832658). Tolypocladium amazonense MS 308 Tolypocladium geodes CBS 723.70 Additional material examined. Thailand, Kalasin Province, Khok Pa Tolypocladium pustulatum MF 5785 Si Community Forest, on Elaphomyces sp., underground, 14 Aug. 2013, Tolypocladium jezoensis K. Tasanathai, W. Noisripoom & A. Khonsanit, BBH37601, culture BCC66578, Purpureocillium lilacinum CBS 284.36 Purpureocillium Purpureocillium lilacinum CBS 431.87 ITS, LSU and tef1 sequences GenBank MN338091, MN337288 and 73/93/100 Drechmeria sinensis CBS 567.95 MN338496; ibid. BBH37602, culture BCC66580 LSU, tef1 and rpb1 sequences 99/100/100 Drechmeria gunnii OSC 76404 Drechmeria GenBank MN337289, MN338497 and MN338494. 96/100/95 Ophiocordyceps longissima EFCC 6814 62/74/- Ophiocordyceps longissima NBRC 108989 Notes — Tolypocladium flavonigrum is a rare species in Ophiocordyceps sobolifera KEW 78842 Thailand found only in Khok Pa Si Community Forest, Kalasin Ophiocordyceps yakusimensis HMAS 199604 Ophiocordyceps 85/86/100 Ophiocordyceps sinensis EFCC 7287 province. Compared with other species occurring on Elapho- Ophiocordyceps sinensis ARSEF 6282 myces sp., T. flavonigrum shows similarity to T. fractum (Mains 97/93/100 Polycephalomyces formosus ARSEF 1424 Polycephalomyces ramosopulvinatus EFCC 5566 Polycephalomyces 1957) in the colour and shape of the fertile head as well as in Polycephalomyces nipponicus BCC 2325 the size and shape of the ascospores but differ in the size of Cordyceps militaris OSC 93623 perithecia and asci. Tolypocladium flavonigrum produces elon- Cordyceps kyusyuensis EFCC 5886 gate, ovoid perithecia and asci, which are broader than T. frac- 50 changes tum (500–600 × 220–260; 300–480 × 5–6 µm, respectively). Phylogenetic tree with T. flavonigrum constructed from a combined dataset Tolypocladium japonicum (Mains 1957) possesses a clavate comprising LSU, tef1 and rpb1. The phylogenetic tree was analysed using fertile head and is distinct from all known species on truffles, Maximum parsimony (MP), Maximum likelihood (ML) and Bayesian infer- ence. The MP analysis was conducted on the combined data set using PAUP while both T. capitatum (Mains 1957) and T. longisegmentum v. 4.0b10 (Swofford 2003), adopting random addition sequences (100 replica- possess a yellowish stipe and reddish brown fertile part of the tions), with gaps being treated as missing data. A bootstrap (BP) analysis was stromata, and differ mainly by the length of their part-spores: performed using the maximum parsimony criterion in 1 000 replications. The T. longisegmentum has the longest part-spores (40–65 × 4–5 ML analysis was run with RAxML-VI-HPC2 v. 8.2.12 (Stamatakis 2014) under µm) followed by T. capitatum, T. japonicum and T. flavonigrum a GTR model, with 1 000 bootstrap replicates. Bayesian phylogenetic infer- (8–32 × 2.5–3; 10–18 × 2.5–4; 2–5 × 1.5–2 µm), respectively. ence was calculated with MrBayes v. 3.2.6 (Ronquist & Huelsenbeck 2003), with 5 M generations and under the same model. Numbers at the signiﬁcant nodes represent MP bootstrap support values/RAxML bootstrap support Colour illustrations. Type locality – a small plot in Khok Pa Si Community values/Bayesian posterior probabilities (BPP) times 100. Thickened lines Forest. Stroma on Elaphomyces sp.; immersed, elongate ovoid perithecia; in the tree represent 99–100 % bootstrap support values and 99–100 BPP. part of asci showing asci tips; ascospore; part-spores; colonies on PDA; Supplementary material conidiophores with conidia; conidia. Scale bars =10 mm (stromata and plate culture), 100 µm (perithecia), 10 µm (asci and ascospore), 5 µm (part-spores, FP1175 List of species and GenBank accessions numbers of sequences conidiophores with conidia and conidia). used in this study.

Kanoksri Tasanathai, Wasana Noisripoom, Artit Khonsanit & Jennifer Luangsa-ard, Plant Microbe Interaction Research Team, Bioscience and Biotechnology for Agriculture, BIOTEC, 113 Thailand Science Park, Pathum Thani 12120, Thailand; e-mail: [email protected], [email protected], [email protected] & [email protected]