Mycologia, 102(6), 2010, pp. 1383–1388. DOI: 10.3852/10-035 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897 The species of Coniolariella Rasoul Zare1 Bita Asgari The sequences were aligned with the pairwise alignment option in GeneDoc (Nicholas and Nicholas 1997). Three species described by Asgari and Zare (2006) and Asgari et al. (2007) in addition to others from GenBank (TABLE I) were included in the analyses of partial LSU (D1–D2, 393–397 bp) and ITS (444–515 bp) phylogeny with parsimony and neighbor joining analyses in MEGA4 (Tamura et al. 2007, see also Asgari and Zare 2010). (Neighbor joining cladograms based on partial LSU and ITS sequences are shown in FIGS 1, 2.) Sequences were submitted to GenBank (TABLE I) and alignments to TreeBASE (SN4942). Coniolaria murandii is validated in Coniolariella as C. macrothecia. Five species are now recognized in Coniolariella as follows. Department of Botany, Iranian Research Institute of Plant Protection, P.O. Box 1454, Tehran 19395, Iran Walter Gams Molenweg 15, 3743CK Baarn, the Netherlands Abstract: Morphological and molecular analyses demonstrate that Coniolariella gamsii and Coniolaria murandii are distinct species. The latter species is validated here as Coniolariella macrothecia. A key to the five species of the genus is provided. Key words: epiphytic fungi, Nodulisporium, taxonomy, Xylariaceae Coniolariella D. Garcı́a, Stchigel & Guarro 2006, recently segregated from Coniochaeta (Coniochaetales), was introduced for species with affinities to Xylariales (Garcı́a et al. 2006) and replaced the previous invalidly published (ICBN Art 36.1) name Coniolaria Seigle-Mur., Guiraud, Steiman & Sage 1995, typified by C. murandii (Seigle-Murandi et al. 1995). The monotype was C. gamsii (Asgari & Zare) Garcı́a, Stchigel & Guarro, with Coniolaria murandii as its purported synonym. Coniolariella accommodates species having asci lacking visible apical structures and polyblastic conidiogenesis. In a further molecular study Checa et al. (2008) added the new species C. hispanica and the new combination C. limoniispora, under which they recognized C. gamsii and Rosellinia australis Sacc. & Trotter as varieties. In the meantime Asgari et al. (2007) introduced another related species, Coniochaeta ershadii, without molecular analysis. In the two molecular studies only an extype strain (LCP 04.5004) of Coniolaria murandii, identified as C. gamsii, was used but no authentic isolates provided by Asgari et al. (2007). It appeared doubtful that the synonymy of Coniolaria murandii and Coniolariella gamsii was correct and re-examination of type material of both taxa confirmed this suspicion. Because the study by Checa et al. (2008) was based only on LSU sequences an attempt to obtain a living culture of C. limoniispora was made to include it in our ITS tree (FIG. 2), but this was not successful. Therefore the conclusions are based only on LSU sequences (FIG. 1). Coniolariella gamsii (Asgari & Zare) Garcı́a, Stchigel & Guarro (2006: 1285), holotype of Coniolariella. Basionym: Coniochaeta gamsii Asgari & Zare, Nova Hedwigia 82:228 (2006). Coniolariella ershadii (Zare, Asgari & W. Gams) Zare, Asgari & W. Gams, comb. nov. Mycobank MB516539. Basionym: Coniochaeta ershadii Zare, Asgari & W. Gams, Nova Hedwigia 84:177 (2007). Coniolariella hispanica Checa, Arenal & J.D. Rogers (2008: 797). Coniolariella limoniispora (Ellis & Everh.) Checa, Arenal & J.D. Rogers (2008: 797). Basionym: Rosellinia limoniispora Ellis & Everh., Proc. Acad. Nat. Sci. Phila. 46:326 (1894). 5 Coniolariella limoniispora var. australis Checa, Arenal & J.D. Rogers (2008: 797), based on Rosellinia australis Sacc. & Trotter 1913 [non Speg. 1909]. Coniolariella macrothecia Zare, Asgari & W. Gams, sp. nov. FIG. 3 MycoBank MB516540. 5 Coniolaria murandii Seigle-Mur., Guiraud, Steiman & Sage, Crypt. Bot. 5:347 (1995); nom. inval., Art. 36.1. Coniolariellae gamsii similis sed ascomatibus majoribus (vulgo 800–1200 mm diam) crassitunicatis distinguenda. Anamorphe Nodulisporii similis, conidiis 7–12 3 2–2.5 mm. Colonies fast-growing, floccose, plane, with low and white aerial mycelium, soon becoming pale gray with Submitted 14 Feb 2010; accepted for publication 10 Apr 2010. 1 Corresponding author. E-mail: simplicillium@yahoo.com 1383 Mycologia myco-102-06-13.3d 22/9/10 01:25:29 1383 Cust # 10-035R 1384 MYCOLOGIA FIG. 1. NJ tree based on partial LSU-rDNA (D1–D2) sequences. Bootstrap values .50% (1000 replicates) shown above branches (those of parsimony analysis are shown in brackets below branches). Coniocessia nodulisporioides is outgroup. Col. 5 Coniolariella, Con. 5 Coniochaeta, X. 5 Xylaria. the production of ascomata, reverse pale buff; ascomata superficial, globose, solitary or in small groups, dark brown to black, 800–1200(–1500) mm, with short ostioles, up to 50 mm long and 100 mm wide; peridium 170–250 mm thick, pseudoparenchymatous, two-layered, outer part of several layers of dark brown, thick-walled, anastomosing and interwoven hyphae (textura intricata), inner part of several layers of hyaline to pale olive-brown, thin-walled, subglobose to angular cells, 5–15 mm diam (textura epidermoidea or angularis); paraphyses numerous, filiform, septate, simple, slightly undulate, considerably longer than the asci (150–230 mm), 7–10 mm wide at the base, tapering to 1.5–2.5 mm at the apex; asci cylindrical, slightly tapering toward the base, eight-spored, 100– Mycologia myco-102-06-13.3d 22/9/10 01:26:20 1384 130 3 12–16 mm; ascospores exuded as a large, globose to irregularly shaped mass at the ostiole, obliquely uniseriate in the ascus, hyaline at first, soon turning dark pistachio green and finally dark chocolate brown and black, ellipsoidal fusoid, with a protrusion at one or both ends, 16–20 3 9–12 mm; immature ascospores with a distinct large guttule when mounted in water; germ slit straight to slightly sinuous at one end (FIG. 3f, g, k–l). Octahedral crystals present in the culture (FIG. 3u, v). Holotype: IRAN 14439 F (ex-type strain LCP 04.5004, IRAN 1637 C, CBS 125772), isolated from soil at Monument Valley area, Colorado, USA, by F. Seigle-Murandi, 1995. Anamorph: nodulisporium-like. Cust # 10-035R ZARE ET AL.: SPECIES OF CONIOLARIELLA 1385 FIG. 2. NJ tree based on ITS (rDNA) sequences. Bootstrap values . 50% (1000 replicates) shown above branches (those of parsimony analysis are shown in brackets below branches). Coniochaeta velutinosa is outgroup. Col. 5 Coniolariella, R. 5 Rosellinia. Conidiophores produced predominantly on ascomatal initials, variable in length, (15–)25–85(–120) 3 2–3 mm, macronematous, mononematous, mostly simple (rarely branched), smooth, septate, hyaline or pale olive-brown; conidiogenous cells monoblastic or polyblastic, integrated, terminal or intercalary, with sympodial elongation, visible as short, less than 1 mm long, inconspicuous denticles bearing blastoconidia solitarily and leaving a distinctly pigmented scar; conidia holoblastic, hyaline to pale olive, smooth, oblong, cylindrical with rounded apex and attenuated and truncated base, 7–12 3 2–2.5 mm (FIG. 3m–t). Remarks. This species is distinguished from C. gamsii by having larger ascomata with a much thicker peridium. In the absence of information relating to the etymology of the original epithet murandii a different, descriptive name for this taxon is introduced based on the largest ascomata thus far known in the genus. FIVE SPECIES OF CONIOLARIELLA ARE TO BE DISTINGUISHED AS FOLLOWS: 1. Ascomata non-ostiolate; paraphyses absent in mature ascomata; ascospores ellipsoidal to slightly Mycologia myco-102-06-13.3d 22/9/10 01:26:28 1385 19. 2. 29. 3. 39. 4. 49. inaequilateral without protruding ends, 16–17.5 3 9–9.5 mm . . . . . . . . . . . . . . . . . . . . . C. hispanica Ascomata ostiolate; paraphyses present in mature ascomata; ascospores with protruding ends . . . . . 2 Ascomata 200–500 mm diam, covered with bluntended setae; peridium 100–120 mm thick; asci 110– 150 3 10.5–12 mm; ascospores 16–18 3 9.5– 10.5 mm, with distinctive protruding ends up to 1.7–2.5 mm long . . . . . . . . . . . . . . . . . . C. ershadii Ascomata larger, without setae; asci slightly shorter (,130 mm); ascospores on average wider with slightly protruding ends . . . . . . . . . . . . . . . . . . . 3 Ascomata globose to ovate, often flattened or depressed above, smooth or granular—roughened; peridium 80–110 mm thick; ascospores 16–18 3 10.5–12 mm; anamorph sporothrix-like . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. limoniispora Ascomata globose to subglobose, not flattened or depressed above, smooth; anamorph nodulisporium-like . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Ascomata 500–800 mm diam; peridium 80–110 mm thick; asci 110–130 3 12–14 mm; ascospores 16–19 3 6–11 mm . . . . . . . . . . . . . . . . . . . . . . . C. gamsii Ascomata 800–1200 mm diam; peridium 170– 250 mm thick; asci 100–130 3 12–16 mm; ascospores 16–20 3 9–12 mm . . . . . . . . . . . . . . . C. macrothecia Cust # 10-035R 1386 MYCOLOGIA TABLE I. Isolates used in phylogenetic analyses Sequences Species Col. ershadii Col. gamsii Col. gamsii Col. hispanica Col. limoniispora Col. limoniispora var. australis Col. macrothecia Con. Con. Con. Con. Con. Con. Con. Con. Con. Con. boothii cruciata discoidea hansenii ornata ostrea punctulata savoryi tetraspora velutinosa Con. verticillata Coniocessia nodulisporioides Daldinia fissa Nodulisporium sp. Nodulisporium sp. R. arcuata R. buxi R. buxi R. necatrix X. badia X. escharoidea X. hypoxylon X. mellissii X. psidii Strain Source LSU ITS Co48, IRAN 972C, CBS 119785 (T) Co27, IRAN 842C, CBS 114379 (T) Co28, IRAN 907C, CBS 117677 Co125, ATCC MYA4453, CBS 124506, TAH 34624 (T) 08fa (R108) Twig of Pistacia vera, Iran GU553331 GU553328 Leaf of Hordeum vulgare, Iran GU553329 GU553325 Leaf of Hordeum vulgare, Iran — GU553326 Leaf of Eryngium campestre, Spain EF489465 GU553323 Vitis vinifera, USA EF489469 — AH 24323 (R154AH) Co127, LCP 04.5004, IRAN 1637 C, CBS 125772 (T) CBS 381.74 (T) FMR 7409 CBS 158.80 (T) CBS 885.68 FMR7415 CBS 507.70 FMR 7408 CBS 725.74 (T) FMR 8167 Co29, IRAN 843C, CBS 117678 (T) CBS 816.71 (T) Twig of Nicotiana glauca, Spain Soil, USA EF489464 AJ875233 AY908997 GU553324 Soil, India Soil, Nigeria Soil, Japan Dung of rabbit, the Netherlands Soil, Russia Twig of Larrea sp., USA Soil, Argentina Wood of Juniperus scopulorum, UK Soil, Spain Leaf of Hordeum vulgare, Iran AJ875226 AJ875222 AJ875230 AJ875223 AJ875228 AJ875227 AJ875231 AJ875229 AJ875225 GU553330 — — — — — — — — — GU553327 Soil, the Netherlands AJ875232 — CBS 281.77 (T) 35524C Et0502 Soil, Jordan Betula sp., Finland Epichloë typhina on Anthoxanthum odoratum ? Japan? ? ? A dead broad-leaf tree, Japan Thailand? Soil, Thailand AJ875224 — — — AF176979 EF600033 — — — — — AB376711 AB376822 AF280629 AB017660 AY909000 DQ272572 AB430453 — — ? Wood, Thailand Thailand? AY327481 AB376710 AB376698 — — — JP807 CBS 347.29 ATCC 32869 R153A W539 BCC 1190 BCC 23634, NBRC 104736 GB6391 BCC 1186 BCC 1127 Underlined sequence numbers are generated in this study, others are from GenBank. Col. 5 Coniolariella, Con. 5 Coniochaeta, R. 5 Rosellinia, X. 5 Xylaria. (T), ex-type strain; ATCC, American Type Culture Collection, USA; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; LCP, Laboratoire de Cryptogamie, Paris, France; IRAN, Herbarium Ministerii Iranici Agriculturae, Tehran, Iran; BCC, BIOTEC Culture Collection, Thailand Biodiversity Center; others are not registered abbreviations. Mycologia myco-102-06-13.3d 22/9/10 01:26:33 1386 Cust # 10-035R ZARE ET AL.: SPECIES OF CONIOLARIELLA 1387 FIG. 3. Coniolariella macrothecia. Teleomorph: a, b. ascomata and masses of ascospores (top view); c. section of ascoma showing two-layered peridium; d. peridium outer layer; e. peridium inner layer; f, g. immature asci containing ascospores with distinct guttule; h. mature ascus; i, j. paraphyses; k. mature ascospores with protruding ends; l. germ slits. Anamorph: m–t. conidiophores, conidiogenous cells and conidia; u, v. crystals. All mounts were prepared in water. Bars: a 5 1000 mm; b 5 200 mm; c 5 50 mm; j 5 20 mm; d–i, k–v 5 10 mm; f, r–v 5 DIC. Mycologia myco-102-06-13.3d 22/9/10 01:26:34 1387 Cust # 10-035R 1388 MYCOLOGIA ACKNOWLEDGMENTS This study was supported in part by a research grant from Iranian National Science Foundation (INSF). The authors are indebted to Dr Joëlle Dupont (France) for supplying the ex-type culture (LCP 04.5004) of Coniolaria murandii and to Dr Gerard Verkley (the Netherlands) for supplying the ex-type culture (CBS 124506) of Coniolariella hispanica. LITERATURE CITED Asgari B, Zare R. 2006. Two new Coniochaeta species from Iran. Nova Hedwig 82:227–236. ———, ———. 2010. Two new species of Preussia from Iran. Nova Hedwig 90:533–548. ———, ———, Gams W. 2007. Coniochaeta ershadii, a new species from Iran, and a key to well documented Coniochaeta species. Nova Hedwigia 84:175–187. Mycologia myco-102-06-13.3d 22/9/10 01:27:22 1388 Checa J, Arenal F, Blanco N, Rogers JD. 2008. Coniolariella hispanica sp. nov. and other additions to Coniolariella. Mycol Res 112:795–801. Garcı́a D, Stchigel AM, Cano J, Calduch M, Hawksworth DL, Guarro J. 2006. Molecular phylogeny of Coniochaetales. Mycol Res 110:1271–1289. Nicholas KB, Nicholas HB Jr. 1997. GeneDoc: a tool for editing and annotating multiple sequences alignments. http://www.psc.edu/biomed/genedoc. Distributed by the authors. Seigle-Murandi F, Guiraud F, Steiman R, Sage L. 1995. Coniolaria: a new genus of Ascomycetes—description of Coniolaria murandii sp. nov. from soil of Monument Valley in Colorado (USA). Crypt Bot 5:346–350. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 10.1093/molbev/ msm092. Cust # 10-035R