STUDIES IN MYCOLOGY 50: 149–157. 2004. Hormonema carpetanum sp. nov., a new lineage of dothideaceous black yeasts from Spain Gerald F. Bills*, Javier Collado, Constantino Ruibal, Fernando Peláez and Gonzalo Platas Centro de Investigación Básica, Merck Sharp and Dohme de España, Josefa Valcárcel 38, Madrid, E-28027, Spain *Correspondence: G.F. Bills, gerald_bills@merck.com Abstract: Strains of an unnamed Hormonema species were collected from living and decaying leaves of Juniperus species, plant litter, and rock surfaces in Spain. Strains were recognized and selected based on their antifungal activity caused by the triterpene glycoside, enfumafungin, or based on morphological characteristics and ITS1-5.8S-ITS2 rDNA (ITS) sequence data. Examination of 13 strains from eight different sites demonstrated that they share a common set of morphological features. The strains have identical or nearly identical sequences of rDNA from ITS regions. Phylogenetic analyses of ITS region and intron-containing actin gene sequences demonstrated that these strains comprised a lineage closely allied to, but distinct from, Hormonema dematioides, and other dothideaceous ascomycetes with Hormonema anamorphs. Therefore, a new species, Hormonema carpetanum, is proposed and illustrated. The strains form a pycnidial synanamorph that resembles the coelomycete genus Sclerophoma in agar culture and on sterilized juniper leaves. Taxonomic novelty: Hormonema carpetanum Bills, Peláez & Ruibal sp. nov. Key words: Dothideales, endophyte, enfumafungin, Kabatina, Rhizosphaera, rock-inhabiting fungi, Sclerophoma, Sydowia. INTRODUCTION Enfumafungin is a hemiacetal triterpene glycoside that is produced in fermentations of a Hormonema sp. associated with living leaves of Juniperus communis L. (Liesch et al. 1998, Peláez et al. 2000, Schwartz et al. 2000). Enfumafungin is one, among several, new fungal triterpenoid glycosides that were discovered because of their potent in vitro antifungal activity. The mode of the antifungal action of enfumafungin and other fungal triterpenoid glycosides was determined to be inhibition of fungal cell wall glucan synthesis by their specific action on (1,3)- -D-glucan synthase (Onishi et al. 2000, Peláez et al. 2000). Three enfumafungin-producing Hormonema Lagerb. & Melin strains from the province of Madrid were compared with ascomycetes that have Hormonema anamorphs, e.g., Sydowia polyspora (Bref. & Tavel) E. Müll., Kabatina R. Schneid. & Arx species, known Hormonema species, and other black yeastlike fungi, and were judged to represent an unnamed Hormonema species (Peláez et al. 2000). However, the Hormonema strains were not formally described as a new species. During recent years, additional strains of the same Hormonema species were collected from living and decaying leaves of Juniperus species, plant litter, and rock surfaces in Spain (Table 1). Some of the strains were recognized and selected based on their antifungal activity caused by enfumafungin, while others were recognized based on morphological characteristics and ITS1-5.8S-ITS2 rDNA (ITS) sequence data. Examination of 13 strains from 8 different sites demonstrated that they share a set of morphological features common to the enfumafungin-producing Hormonema sp. The strains have identical or nearly identical sequences of rDNA from ITS regions. Phylogenetic analyses of ITS region and actin gene sequences demonstrated that these strains comprised a lineage closely allied to, but distinct from, Hormonema dematioides Lagerb. & Melin, and other dothideaceous ascomycetes with Hormonema anamorphs. In this report we describe the set of isolates as a new species, Hormonema carpetanum, observe that the strains form a pycnidial synanamorph in agar culture, and present an expanded analysis of their molecular, morphological, and ecological characteristics. MATERIALS AND METHODS Isolates Living cultures of H. carpetanum are maintained in the Merck Research Laboratories Microbial Resources Culture Collection in Rahway, New Jersey, U.S.A. (MRL), the Centro de Investigación Básica, Merck Sharp and Dohme in Madrid (CIBE), and unless indicated otherwise (Table 1). Reference strains of Hormonema, Kabatina and Sydowia species were obtained from the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. 149 BILLS ET AL. Table 1. Details pertaining to isolates of Hormonema carpetanum studied. Isolate F131395 F138213 F0599041 F0594611 MF61671, ATCC 74360, IMI 392072 F154715 F154786 TRN24 TRN252, CBS 115712 TRN31, IMI 392073 TRN40, CBS 115713 TRN201 TRN278 1 Sequence accession No. (ITS, actin) AY616203, AY616225 Geographic origin Host or substratum Circo de Gredos, Ávila, Spain AY616200, AY616226 AY616210, AY616224 AY616209, AY616223 AF182375, AY616222 Sierra de Rubión, Covarrubias, Burgos, Spain Navalquejigo, Madrid, Spain Navalquejigo, Madrid, Spain Navalquejigo, Madrid, Spain Living leaves, Juniperus communis subsp. nana Living leaves, Juniperus oxycedrus Living leaves, Juniperus communis Living leaves, Juniperus communis Living leaves, Juniperus communis AY616207, AY616227 AY616208, AY616228 AY616202, AY616220 AY616205, AY616217 Trevélez, Granada, Spain Ossa de Montiel, Albacete, Spain Cancho Gordo, La Cabrera, Madrid, Spain Cancho Gordo, La Cabrera, Madrid, Spain Leaf litter, Cistus albidus Leaf litter, Juniperus sabina Granite Granite AY616206, AY616218 AY616201, AY616221 AY616204, AY616219 AY616199, AY616216 Cancho Gordo, La Cabrera, Madrid, Spain Cancho Gordo, La Cabrera, Madrid, Spain La Tejera, Checa, Guadalajara, Spain Atazar, Madrid, Spain Granite Granite Sandstone Slate Strains previously characterised in Peláez et al. (2000). 2Holotype and ex-type culture. Morphology All isolates were cultured in at least three different media to study their macro- and microscopic characters. The set of media used for characterization was based on their previous efficiency in the induction of sexual or asexual states, and included: potatodextrose agar (PDA, Difco), oatmeal agar (OA, Difco), and Czapek-yeast extract agar (CYA) (Pitt & Hocking 1997). For some isolates, additional observations were made on cornmeal agar (Sigma-Aldrich) and potato-carrot agar (Gams et al. 1998). Colony diameter, texture, pigmentation, margin appearance, exudates, and colours in the descriptions were recorded after 2 wk at 22 ºC, unless noted otherwise. Colour designations, e.g., 4F6–2, are from Kornerup and Wanscher (1978) and those in capital letters are from Ridgway (1912). Pycnidial conidiomata were induced in a subset strains by inoculating flasks of Sabouraud’s maltose broth containing autoclaved fresh leaves of Juniperus oxycedrus L. with four 5mm agar discs of mycelia and conidia. Flasks were incubated 5 d at 22 ºC at 220 rpm on a rotary shaker. Mycelia-covered leaves were removed from liquid cultures with forceps and incubated on water agar. Additional observations on pycnidial conidiomata were made from 3- to 6-wk-old cultures on PDA and OA. Procedures for isolating strains from rock surfaces were described in Ruibal et al. (2005). Microscopic features were evaluated by 1) observing structures mounted in 5 % KOH; 2) growing fungal colonies on cover glasses immersed in dilute malt extract agar (0.5 % malt extract Difco, 1.5 % agar) and supported on 2 % water agar. Colonies on cover glasses were fixed in 95 % ethanol, mounted in 5 % KOH, and photographed. DNA extraction, amplification, DNA analyses DNA extraction was performed by the methods described in Peláez et al. (1996). The first amplification of both internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of these isolates was per- 150 formed using primers 18S-3 (5’-GAT GCC CTT AGA TGT TCT GGG G-3’) and ITS4A (Larena et al. 1999). To further test relationships at the interand intraspecific level, an intron-containing portion of the actin gene was amplified using primers ACT512F and ACT-783R (Carbone & Kohn 1999). Polymerase chain reactions were performed following standard procedures (5 min at 93 ºC followed by 40 cycles of 30 s at 93 ºC, 30 s at 53 ºC and 2 min at 72 ºC) with Taq DNA polymerase (QbioGene) following the procedures recommended by the manufacturer. Amplification products (0.1 g/mL) were sequenced using the BigDye Terminators version 1.1 (Applied Biosystems, Foster City, CA) following the procedures recommended by the manufacturer. For all the amplification products, each strand was sequenced with the same primers used for the initial amplification. Separation of the reaction products by electrophoresis was performed in an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, CA). Partial sequences were assembled manually, and a consensus sequence was generated. Sequence matching with public or proprietary databases was performed with BLAST2N and FastA (GCG WISCONSIN PACKAGE Version 10.3UNIX, Accelrys Inc). A selection of the best matching sequences were aligned manually using GENEDOC (Nicholas et al. 1997). Phylogenetic relationships were determined from the aligned sequences using PAUP version 4 (Swofford 2000). Nucleotide substitutions were treated as unordered, unweighted characters. Maximum parsimony trees were inferred using the heuristic search options with stepwise addition and the tree bisection reconnection algorithm. Data were resampled with 1000 bootstrap replicates by using the heuristic search option of PAUP. The percentage of bootstrap replicates that yielded greater than 50 % for each group was used as a measure of statistical confidence. Consensus trees were calculated using 50 % majority rule. Sequence alignments and phylogenetic trees were deposited in A NEW HORMONEMA FROM SPAIN TreeBase as accession numbers S1171, M2027 and M2028. In addition to sequences of Hormonema carpetanum listed in Table 1, new sequences of the ITS1, ITS2 internal transcribed spacers and the 5.8S rDNA gene obtained in this study are: Hormonema prunorum (Dennis & Buhagiar) Herm.-Nijh. CBS 933.72 (AY616213), Kabatina juniperi R. Schneid. & Arx CBS 239.66 (AY616211), CBS 466.66 (AY616212). New sequences of actin gene fragment from H. carpetanum are listed in Table 1. In addition, the fragment was sequenced from Sydowia polyspora CBS 128.66 (AY616214) and Kabatina juniperi CBS 239.66 (AY616215). The following sequences, largely derived from previous studies on dothideaceous fungi of the Aureobasidium and Hormonema complexes (de Hoog et al. 1999, Yurlova et al. 1999, Hambleton et al. 2003), were obtained from GenBank in order to supplement analyses of newly obtained sequences; Hormonema dematioides AJ278925, AJ278925, AJ278926, AJ278927, AJ278928, AJ278929, AJ278939, AF013228, AF462439, AY160202, H. macrosporum Voronin AJ244247, Rhizosphaera kalkhoffii Bubák AF01321, Scleroconidioma sphagnicola Tsuneda, Currah & Thormann AY220610, Sclerophoma pythiophila (Corda) Höhn. AF462438, Sydowia polyspora AJ244262. RESULTS Phylogenetic analysis Sequencing of the rDNA of the 13 isolates of H. carpetanum revealed that the ITS1-5.8S-ITS2 gene fragment was 510 nucleotides long. The percentage homology among isolates of the H. carpetanum clade (Fig. 1) ranged between 99 to 100 %. The lengths of the actin gene fragments from H. carpetanum varied from 237 to 232 nucleotides among the 13 isolates. Percentages of homology of the actin gene fragments ranged from 92 to 100 % among the isolates, with isolate TRN278 having the highest sequence divergence (Fig. 2). To estimate intergeneric distances, the actin gene was also sequenced in Sydowia polyspora CBS 128.64 and Kabatina juniperi CBS 239.66. Attempts at amplification and sequencing of the actin gene fragment of Kabatina juniperi CBS 466.66 and Hormonema prunorum CBS 933.72 yielded chromatograms with poorly defined nucleotide sequences, suggesting the presence of more than one actin sequences within the same isolate (data not shown). Morphology, blast search results, and alignments of the ITS1-5.8S-ITS2 rDNA clearly associated H. carpetanum with species classified among the Dothideales, especially a complex of fungi that have in common Hormonema conidial states (Peláez et al. 2000). Recognition and differentiation of H. carpeta- num as a distinct and monophyletic lineage was well supported by parsimony analysis of both genes fragments (Figs 1, 2). In both single-gene genealogies, the isolates formed a discrete group supported in 90 % and 100 % of the bootstrap-resampled ITS15.8S-ITS2 and actin datasets, respectively (Figs 1, 2). Small infraspecific differences were evident among isolates of H. carpetanum, and infraspecific grouping varied depending on the gene fragment analysed. Fig. 1. Relationships of Hormonema carpetanum isolates and selected reference strains inferred by maximum parsimony consensus of aligned sequences of the ITS15.8-ITS2 rDNA. Statistical support (1000 bootstrap) values of > 50 % indicated at branch points. Tree parameters: total characters = 528, constant characters = 454, variable characters parsimony-uninformative = 23, variable characters parsimony-informative = 51, tree length = 103, consistency index (CI) = 0.777 and retention index (RI) = 0.932. Outgroup taxon: R. kalkhoffii. Neither gene fragment grouped isolates of H. carpetanum according to their origins from living plants, plant litter, or rock surfaces (Figs 1, 2). Isolates referable to Sydowia polyspora and its H. dematioides and Sclerophoma pythiophila synanamorphs also formed a distinct and well supported lineage parallel to that of H. carpetanum (Fig. 1). The infraspecific variation among the ITS1-5.8S-ITS2 dataset of H. carpetanum was similar to the differences observed among the same dataset from isolates referable to Sydowia polyspora and its synanamorphs (Fig. 1). The homology among isolates of the S. polyspora–H. dematioides clade ranged between 97 to 99.5 %. Pairwise comparisons of sequence homologies between strains of the H. carpetanum and the S. polyspora–H. dematioides clade ranged between 92 to 94 %, and most of the nucleotide differences between strains of the two clades were concentrated in the ITS2 region. 151 BILLS ET AL. Fig. 2. Relationship of Hormonema carpetanum isolates and selected reference strains inferred by maximum parsimony consensus of aligned sequences of an introncontaining fragment of the actin gene. Statistical support (1000 bootstrap) values of > 50 % indicated at branch points. Tree parameters: total characters = 248, constant characters = 152, variable characters parsimonyuninformative = 78, variable characters parsimonyinformative = 25, tree length = 142, consistency index (CI) = 0.915 and retention index (RI) = 0.750. Outgroup taxon: S. polyspora. The relatively low bootstrap values within the S. polyspora–H. dematioides and H. carpetanum species clusters suggested that further delineation of isolates based on the ITS1-5.8S-ITS2 dataset alone is unreliable. However, some infraspecific grouping of H. carpetanum strains was apparent based on actin gene sequences (Fig. 2). Taxonomic Part Hormonema carpetanum Bills, Peláez & Ruibal, sp. nov. MycoBank MB500039. Figs 3–21. = Hormonema sp., Peláez et al., Syst. & Appl. Microbiol. 23: 333. 2000. Synanamorph: Sclerophoma sp. Etymology: Carpetanus (Latin), in reference to the ancient Roman name for the Central Cordillera of Spain. Species H. dematioides similis, sed conidiis maioribus et serie ex nucleicis acidis differt. Coloniae radiatim rugulosae in agaro avenae farinae vel tuberum solani dextrosato; constantes praecipue ex hyphis submersis radialiter exten- 152 sis; mycelio aerio absente vel exiguo, pycnidiis formatis in ca. 3–4 hebdomadis in vel sub agari superficie, plerumque cum massa humecta conidiorum e conidiogenesi myceliali accumulatorum in guttis vel filis radialibus olivaceo-nigris ad atris. Conidiophora absentia in mycelio. Cellulae conidiogenae holoblasticae, incorporataae, intercalares, mycelio similes. Conidia ad 7–14(–16) µm longa, ad 3–6 µm lata, hyalina ad pallide olivaceo-grisea. Conidiomata pycnidialia formata in vitro, similia generi Sclerophoma, ad 350 µm diam, globosa vel subglobosa, unilocularia, solitaria ad gregaria, olivaceo-brunnea ad atra, glabra vel setosa, initiata e massa stromatica cellularum isodiametricarum, strato tenui hypharum obtecta. Cellulae conidiogenae phialidicae, isodiametricae, 8–15 um diam, unum vel plura conidia ex uno vel pluribus aegre distinctis ostiolis proferentes vel per conversionem ipsorum conidia praebentes. Conidia pycnidialia ellipsoidea ad late ellipsoidea, aliquando curvata vel constricta in parte media, glabra, attenuata ad basim, hyalina usque ad atrogriseo-brunnea, 8–14(–17) 4–6 µm. Inventa in foliis Juniperi speciei superficialiter disinfectis, plantarum residuis et superficie saxorum in Hispania centrali: Holotype exsiccatum specimen herb. CBS H-13135, (CBS H-13136 isotype). Et cultura viva TRN 25 = CBS 115712. Mycelium consisting of torulous, wide, often thickwalled, hyaline to dark olive to olive black, shortcylindrical to subglobose cells, often with hyphal cells longitudinally septate, in age sometimes developing groups of muriform cells, with individual cells up to 22 µ m diam, generally sparsely branched, but sometimes becoming more branched with age. Conidiophores absent on mycelia. Conidiogenous cells holoblastic, integrated, intercalary, usually not differentiated from main axes of vegetative mycelium, occasionally arising from an undifferentiated lateral cell or filament, sometimes with a faint pore or slightly protruding annellate scar evident at the conidiogenous locus, often abundant along the entire radius of young extending hyphae, less so on older, contorted and melanized hyphae. Conidia up to 7– 14(–16) µm long, up to 3–6 µ m wide, aseptate, or rarely 1-septate in old cultures, usually ellipsoidal and tapered towards the base, but quite variable, occasionally pyriform or subglobose, smooth, often with one or more budding scars, hyaline to pale olivaceous-grey, often budding to produce 1 or 2 secondary conidia, accumulating in yeast-like masses along radial axes of vegetative hyphae. A NEW HORMONEMA FROM SPAIN determinate, short cylindrical to isodiametric, 8–15 µm diam, thin- to thick-walled, hyaline to pale, giving rise to one or more conidia, through one or more poorly defined openings or by conversion of the cell’s internal contents directly into to a conidium, collapsing after conidia are released. Pycnidial conidia ellipsoid to broadly ellipsoid, sometime curved, indented on one side or constricted at the centre, smooth, usually tapered toward base, occasionally with eccentric basal scar, hyaline to dark greyish brown, 8–14(–17) × 4–6 µm. Figs 3–6. Hormonema carpetanum in agar culture after two wks. 3. TRN40, on potato-dextrose agar. 4. TRN40, on oatmeal agar. 5. F059461, on Czapek yeast extract agar. 6. F059461, on oatmeal agar. Figs 7–9. Mycelial conidiogenesis and conidia of Hormonema carpetanum. 7, 8. F131395, slide culture on dilute malt agar. 9. TRN 31, slide culture on dilute malt agar. Scale bars = 10 µm. Pycnidial conidiomata in agar or on sterilized leaves up to 350 µm diam, globose to subglobose, unilocular, solitary to gregarious, rarely confluent in age, shiny, dark olive-brown to black, smooth, or with scanty 30–75 µ m long, basally up to 7 µ m wide setae; conidiomata initiating as stromatic masses of broadly cylindrical to isodiametric cells, enveloped by a thin layer of hyphal filaments, dehiscing by irregular erosion of upper layers, accumulating dark brown masses of moist conidia when mature. As basal stromatic cells of conidiomata accumulate and expand, upper layers of stromatic cells convert into conidiogenous cells. Conidiogenous cells phialidic, Cultural characteristics: Colonies on PDA differed in radial extension depending on the isolate, ranging from 21–37 mm diam, with margin submerged, fimbriate, usually radially rugulose or slightly furrowed, often slightly depressed toward the centre, consisting predominantly of submerged, radially extending hyphae, shiny to moist, during the first few weeks, aerial mycelium absent to scant, but abundant aerial filamentous mycelium may emerge with prolonged incubation (>1 mo), in all strains scattered to gregarious pycnidia formed in about 3–4 wks at or below the agar surface, usually with masses of moist conidia from mycelial conidiogenesis accumulating in moist drops or in radial strands, pale olive-brown to dark olive, Yellowish Olive, Dark Olive, Dark Greenish Olive, 3F6–2, 4F6–2, or dark olive-grey, Dark Greyish Olive, Olivaceous-Black (1), 30F5–2, eventually becoming black. Colonies on OM ranged from 24–32 mm diam; depending on the isolate, submerged to appressed, radially furrowed, silky to shiny, moist, dark olive, olivaceous-black to black, in most strains scattered pycnidia formed at or below the agar surface in about 3–4 wks. Colonies on CYA 17–21 mm, raised, rugulose, with radially extending yeast-like hyphae, often with hyaline to buff watery zones or sectors mixed with dark olivaceous-black zones. Growth was poor on media with dilute starch as the primary carbon source, e.g., cornmeal agar or potato-carrot agar. No growth was observed at 37 °C. Habitat: Isolated from surface-sterilized leaves of Juniperus species, plant litter, and rock surfaces. Known distribution: Spain, from Burgos southward to Granada and from Ávila eastward to Guadalajara. Specimens examined: Living cultures listed in Table 1. Spain, Madrid, La Cabrera, Cancho Gordo, isolated from surface of granite formation, Aug. 2001, dried holotype culture with mycelial conidia and pycnidia conidiomata and ex-holotype culture TRN25 = CBS 115712. 153 BILLS ET AL. Figs 10–14. Pycnidia of Hormonema carpetanum. 10. ATCC 74360, pycnidia on autoclaved leaves of Juniperus oxycedrus. Scale bar = 500 µm. 11. TRN31, pycnidia on autoclaved leaves of J. oxycedrus. Scale bar = 500 µm. 12. TRN201, pycnidia from edge of oatmeal agar plate. Scale bar = 500 µm. 13. TRN201, pycnidia from edge of oatmeal agar plate. 14. Scale bar = 250 µm. TRN24, pycnidial initial from potato dextrose agar. Scale bar = 50 µm. DISCUSSION The genus Hormonema, typified by H. dematioides, has been applied to melanized filamentous fungi that produce slimy, yeast-like conidia that are formed basipetally in a non-synchronous manner from one or few loci on cells of undifferentiated vegetative hyphae. Percurrent conidiogenous loci in Hormonema has served to distinguish the genus from similar fungi classified in Aureobasidium that produce conidia synchronously from the conidiogenous loci (Hermanides-Nijhof 1977, de Hoog & Yurlova 1994). The differences in modes of conidiogenesis and cluster analysis of ITS1 and ITS2 sequences clearly separated fungi with Aureobasidium anamorphs from those with Hormonema anamorphs (de Hoog et al. 1999, Yurlova et al. 1999). Furthermore, other studies based on ITS data have consistently 154 segregated strains of the S. polyspora–H. dematioides complex into well-supported clades (Yurlova et al. 1999, Hambleton et al. 2003). Peláez et al. (2000) used a combination of mycelial features, conidiogenesis, and ITS1-5.8S-ITS2 sequences to place the then unnamed H. carpetanum among dothideacous fungi with Hormonema anamorphs. Our reanalysis of an expanded data set, which includes more isolates and more new sequences of related fungi from public databases, reconfirms the conclusion that the new species is a close, but distinct relative of H. dematioides, Kabatina species, Rhizosphaera species, and the recently described Scleroconidioma sphagnicola (Hambleton et al. 2003). The pattern of synanamorphy of H. carpetanum is consistent with patterns of closely related fungi, viz. pycnidial conidiomata forming on plant surfaces, or sometimes in culture, and a mycelial conidial state (Hormonema) associated with vegetative growth. A NEW HORMONEMA FROM SPAIN Figs 15–21. Pycnidial conidiogenous cells and conidia of Hormonema carpetanum. 15. F131395, young stromatic cells and conidiogenous cell (arrow). 16. F131395, young stromatic cells and conidiogenous cell (arrow). 17. TRN201, stromatic cells and conidiogenous cell (arrow), note collapsed stromatic cells. 18. TRN201, stromatic cells and conidiogenous cells (arrows), note collapsed stromatic cells. 19. TRN201, stromatic cells and conidiogenous cells (arrows). 20. F131395, conidiogenous cell (arrow). 21. TRN31, pycnidial conidia produced on autoclaved leaves of J. oxycedrus. Scale bars = 10 µm. In the preliminary description of H. carpetanum (Liesch et al. 1998, Peláez et al. 2000), pycnidial conidiomata were not mentioned probably because strains were not incubated for extended intervals on a variety of media. It is also possible that conidial masses from pycnidial initials were overlooked and confused with accumulated masses of dark conidia from vegetative hyphae. Sequence similarities to other species with pycnidial synanamorphs led us to suspect that H. carpetanum should produce a pycnidial state, at least on host tissue. We were able to artificially induce pycnidia on autoclaved leaves of J. oxycedrus (Figs 10, 11). Once pycnidia were recognized on plant tissue, masses of stromatic cells at and below the agar surface were recognized as pycnidial initials (Fig. 14), and sporulating pycnidia were easily distinguished in 3–4 wk-old cultures on PDA and OA (Figs 12, 13). The development of both pycnidial conidiomata and 155 BILLS ET AL. the Hormonema state of Sydowia polyspora have been observed in vitro (Sutton & Waterston 1970). In a monographic revision of the genus Hormonema, the possibility of in vitro production of acervuli and pycnidia by Hormonema species was acknowledged (Hermanides-Nijhof 1977), however, in vitro production of pycnidia by strains of H. dematioides was not described as a component of its in vitro life cycle. Pycnidial development and morphology of H. carpetanum differs from that described in species of Rhizosphaera, Kabatina, Scleroconidioma, and Sclerophoma. Rhizosphaera species have discreet, phialidic conidiogenous cells, which are produced in intercalary or terminally on chains of cells arising from the pycnidial wall (Gourbière & Morelet 1979, Gourbière & Morelet 1980, Sutton 1980, Butin & Kehr 2000). In Kabatina, erect phialidic conidiogenous cells form on the upper layer of a stroma-like acervulus (Sutton 1980). In Scleroconidioma, the outer surface layer of stromatic conidiomata are converted into conidiogenous cells and produce conidia through percurrently proliferating cells or phialides (Tsuneda et al. 2000, Tsuneda et al. 2001). The conidiomata of H. carpetanum seem most similar to those of Sclerophoma pythiophila in which the cells of the stromatic tissue, consisting of a thick-walled textura angularis, function as conidiogenous cells. Descriptions of S. pythiophila describe the cells of the stromatic tissue which function as phialides. As conidiogenesis cells produce conidia, they accumulate in the upper regions of the conidiomata and are released by an irregular erosion of the conidiomatal wall or membrane (Sutton & Waterston 1970, Sutton 1980, Butin & Peredo 1986). In H. carpetanum, the stromatic cells of the pycnidia (Figs 15, 16) produce conidia blastically or internally (Figs 15–20). As the stromatic cells mature and collapse (Figs 17–20), conidia are released via erosion of the pycnidial membrane and accumulate as a moist mass (Figs 10, 12, 13). However, pycnidial conidial dimensions in H. carpetanum mostly are in the range of 8–14 × 4–6 µ m, and therefore larger than those reported for S. pythiophila which have been recorded as 4–8 × 2–3 µm. Pycnidia of S. pythiophila are described as either multilocular or unilocular, while thus far in vitro only unilocular conidiomata have been observed in H. carpetanum. Furthermore, mycelial conidial dimensions in H. carpetanum often range up to 14 µ m, while conidia of H. dematioides have been reported to be up to 12 µ m (Hermanides-Nijhof 1977, de Hoog et al. 2000). We conclude that the pycnidial conidiomata of H. carpetanum could be accommodated in Sclerophoma, but we prefer not to formally name this state of the life cycle because it will likely be accompanied by the prevailing Hormonema state. Tsuneda et al. (2004) described a new genus and species, Endoconidioma populi Tsuneda, Hambleton & Currah to accommodate a pycnidial fungus with a 156 closed peridium and pycnidial locule filled with conidiogenous cells that form conidia endogenously. A Hormonema-like synamorph is produced by the mycelia in culture. ITS sequence data indicated that the E. populi is a very close relative of H. carpetanum. Comparison of the morphological descriptions E. populi and H. carpetanum indicated the two are distinct and different fungi; Tsuneda et al. (2004) hypothesized, based on similarities in ITS sequences, that the yet undescribed Hormonema strain ATCC 74360 was congeneric with Endoconidioma. Most fungi with Hormonema anamorphs described to date are associated with living plants (HermanidesNijhof 1977, Ramaley 1992, Middlehoven & de Hoog 1997, Tsuneda et al. 2000). Most notable among these fungi are a complex of Rhizosphaera and Kabatina species associated with needle diebacks in conifers (Gourbière & Morelet 1980, Sutton 1980, Martínez & Ramírez 1983, Butin & Kehr 2000), and Sydowia polyspora and its synanamorphs (Sutton & Waterson 1970, Sutton 1980, Butin & Peredo 1986). However, examination of the list of isolates of H. carpetanum (Table 1) suggests on one hand, a pattern of ecological specialization with regard to foliage of Juniperus species, yet on the other, the ability to at least survive on, and perhaps colonize, dead plant litter, and rock surfaces. Presumably the ability to survive in stone surfaces is related to heavily melanized vegetative cells or cell aggregates. Such cells could survive the high radiation and desiccation of these exposed environments, act as vegetative propagules, and perhaps propagate and disperse by budding yeast cells during favourable conditions. Trimmatostroma abietis (Butin et al. 1996), a fungus that grows epiphytically on living conifer needles and forms stromatic conidiomata on senescent needles of Abies and Pinus species, exhibits a similar pattern of distribution. The fungus has also been isolated from man-made and natural stone surfaces as well as being observed in certain traumatic human mycoses. The pattern of distribution is also reminiscent of that of H. dematioides, where the mycelial conidial state is frequently observed as an epiphyte and endophyte of various conifer species, isolated from dead conifer wood, and occasionally recovered from human and animal infections (Hermanides-Nijhof 1977, de Hoog et al. 2000). ACKNOWLEDGEMENTS We thank José Manuel Ruiz Vila for his scholarly assistance in preparation of the Latin diagnosis. Portions of this work were derived from the doctoral dissertation of C. Ruibal to be submitted to the Faculty of Science, Universidad Autónoma de Madrid. A NEW HORMONEMA FROM SPAIN REFERENCES Butin H, Kehr R (2000). Rhizosphaera pseudotsugae sp. nov. and related species. Mycological Research 104: 1012–1016. Butin H, Pehl L, Hoog GS de, Wollenzien U (1996). Trimmatostroma abietis sp. nov. (hyphomycetes) and related species. Antonie van Leeuwenhoek 69: 203–209. Butin H, Peredo HL (1986). Hongos parásitos en coníferas de América del Sur con especial referencia a Chile. Bibliotheca Mycologica 101: 1–100. Carbone I, Kohn LM (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553–556. Gams W, Hoekstra ES, Aproot A (1998). CBS course of mycology. Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. Gourbiere F, Morelet M (1979). Le genre Rhizosphaera Mangin et Hariot 1. R. oudemansii et R. macrospora. Revue de Mycologie 43: 81–95. Gourbiere F, Morelet M (1980). Le genre Rhizosphaera Mangin et Hariot 2. R. pini, R. kobayashi et R. kalkhoffi. Cryptogamie Mycologie 1: 69–81. Hambleton S, Tsuneda A, Currah RS (2003). Comparative morphology and phylogenetic placement of two microsclerotial black fungi from Sphagnum. Mycologia 95: 959–975. Hermanides-Nijhof EJ (1977). Aureobasidium and allied genera. Studies in Mycology 15: 141–177. Hoog GS de, Yurlova, NA (1994). Conidiogenesis, nutritional physiology and taxonomy of Aureobasidium and Hormonema. Antonie van Leeuwenhoek 65: 41–54. Hoog GS de, Zalar P, Urzi C, Leo FD, Yurlova NA, Sterflinger K (1999). Relationships of dothideaceous black yeasts and meristematic fungi based on 5.8S and ITS2 rDNA sequence comparison. Studies in Mycology 43: 31–37. Hoog GS, Guarro J, Gené J, Figueras MJ (2000). Atlas of Clinical Fungi. 2nd ed. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. Kornerup A, Wanscher JH (1978). Methuen handbook of colour. 3rd ed. Eyre Methuen Ltd., London, U.K. Larena I, Salazar O, González V, Julián MC, Rubio V (1999). Design of a primer for ribosomal DNA internal transcribed spacer with enhanced specificity for ascomycetes. Journal of Biotechnology 75: 187–194. Liesch JM, Meinz MS, Onishi JC, Morris SA, Schwartz RE, Bills GF, Giacobbe RA, Horn WS, Zink DL, Cabello A, Diéz MT, Martín I, Peláez F, Vicente F (1998). Antifungal agent obtained from Hormonema. U.S. Patent 5756472. Martínez AT, Ramírez C (1983). Rhizosphaera oudesmansii (Sphaeropsidales) associated with a needle cast of Spanish Abies pinsapo. Mycopathologia 83: 175–182. Middlehoven WJ, Hoog GS de (1997). Hormonema schizolunatum, a new species of dothiaceous black yeasts from phyllosphere. Antonie von Leeuwenhoek 71: 297–305. Nicholas KB, Nicholas HB, Deerfield DW (1997). GeneDoc: Analysis and Visualization of Genetic Variation. EMBNEW NEWS 4: 14. Onishi J, Meinz M, Thompson J, Curotto J, Dreikorn S, Rosenbach M, Douglas C, Abruzzo G, Flattery A, Kong L, Cabello A, Vincente F, Peláez F, Díez MT, Martín I, Bills G, Giacobbe R, Dombrowski A, Schwartz R, Morris S, Harris G, Tsipouras A, Wilson K, Kurtz MB (2000). Discovery of novel (1,3)-glucan synthase inhibitors. Antimicrobial Agents and Chemotherapy 44: 368–377. Peláez F, Platas G, Collado J, Díez MT (1996). Infraspecific variation in two species of aquatic hyphomycetes, assessed by RAPD analysis. Mycological Research 100: 831–837. Peláez F, Cabello A, Platas G, Díez MT, González A, Basilio A, Martín I, Vincente F, Bills GF, Giacobbe RA, Schwartz RE, Onishi JC, Meinz MS, Arbruzzo GK, Flattery AM, Kong L, Kurtz MB (2000). The discovery of enfumafungin, a novel antifungal compound produced by endophytic Hormonema species, biological activity, and taxonomy of the producing organisms. Systematic and Applied Microbiology 23: 333–343. Pitt JI, Hocking AD (1997). Fungi and food spoilage. 2nd ed. Blackie Academic and Professional, London, U.K. Ramaley AW (1992). Tectacervulus mahoniae, Kabatina mahoniae and Selenophoma mahoniae, three new fungi on Mahonia repens. Mycotaxon 43: 437–452. Ridgway R (1912). Color standards and nomenclature. Published by the author, Washington, D.C. Ruibal C, Platas G, Bills GF (2005). Isolation and characterization of melanized fungi from limestone formations in Mallorca. Mycological Progress (in press). Schwartz RE, Smith SK, Onishi JC, Meinz M, Kurtz MB, Giacobbe RA, Wilson KE, Liesch J, Zink D, Horn W, Morris S, Cabello A, Vicente F (2000). The isolation and structure determination of enfumafungin, a triterpene glycoside antifungal agent from the fermenation of a Hormonema sp. Journal of the American Chemical Society 122: 4882–4886. Sutton BC (1980). The Coelomycetes. Commonwealth Mycological Institute, Kew, U.K. Sutton BC, Waterson JM (1970). Sydowia polyspora. C.M.I. Descriptions of pathogenic fungi and bacteria 228: 1–2. Swofford DL (2000). PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4.0b3a. Sinauer Associates, Sunderland, Massachusetts, U.S.A. Tsuneda A, Chen MH, Currah RS (2001). Conidiomatal morphogenesis and pleomorphic conidiogenesis in Scleroconidioma sphagnicola. Mycologia 93: 1164–1173. Tsuneda A, Hambleton S, Currah R (2004). Morphology and phylogenetic placement of Endoconidioma, a new endoconidial genus from trembling aspen. Mycologia 96: 1128-1135. Tsuneda A, Thormann MN, Currah RS (2000). Scleroconidioma, a new genus of dematiaceous Hyphomycetes. Canadian Journal of Botany 78: 1294–1298. Yurlova NA, Hoog GS de, Gerrits van der Ende AHG (1999). Taxonomy of Aureobasidium and allied genera. Studies in Mycology 43: 63–69. 157 BILLS ET AL. 158