Mycologia, 93(4), 2001, pp. 758-763. ? 2001 by The Mycological Society of America, Lawrence, KS 66044-8897

Hypocrea nigrovirens, a new species with a -like anamorph

Priscila Chaverril stroma formed of pseudoparenchymatous or highly The Pennsylvania State University,Department of compacted hyphae. The ascospores are colorless or Plant Pathology, 301 Buckhout Lab., UniversityPark, green, 1-septate, disarticulating early in development Pennsylvania 16802 into two part-ascospores that are symmetric or asym- Gary J. Samuels metric. The anamorphs of are typically United States Department of Agriculture, Agricultural TrichodermaPers. However, stilbella-, -, Research Service, SystematicBotany and Mycology gliocladium-, and -like forms are also Lab., Rm. 304, B-Ol lA, BARC-W,Beltsville, known (Rossman et al 1999). Maryland 20705-2350 Ascospores of Hypocrea are typically colorless but, Elwin L. Stewart as in other ascomycete groups, pigmented ascospores have attracted taxonomic attention. of The Pennsylvania State University,Department of Species Hy- Plant Pathology, 212B Buckhout Lab., University pocrea with green ascospores have been segregated Park, Pennsylvania 16802 into the genera CreopusLink (Link 1833) and Chrom- ocrea Seaver (Seaver 1910), both of which are based Loengrin Umafia on Hypocreagelatinosa (Tode: Fr.) Fr. Neither Creopus National Institute 22- Biodiversity (INBio), Apdo. nor Chromocreahas been widely accepted. 3100, Santo Domingo, Heredia, Costa Rica Polyphyly of the green-spored species of Hypocreahas been re- ported in a recent study (Chaverri et al 1999), prov- ing that Creopusand Chromocreaare artificial genera. Abstract: Hypocrea a new species with a nigrovirens, Doi (1972) erected subsection Creopuswithin sec- gliocladium-like anamorph, was collected on the At- tion Hypocreato accommodate species with green as- lantic coast of Costa Rica. This species is recognized cospores. Within subsection Creopus,Doi (Doi 1972) by dark green, almost black stroma, warts around the designated the artificial group Gelatinosa for species ostiolar opening, conspicuously thickened ascus tip with gliocladium-like anamorphs. The Gelatinosa and conidia formed on gliocladium-like conid- large group also has been proven to have multiple evolu- iophores. tionary origins within Hypocrea (Chaverri and Sa- Words: Key green ascospores, , Hy- muels 2000). pocreales, systematics, There are more than 200 named Hypocreaspecies and related anamorphs, most of which have not been reevaluated since they were described in the 19thand INTRODUCTION early 20th centuries. Only a few Hypocreaspecies have been linked to an anamorph. A new species of Hypocrea Fr. with green ascospores About thirty species of Hypocrea with green asco- and a nearly black stroma was found on decaying spores have been described; however, the number of wood in a low elevation forest on the Atlantic coast Hypocrea and Trichodermaspecies has been underes- of Costa Rica during a biodiversity inventory in Jun timated in early works. Recent papers (Bissett 1991 a, 1999. This new is because it has a species unique very b, Turner et al 1997, Samuels et al 1998, Lieckfeldt dark stroma with a tuberculate surface due to warts et al 1999) show a higher diversity of species in around the ostiolar a thick- opening, conspicuously Trichodermaand Hypocrea,when compared to previ- ened ascus and a that tip gliocladium-like anamorph ous studies (e.g., Seaver 1909a, b, 1910, 1911, Dingley forms conidia. In it forms relatively large addition, 1952, Doi 1972, 1975, 1976, 1978) where the named stromata with fertile in a perithecia culture, phenom- species were many fewer. Therefore, the discovery of enon that is not common in Hypocrea. new species of Hypocrea and Trichodermaare very Hypocrea (Ascomycetes, , Hypocrea- likely a result of intensive collecting in this group. ceae) is characterized by perithecia completely im- mersed in a brightly colored, or rarely dark, fleshy MATERIALSAND METHODS Accepted for publication January 15, 2001. Individual ascospores were isolated immediately after col- 1 Corresponding author, E-mail: [email protected] lection on CMD (Difco cornmeal agar + 2% dextrose) plus

758 CHAVERRI ET AL: HYPOCREANIGROVIRENS, SP. NOV. 759 antibiotic (50% streptomycin and 50% chlortetracycline) jim thick (n = 10), formed of angular cells, dark with the aid of a micromanipulator. Later, single ascospores green, KOH-, (5.5-)7.0-8.0(-10.0) pm diam (n = were isolated from each of two asci from stromata that were 12), densely compacted with walls 0.5-1.0 Jim thick formed in culture. data were tak- Anamorph morphological (n = 10); cells composing the warts around the os- en from cultures grown on CMD. Images were captured tiolar opening dark green, KOH-, angular, 7.5-11.5 (- with the software Scion Image (Scion Corporation, Fred- 20.5) jim diam (n = 28), walls 0.5-1.0 jxm thick (n erick, Maryland). Colony characteristics were taken after ca = Tissue below the stromatal sur- one week from CMD cultures incubated at 20 C, with alter- 10). immediately nating 12 h darkness and 12 h white fluorescent light. Op- face of compact, pseudoparenchymatous cells, col- timum temperature for growth was determined using CMD orless or slightly pigmented, KOH-. Internal tissue at four different temperatures: 15, 20, 28 and 35 C; with below perithecia pseudoparenchymatous, colorless or alternating 12 h darkness and 12 h white fluorescent light. slightly pigmented, KOH-, cells (8.0-)11.5-14.0(- To determine growth rate, a 5-mm disc taken from the edge 20.5) rim diam, walls 0.5-1.0 jim thick (n = 15). Peri- of an actively growing colony was plated on a petri dish thecia closely spaced, subglobose, (179-)189-253(- containing 20 mL of CMD. The disc was placed one centi- 255) jim high (n = 4), (125-)129-150 tjm wide (n meter from the edge of the plate. Ten repetitions were = 4), ostiolar canal (56-)57-78(-81) ,jm (n = 4) done. Measurements of the colony radius were taken every long. Asci cylindrical, (94.5-)103.0-106.5(-117.5) X 24 h at the same time of the for several until the day days (5.0-) 6.0-6.5(-7.0) Ijm (n = 25), with a thickened colony covered the plate. Cultures are preserved at SBML tip, with apical pore; part-ascospores uniseriate. Part- (BPI), and INBio (Costa Rica). ascospores dimorphic, ellipsoidal, sometimes tending Micromorphological measurements of the teleomorph to be at the ascus distal were made from dried material. The specimen was rehy- conical, especially tip, part drated in 3% KOH and then sectioned with the aid of a below the ascus tip ellipsoidal (5.5-)6.0-6.5(-7.0) X freezing microtome. Summary statistics of micromorphol- (5.0-)5.5-6.0 jm, proximal part wedge-shaped (5.0-) ogy were made on the basis of thirty measurements except 5.5-6.0(-7.5) X (4.0-)4.5-5.0(-5.5) ,jm, dark olive where noted. green, conspicuously spinose. Colonies on CMD col- To determine the genetics of sexuality, 13 and 14 part- orless and odorless, aerial mycelium lacking. Opti- ascospores were taken from each of two asci, respectively. mum temperature for growth on CMD 28 C; growing The part-spores were removed without respect to their po- 4 mm/d at 15 C, 11 mm/d at 20 C, 14 mm/d at 28 sition in the ascus. These were plated singly on potato dex- C and no growth at 35 C. Conidiophores scarce, scat- trose and incubated at 20 C with 12 h darkness agar (Difco) tered near the edges of the petri dish, nearly invisi- and 12 h cool white fluorescent Formation of peri- light. ble, KOH-, arising from a stalk cell, multiseptate, thin thecia was noted. walled, smooth, (123-) 124-171(-173) X 8-15(-16) pim, with one or two branches. Phialide branching RESULTS penicillioid (gliocladium-like), 2-level verticillate sometimes biverticillate, with few phialides (8-12), Hypocrea mngrovirens Chaverri et Samuels, sp. nov. producing a globose, watery green conidial mass; FIGS. 1-10 penicillus (0.02-) 0.04-0.05(-0.09) mm wide. Phiali- Stromata solitaria vel gregaria, pulvinata, nigrovirentia, des cylindrical, sometimes swollen in the middle, ostiolo (0.3-)0.4-0.7(-1.0) mm, superficie tuberculata, (9.0-)13.5-15.5(-20.5) ,pm long, (3.0-) 3.5-4.0 (-5.5) dum Perithecia dense juvenia conspicuo. gregaria, subglo- jm at the widest, (2.0-)2.5-3.0(-4.5) jpm at the base, bosa. Asci cylindrici, (94.5-)103.0-106.5(-117.5) X (5.0-) length/width ratio (2.5-) 3.5-4.0 (-6.0), never 6.0-6.5 (-7.0) iLm,apice incrassato, poro apicali praediti. As- hooked or sinuous, axis of phialide (2.0-) 3.0-3.5(- cosporae bicellulares, ad septum disarticulatae; parte distali wide; collarette flared, ellipsoideae, (5.5-)6.0-6.5(-7.0) X (5.0-)5.5-6.0 p,m, parte 4.5) |jm slightly pigmented metulae proximali cuneiformes (5.0-)5.5-6.0(-7.5) X (4.0-) 4.5- green; intercalary phialides common, not 5.0(-5.5) um, olivaceae, conspicue verrucosae. Status ana- common, cylindrical, (10.0-)13.5-15.5(-20.5) X 4.0- = morphosis Trichoderma,gliocladio similis. Conidia ellipso- 4.5(-5.5) jm (n 15). Conidia broadly ellipsoidal, idea, glabra, viridia, (4.0-)6.0-6.5(-9.0) X (3.5-)4.5-5.0(- green, smooth to slightly spinulose, (4.0-)6.0-6.5(- 6.0) jim. 9.0) X (3.5-)4.5-5.0(-6.0) ,jm, conidium length/ Stromata solitary and scattered, sometimes in pairs, width ratio 1.0-1.5. Chlamydospores not observed. pulvinate, dark green to almost black, KOH-, nearly Perithecia formed in culture within ca 10 d on CMD circular in outline, (0.3-)0.4-0.7(-1.0) mm diam (n and PDA. = 11), at most only slightly constricted at base, sur- Anamorph. Trichodermasp. (gliocladium-like). face rough, conspicuously tuberculate from perithe- Habitat. On decorticated wood. cial apices, 5-11 (-15) perithecia per stroma (n = 6), Known distribution. Atlantic coast of Costa Rica. ostiolar openings visible when fresh, dark green near- Known only from the type location and collection. ly black, with warts. Stroma surface (16-) 18-22 (-25) Specimensexamined. COSTA RICA. LIMON: Puerto Viejo, 760 MYCOLOGIA

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FIGS.1-10. Hypocrea nigrovirens (from HOLOTYPE). 1. Top view of stroma. 2. Longitudinal section of stroma. 3. View of warts, tissue of the surface of the stroma and tissue between perithecia. 4. View of ostiolar opening and cells composing the warts. 5. Internal tissue of stroma. 6, 7. Asci with ascospores. Note roughness on ascospores depicted in one ascus in FIG. 7. 8, 9. Conidiophores. 10. Conidia. Bars: 1, 2 = 0.25 mm; 3 = 35 mm; 4, 5 = 20 ,um; 6, 8 = 10 jum;7, 10 = 5 Jim;9 = 50 ,um. CHAVERRI ET AL: HYPOCREANIGROVIRENS, SP. NOV. 761

Refugio Nacional de Vida Silvestre Gandoca-Manzanillo, on DISCUSSION decorticated wood, 8 Jul 1999, L. Umana (HOLOTYPE: BPI, GJ.S. 99-64; ISOTYPE: INBio-Costa Rica). Hypocrea nigrovirens was found on decorticated wood in a disturbed forest dominated by palms, ca 100 m KEYTO SPECIESOF HYPOCREAWITH from the ocean on the Atlantic coast of Costa Rica. GLIOCLADIUM-LIKEANAMORPHS This species differs from other Hypocrea species in the warts around the ostiolar opening and large co- 1. Perithecia immersed in an effuse subiculum, part-asco- nidia formed from penicillately branched conidio- spores hyaline, conidia hyaline, growing on Aphyllo- phores. Hypocrea nigrovirens is further distinguished phorales . . . "Hypocrea" pallida Ellis & Everh.-complex in Hypocrea by the exceptionally thickened ascus tip, (see Doi and Yamatoya1989) the rather and the to 1. Perithecia immersed in a well-developed stroma, part- large ascospores ability produce in culture. ascospores green or hyaline, conidia green or hya- perithecia line, growing mainly on decaying woody substrata . . 2 Recent phylogenetic studies (Chaverri and Sa- 2. Part-ascospores green ...... 3 muels 2000) based on sequence data from rDNA 2. Part-ascospores hyaline ...... 5 place H. nigrovirens within Hypocrea, and within a 3. Stroma color dark-green almost black, surface con- major clade containing other species of Hypocrea with spicuously tuberculate ...... H. nigrovirens green ascospores and gliocladium-like anamorphs. In 3. Stroma color yellow to ochre, surface not tubercu- this study, H. nigrovirens differs from other Hypocrea late ...... 4 species by more than 40 nucleotides. We do not pre- 4. Ascospores, distal 3.2-4.1 X 2.9-3.2 L.m,proximal sent our molecular data here because most of the 3.8-4.6 X 2.8-3.1 jim, stroma to yellow-ochre species in this clade have not been named. Work on brown, surface smooth ...... of this clade is in progress...... H. luteovirensDoi (Doi 1972) Gliocladium-like morphology in the Hypocreales 4. Ascospores, distal 3.7-4.6 X 3.5-3.9 JLm,proximal has been to result from evolution 3.7-4.7 X 2.9-3.7 jlm, stroma yellow, surface with proven convergent perithecial protuberations ... H. gelatinosa (Tode: (Rehner and Samuels 1994). The type species, Glio- Fr.) Fr. (Webster 1964, Bissett 1991b) cladium penicillioides Corda, is the anamorph of 5. Conidia hyaline ...... 6 Sphaerostilbella (Henn.) Sacc. & D. Sacc. (Seifert 5. Conidia green ...... 7 1985) and is very close in morphology to the Gliocla- 6. Conidia 2.3-3.6 X 1.3-1.9 jLm, aleuriospores dium anamorph of Hypocrea pallida. These fungi are abundant, stroma ochraceous ...... fungicolous, occurring on aphyllophores. The ana- ...... H. peruviana Doi (Doi 1976) morph of H. nigrovirens can easily be distinguished 6. X Conidia 3.5-6(-8.5) 3-3.5(-4) Ijm, aleurio- from G. penicillioides and its relatives. absent, stroma ...... spores golden-yellow Gliocladium-like morphology is polyphyletic within ... H. psychrophilaMuller et al (Muller et al 1972) Hypocrea (Chaverri and Samuels 2000). Other species 7. Conidia pale green, phialides 6-16 X 2-3 Jim, chla- with gliocladium-like anamorphs and green asco- mydospores absent, stroma pale yellow-ochre .... spores have been described, viz. H. gelatinosa and H. ... H. argillacea Phil. & Plowr. (Phillips and Plowright luteovirens Doi. In addition to the "H. 1885, description of anamorph in Doi 1972) "pallida-com- H. H. H. H. 7. Conidia green, phialides 4-11 X 1.5-2 Jim, chla- plex; argillacea, lutea, peruviana, psy- mydospores present or absent, stroma white or yel- chrophila and H. sublutea have colorless ascospores low-ochre ...... 8 and gliocladium-like anamorphs. Among the known 8. Stroma white, phialides 8-11 X 2 Jim, chlamydo- anamorphs of Hypocrea species that have green as- spores abundant, anamorph regular "Gliocladi- cospores, the anamorph of H. nigrovirens most um" viride .... H. lutea (Tode) Petch (Petch 1937, strongly resembles that of H. gelatinosa (Webster description of anamorph in Doi 1966) 1964, Bissett 1991b) in forming conidia in a clear 8. Stroma yellow-ochre, phialides 4-6 X 1.5-2.1 JLm, drop of green liquid at the tip of a well defined pen- intermediate chlamydospores absent, anamorph icillus. However, branching of the conidiophore of between gliocladium-like and trichoderma-like . . H. nigrovirens is simple while the anamorph of H...... H. sublutea Doi (Doi 1971) gelatinosa is more complex. Moreover, conidia of the new species are much larger than in H. gelatinosa Genetics of sexuality.-After single part-spore isola- (2.5-4.5 X 2.0-3.1 iLm,Bissett 1991b). tions from different asci, fertile perithecia and conid- Although we have not undertaken a complete ia formed in approximately half of the single spore study of sexual compatibility, the preliminary results cultures from each ascus, but not more than eight. that we present here are suggestive of the phenom- Only conidia formed in the cultures derived from the enon of mating-type switching. This phenomenon rest of the part-spores of each ascus. was first reported for Hypocrea spinulosa Fuckel, a 762 MYCOLOGIA

member of the Hypocreaceae, but has since been . 1971. Some species of the Hypocrea.Bull Natl found in a wide diversity of pyrenomycetes (see re- Sci Mus 14:387-400. . Revision of the with cultural ob- view in Perkins 1987). In mating-type switching, half 1972. Hypocreales servations. IV. The and its allies in of the ascospores are self sterile while the other half genus Hypocrea Ja- pan (2). Enumeration of the species. Bull Natl Sci Mus are self-fertile. The self fertility has been explained 15:649-751. by a unidirectional reversal of mating type within the . 1975. Revision of the Hypocreales with cultural ob- In H. mutants were mycelium. spinulosa, auxotrophic servations. VII. The genus Hypocreaand its allied gen- utilized to demonstrate that the self sterile colonies era in South America (1). Bull Natl Sci Mus Ser B (Bot) were able to cross with the apparently self fertile col- 1:1-33. onies. Although the possibility of mating-type switch- . 1976. Revision of the Hypocreales with cultural ob- ing has been suggested for other Hypocrea species servations. IX. The genus Hypocrea and its allied gen- (Canham 1969, Samuels and Lodge 1996), a thor- era in South America (2). Bull Natl Sci Mus Ser B (Bot) ough genetic analysis of these fungi has not been un- 2:119-131. dertaken. In vitro perithecial production in Hypocrea . 1978. Revision of the Hypocreales with cultural ob- servations. XI. Additional notes of and its al- is uncommon. Among the species that do produce Hypocrea lies in Japan (1). Bull Natl Sci Mus Ser B (Bot) 4:19- perithecia under artificial conditions, only one (H. 26. jecorina Berk. & Broome) exhibits a normal bipolar , Yamatoya K. 1989. Hypocrea pallida and its allies et al compatibility system (Lieckfeldt 2000). (Hypocreaceae). Mem New York Bot Gard 49:233-242. Lieckfeldt E, Kullnig C, Samuels GJ, Kubicek CP. 2000. Sex- ually competent, sucrose- and nitrate-assimilating ACKNOWLEDGMENTS strains of Hypocrea jecorina () from The authors are grateful to the National Biodiversity Insti- South American soils. Mycologia 92:374-380. tute (INBio, Costa Rica) for the organization and funding , Samuels GJ, Nirenberg HI, Petrini 0. 1999. A mor- of this collecting trip. In addition, this study was further phological and molecular perspective of Trichoderma financed by the National Science Foundation (PEET) grant viride. is it one or two species? Appl Environ Microbiol 9712308 "Monographic Studies of Hypocrealean Fungi: Hy- 65:2418-2428. pocrea and Hypomyces."We also acknowledge the comments LinkJHF. 1833. Handbuch zur Erkennung der nutzbarsten of Drs. Amy Y. Rossman, David M. Geiser and Ms. Nancy G. und am haufigsten vorkommenden Gewachse. Part 3. Wenner, offered in preparation and revision of this manu- Berlin: In der Haude und Spenerschen Buchhandlung script. We thank Dr. Orlando Petrini for correcting the Lat- (SJ. Joseephy). 3:349. in description. The assistance of the INBio parataxonomists Muller E, Aebi B, Webster J. 1972. Culture studies on Hy- Enia Navarro and Milton Umafia in our fieldwork in Costa pocrea and Trichoderma.V. Hypocrea psychrophylasp. Rica is greatly appreciated. nov. Trans Br Mycol Soc 58:1-4. Perkins DD. 1987. Letter to the editor. Mating-type switch- ing in filamentous ascomycetes. Genetics 115:215-216. LITERATURE CITED Petch T. 1937. Notes on British Hypocreaceae. III. J Bot 75: 217-231. Bissett J. 1991a. A revision of the genus Trichoderma.II. Phillips W, Plowright CB. 1885. New and rare British fungi. Infrageneric classification. Can J Bot 69:2357-2372. Grevillea 13:79. . 1991b. A revision of the genus Trichoderma.III. Sec- Rehner SA, Samuels GJ. 1994. Taxonomy and phylogeny of tion Pachybasium. Can J Bot 69:2373-2417. Gliocladium analyzed from nuclear large subunit ribo- Canham SC. 1969. Taxonomy and morphology of Hypocrea somal DNA sequences. Mycol Res 98:625-634. citrina. Mycologia 61:315-331. Rossman AY, Samuels GJ, Rogerson CT, Lowen R. 1999. Chaverri P, Samuels GJ. 2000. Convergent evolution of Glio- Genera of , Hypocreaceae and Nectri- cladium morphology in Hypocrea. Abstract. Inoculum. aceae (Hypocreales, Ascomycetes). Stud Mycol 42:1- Newsletter of the Mycological Society of America. My- 248. cologia 51 (suppl):24. Samuels GJ, Lodge DJ. 1996. Three species of Hypocreawith , , Stewart EL. 1999. Ascospore color is not a stipitate stromata and Trichodermaanamorphs. Mycol- useful generic character in Hypocrea.Abstract. In: Ab- ogia 88:302-315. stracts. XVI International Botanical Congress, St. Louis, , Petrini 0, Kuhls K, Lieckfeldt E, Kubicek C. 1998. Missouri (Aug 1-7, 1999). XVI International Botanical The Hypocrea schweinitzii complex and Trichoderma Congress: St. Louis. 308 p. sect. Longibrachiatum.Stud Mycol 41:1-54. DingleyJM. 1952. The Hypocreales of New Zealand. III: the Seaver FJ. 1909a. The Hypocreales of North America. I. My- genus Hypocrea. Trans Roy Soc New Zealand 79:323- cologia 1:41-76. 337. . 1909b. The Hypocreales of North America. II. My- Doi Y. 1966. A revision of Hypocreales with cultural obser- cologia 1:177-207. vations. I. Some Japanese species of Hypocrea and Po- . 1910. The Hypocreales of North America. III. dostroma. Bull Natl Sci Mus 9:345-357. 1910b. Hypocreales. North Am Fl 3(1):1-88. CHAVERRI ET AL: HYPOCREANIGROVIRENS, SP. NOV. 763

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