Mycologia, 88(2), 1996, pp. 302-315. © 1996 by The New York Botanical Garden, Bronx, NY 10458-5126

Three species of Hypocrea with stipitate stromata and Trichoderma anamorphs

Gary J. Samuels United States Department of Agriculture, Agriculture Research Service, Systematic Botany and Mycology Laboratory, Rm. 304, B-011A, BARC-W, Beltsville, Maryland 20705

D. J. Lodge Centerfor Forest Mycology Research, United States Department of Agriculture, Forest Service, Forest Products Lab., PO Box B, Palmer, Puerto Rico 00721

Abstract: Hypocrea brevipes, H. poronioidea and H. capitata, a new species, were grown in pure culture from individual part-ascospores. Each produced a Trichoderma anamorph, but none of the anamorphs were referable to any named Trichoderma aggregate species. Stromata with fertile ascospores formed in cultures derived from single part-ascospores of H. po­ronioidea. In addition, a hyphomycetous, Acremon­ium-like synanamorph with hyaline conidia borne in slime formed in cultures of H. poronioidea. The dis­tribution of H. poronioidea is extended from the Americas to Africa. These Hypocrea species and their anamorphs are described.

Key Words: Hypocreales, Podocrea,Podostroma, sys­tematics

INTRODUCTION

Three Hypocrea Fr. species having stipitate-capitate stromata were collected recently in tropical habitats. These species are H. brevipes (Mont.) Sacc., H. poron­ioidea Möller, and the new species H. capitata. Hy­pocrea poronioidea has been reported once since its original description (Möller 1901), as Podostroma or­biculare Chardón (Chardón, 1921), but has not been redescribed in modern terms. Hypocrea brevipes was redescribed by Doi (1975, 1979). Trichoderma Pers. anamorphs developed in colonies derived from as­cospores of each. In addition, a synanamorph having hyaline conidia borne in colorless slime formed in cultures of H. poronioidea. Neither H. brevipes nor H. poronioidea have previously been linked to ana­morphs. These Hypocrea species and their anamorphs are described, and the relationship of Hypocrea to Po­dostroma P. Karst. is discussed.

Accepted for publication October 31, 1995.

The Hypocrea species discussed in the present work could be, or have been, included in Podostroma or Podocrea (Sacc.) Lindau on account of their having stalked stromata. Saccardo (1883) included H. brevi­pes in Hypocrea subg. Podocrea Sacc., and later (Sac­cardo and Saccardo, 1905) included both H. brevipes and H. poronioidea in Podocrea. Doi (1975, 1979) in­cluded H. brevipes in Podostroma. Moreover, a syn­onym of H. poronioidea is P. orbiculare. In Podostroma stromata comprise a stipe surmounted by a distinct, fertile cap. In Podocrea the stroma is cylindrical and not capitate. Atkinson (1905) synonymized the later genus, Podocrea, with PodostromaJ and this synonymy has generally been followed. Rogerson (1970) ac­cepted Podostroma. Doi (1975, 1979) provisionally ac­cepted Podostroma, doubting that it could be main­tained as a genus distinct from Hypocrea (see Doi, 1975).

Within Hypocrea there is nearly continuous varia­tion in form that includes stromata that are indefi­nitely effused, pulvinate but broadly attached, discoi­dal and attached at a central point, or clavate to stip itate-capitate. Among the species that have Trichoder­ma. anamorphs, there is no obvious morphological or biological hiatus that could support generic separa­tion on the basis of stromal form, and the clavate or stipitate condition per se does not imply a dose rela­tionship among those species that posess it. Although stromata of H. poronioidea, H. capitata, and H. brev­ipes are morphologically similar, stromata of H. brev­ipes are anatomically distinct from the others. Hypo­crea brevipes and Podostroma eperuae Rogerson & Sa­muels (Rogerson and Samuels, 1992), a species with a clavate stroma, share the feature of unusual elon­gated or brick-like cells at the stroma surface, but otherwise have little in common. While our under­standing of stromal anatomy in Hypocrea is far from complete, we suspect that these brick-like cells have resulted from the extension of the stroma and do not indicate any relationship.

Anamorphs have suggested relationships among genera and species of the Hypocreales (Samuels and Seifert, 1987). Few species of Podostroma have been grown in pure culture and linked to anarnorphs. At­kinson (1905) obtained stromata of P. alutaceum (Pers.) G.F. Atk. in agar culture tubes, but did not note the presence of an anamorph. Tubaki (1958)

302

SAMUELS AND LODGE: HYPOCREA

and Doi (1967, 1973) have connected Podostroma species to Trichoderma or to Verticillium-like ana­morphs, respectively.

The Trichoderma anamorphs of the three Hypocrea species discussed in the present paper cannot be placed into any of the aggregate species defined by Rifai (1969), although they bear some similarity to T. hamatum- aggr., or to species included in Trichoderma sect. Pachybasium Bissett (Bissett, 1991b) or section Trichoderma (Bissett, 1991a). The anamorph of H. capitata is characterized by having ampulliform to la­geniform phialides clustered on a rather broad main axis; additionally, sterile branches sometimes extend from conidiophores (FIG. 55). This anamorph could be placed in sect. Pachybasium. The anamorph of H. poronioidea is morphologically more diverse. In part it could be included in sect. Pachybasium because of its clustered, ampulliform phialides (FIG. 58). How­ever, it also produces more loosely aggregated whorls of lageniform phialides (FIG. 59) which indicate sect. Trichoderma. The Trichoderma anamorph of H. brev­ipes, with its loose verticils of lageniform phialides is better placed in T. sect. Trichoderma. Various Hypo­crea species have been linked to each of these sec­tions (Bissett, 1991a,b; Doi and Doi, 1979; Domsch et al., 1980), but the sections-whichare derived from Rifai’s (1969) aggregate species-are based en­tirely on form. Our knowledge of Hypocrea and Trich­oderma is not sufficiently advanced to enable predic­tion of an aggregate species (Rifai, 1969) or section (Bissett, 1991a) of Trichoderma, or type of Hypocrea from, respectively, an unknown Hypocrea or Tricho­derma.

A remarkable feature of cultures of H. poronioidea was the formation of a hyphomycetous synanamorph in association with incipient stromata in self-fertile cultures. Conidia of this synanamorph are colorless and held in conspicuous drops of liquid at the tips of Acremonium-like conidiophores. Conidia are ca­pable of germination so that while these may have a spermatial role, they are also capable of dissemina­tion. This synanamorph is similar to the anamorphs formed by species of Hypocrea that have effused stro­mata such as H. lactea (Fr.: Fr.) Fr., H. pulvinata (Ri­fai and Webster, 1966) and other similar species (Doi 1972).

MATERIALS AND METHODS

Single ascospores were isolated with the aid of a mi­cromanipulator on cornmeal dextrose agar medium (CMD: Difco cornmeal agar + 2 g/l dextrose) at 20 C. Colony characters were taken from CMD, malt ex­tract agar (ME), and potato dextrose agar (PDA, Dif­co). Colonies were grown at 20-21 C under condi-

WITH STIPITATE STROMATA 303

tions of 12 h darkness/12 h cool white fluorescent light. Colony colors are taken from Kornerup and Wanscher (1978).

Dry specimens were rehydrated briefly in 3% (aq.) KOH and the mounts were then flooded with water. Measurements and photographs were taken of ma­terial mounted in water with the following excep­tions. Perithecial sections were mounted in 100% lac­tic acid. The optical brightener calcofluor (Sigma Chemical Co., 0.05% w/v in sodium phosphate buff­er at pH 8) was used for fluorescence microscopy. Anamorph measurements are taken from CMD.

Four types of microscopy were used in this study. These are indicated in the legends to the illustrations as brightfield (BF), fluorescence (EL), differential in­terference contrast (DIC), and phase contrast (PC).

The conventions KOH+ and KOH- are used. KOH+ indicates that perithecia and/or stromata be­come some color of red in 3% KOH and yellow in 100% lactic acid. KOH- indicates that no red color changes were observed in 3% KOH or 100% lactic acid.

Representative isolates have been deposited in the American Type Culture Collection (ATCC) and the Centraalbureau voor Schimmelcultures (CBS).

DESCRIPTIONS OF THE SPECIES

1. Hypocrea brevipes (Mont.), Saccardo, Michelia 1:

Cordyceps brevipes Montagne, Syll. gen. crypt. 201. 1856. Podocrea brevipes (Mont.) Sacc. & D. Sacc., Syll. Fung. 17: 799. 1905. Podostroma brevipes (Mont.) Seaver, Mycologia

2: 66. 1910.

Anamorph. Trichoderma sp. FIGS. 10-15

Stromata capitate-stipitate to pulvinate and then centrally attached with margins free; stroma some­times appearing to have formed through the lateral fusion of adjacent stromata with more or less conspic­uous fissures at points of joining of adjacent stromata, caps of individual stromata circular in outline, 2-8 mm diam, hemispherical, margin slightly inrolled or not, in colors of dark to grayish brown (6E-F7-8), KOH-, surface glabrous, wrinkled and perithecial elevations not evident or slightly tuberculate from perithecial el­evations, ostiolar openings visible as small viscid dots; undersurface of cap concolorous, slightly velvety or not with velvet continuing on stipe or not. Stipe cen­tral, 1.5-5 mm long, 0.75-1 mm diam, adjacent stipes often appearing to have fused, with fissures demarking the individual stipes, KOH-. Cells of stromal surface,

304 MYCOLOGIA

SAMUELS AND LODGE: HYPOCREA WITH STIPITATE STROMATA 305

FIGS. 10-13. Hypocrea brevipes Trichoderma anamorph on CMD. From van der Gucht & L. De Meester 92-14325b. Scale bars: 10-12 = 50 µm; 13 = 10 µm.

in surface view, angular, 10-15 µm diam, walls 1 µm thick, KOH-. Stromal surface region ca. 50 µm wide, cells pseudoparenchymatous, 6-10 µm diam, walls 1.0-1.5 µm thick, pale yellow, colorless in lactic acid. Tissue below the stromal surface of loosely inter­twined, 3 µm wide, thin-walled hyphae. Cells at sur­face of stipe brick-like and elongated parallel to the long axis of the stipe o not elongated, 15 µm long, 7.5 µm wide, walls 1.5-2.0 µm thick. Perithecia crowd­ed below the stromal surface, elliptic, (n = 13) 245­328 µm high, 128-214 µm wide; cells at perithecial apex around ostiolar opening small and pseudopar­enchymatous, thin-walled, yellow in lactic acid; ostiolar canal 62-104 µm long, periphysate. Tissue below per­ithecia of vertically oriented, brick-like cells to 50 µm long, 10 µm wide, walls ca. 1 µm thick, nodose ele­ments lacking, colorless. Asci cylindrical, (n = 66) (60.0-) 71.8-87.6 (-97.7) × (3.0-) 4.2-5.6(-6.6) µm, apex thickened and with a ring; 8-spored, ascospores uni­seriate and often with overlapping ends, completely filling each ascus. Part-ascospores dimorphic, distal part subglobose to conic, (n = 71) (3.0-) 3.5-4.3 (-4.7) × (2.4) 3.1-3.9 (4.7) µm, proximal part wedge-shaped, (3.0-) 3.6-5.0 (-6.1) × (2.2-) 2.2-3.2 (-3.4) µm; hyaline, finely spinulose.

A few ascospores germinated within 48 h. On CMD

> 9 cm diam within 6 da; aerial mycelium lacking, diffusing pigment lacking; conidia forming in a sin­gle broad, continuous band around the margin, or in 2-3 concentric rings. Aggregates uniformly cot­tony with Fertile branches protruding, easily removed from the agar surface; deep green (27E8) or grayish green (28D6) fading through gray to near white at the margin. Colonies on PDA > 9 cm diam within 6 da, no diffusing pigment formed; conidia formed profusely in dense concentric sings alternating with mycelial. production, grayish- to deep green (28D-E7­8) in the middle, progressively lighter green toward the margin where conidia are nearly white. Colonies on ME > 9 cm within 6 da, no diffusing pigment formed; conidial production much as on PDA but conidia developing more slowly on ME and some­times with yellow pigment in conidia formed around the center of the colony. Main axes of conidiophores formed on CMD 3-4 µm wide, fertile branches 24­35 µm long, infrequently rebranched; phialides aris­ing singly along the length of branches and in cru­ciate whorls of ca. 3 at branch tips. Phialides lageni­form, (n = 30) (3.6-) 5.2-8.2 (-10.5) µm long, (2.4-) 2,7­3.5 (-4.3) µm wide in the middle, (1.5-) 1.9-2.5 (-2.9) µm wide at the base. Hypha or branch immediately subtending each phialide (n = 30) (2.0-) 2.4-3.4

FIGS. 1-9. Hypocrea brevipes. 1. Stroma. 2. Median longitudinal section through a mature perithecium. 3. Cells at stromal surface. 4. Detail of perithecial apex. 5. Detail of stromal suface between perithecia. 6. Internal tissue of stroma below perithecia. 7. Cells at surface of stipe. 8, 9. Asci; ascal apex visible in 9. Fig. 1 = BF 2-9 = DIC; Fig. 9 stained with cotton blue to reveal ascal apex. Figs. 10-13 = PC. All from van der Gucht & L. De Meester 92-14325b. Scale bars: 1, 8, 9 = IO µm; 2 = 100 µm; 3-7 = 50 µm.

306

FIGS. 14-16. Hypocrea brevipes. 14, 15. Trichoderma anamorph on CMD. 16. Ascus and ascospores. FIGS. 17, 18. H. capitata. 17. Ascus and ascospores. 18. Trichoderma anamorph from CMD. 14, 15 from van der Gucht & L. De Meester 92-14325b; 16 from the type; 17, 18 from Rossman 3298. Scale bars = 10 µm.

(-2.9) µm wide. Conidia oblong to ellipsoidal, (n = Known distribution. England (Doi, 1975), French 31) (2.9-) 3.1-3.9 (-4.7) × (2.0-) 2.2-2.8 ()-2.9) µm, lack- Guiana, Japan (Doi, 1975), New Guinea, Puerto Rico. ing a visible basal abscission scar, smooth, green. HOLOTYPE. FRENCH GUIANA. [locality and Chlamydospores not seen. date unknown], [on decorticated wood], Leprieur

Habitat. Decaying wood. 1073 (PC! isotype BPI-Lloyd 715550!).

SAMUELS AND LODGE: HYPOCREA

Additional specimens examined. NEW GUINEA, MADANG PROV.: Ramu Region, road to Bundi, 05°44'N, 145°19'E, on decayed wood, 27 May 1992, K. van der Gucht & L. De Meester 92-14325b (Sa­muels culture 92-76) (BPI, GENT). PUERTO RICO. El Yunque, [host unknown], 4 Dec. 1912, F J. Seaver (BPI 630179).

Notes. Hypocrea brevipes was cultured from the re­cent collection from New Guinea. The above descrip­tion is drawn from the three collections cited above. The stromata of the New Guinea and Puerto Rican collections appear to have resulted from the fusion of two or more adjacent stromata. The type collection consists of only a single stroma that has a longitudi­nally furrowed stipe, but that is not obviously com­pound. The undersurface of the cap, and the stipe of the type collection are slightly velutinous whereas the stromata of the other two collections are either glabrous or, at most, slightly velutinous.

Doi (1975) accepted a wide range in size and mor­phology for H. brevipes, reporting the species from temperate and tropical latitudes,

2. Hypocrea poronioidea A. Möller, Phycom. und As­com. p. 295. 1901. FIGS. 19-40

Podocrea poronioidea (A. Möller) Sacc. & D. Sacc., Syll. Fung. 17: 799. 1905. = Podostroma orbiculare Chardón, Mycolegia 13:

286. 1921.

Anamorph: Trichoderma sp. FIGS. 28-40

Stromata capitate-stipitate to pulvinate and then centrally attached with margins free; cap circular in outline, (2.0-) 3.0-6.5 (-10.0) mm diam, slightly con­vex and margin slightly inrolled, in colors of brown (6E8, 7E7), yellowish brown (5D8, 5E8), light brown (6D8), or brownish gray (6F8), KOH-, surface gla­brous, plane and perithecial elevations not evident, ostiolar openings visible as dark brown or black dots against the lighter brown background; undersurface of cap often lighter or darker in color, apricot yellow to Indian yellow (5B6-7), linoleum brown (5E7), or brown (6E8), KOH-, velvety with velvet continuing on stipe. Stipe central, to 7 mm long, 1.0-2.5 mm diam, or lacking, concolorous with underside of cap, vel­vety. Cells of stromal surface, in surface view, lacking a definite outline or angular, walls ca. 1.5 µm thick. In section, stromal surface clearly differentiated from underlying tissue, crustose, 15-25 µm wide, cells an-

WITH STIPITATE STROMATA 307

gion of the underside of the cap and of the stipe circular to angular, ca. 7 µm diam, thin-walled, pro­ducing hyphae to 100 µm long, ca. 5 µm wide, sep­tate, unbranched through most of the length and with many free ends, reddish brown, not changing color in lactic acid. Perithecia crowded below the stromal surface, oblong, (n = 50) (200-) 237-281(­300) µm high, (76-) 89-131 (-162) µm wide; cells at perithecia1 apex around ostiolar opening inter­twined, short, and conspicuously thick-walled, form­ing “shoulders” on the perithecium as seen in lon­gitudinal section, yellow in lactic acid; ostiolar canal 22-79 µm long, periphysate. Tissue of stromal inte­rior below perithecia densely disposed 5-6 µm wide hyphae with walls conspicuously thickened, ca. 1.5 µm; swollen or nodose elements lacking. Asci cylin­drical, (n = 172) (41-) 51-69 (-85) × (2.5-) 3.0-4.6 (­6.4) µm, apex thickened and with a ring; 8-spored: ascospores at first uniseriate but at maturity becom­ing transversely oriented or overlapping, completely filling each ascus. Part-ascospores dimorphic, distal part globose to subglobose, (n = 185) (1.9-) 2.6-2.9 (-3.2) × (1.5-) 1.9-2.5 (-2.8) µm; proximal part ob­long, (2.0-) 2.5-5.3 (-4.5) × (1.4-) 1.7-2.1 (-2.5) µm; ma­turing slowly in the ascus, hyaline, ultimately becom­ing finely spinulose.

Ascospores germinating in low percentage within 36 h. Colonies on CMD and ME > 9 cm diam within 6 da; aerial mycelium and pigment lacking, conidia forming only after 10 da on CMD, in 1-3 mm diam pulvinate tufts at the colony margin. Colonies on PDA > 9 cm diam within 6 da; aerial mycelium scant, pigment lacking; conidial production beginning within 6 da. Conidia on ME and PDA forming in more or less uniformly distributed pulvinate tufts that are cottony and that have protruding branches that are fertile to the tip as well as with long protruding branches that are fertile only at the tip; on CMD, ME and PDA deep green (28D8 to 30D8) or pea green (29D5); conidial production on CMD is considerably less than on ME or PDA. Main axes and lateral branches 1.8-3.2 µm wide; phialides arising singly from the main axis or lateral branches, or arranged in ‘cruciate’ whorls of 3-5 at the tips of short branch­es, or more densely clustered, tending to be botryose. Phialides lageniform and widest in the middle, (n = 40) (3.6-) 4.2-7.4 (-10.0) µm long, (1.9-) 2.1-2.7 (-3.1) µm wide in the middle, (1.0-) 1.2-l.8 (-2.3) µm wide at the base. Hypha or branch immediately subtend­ing each phialide (n = 30) (1.8-) 2.2-3.0 (-3.2) µm

gular and ca. 7 µm diam, or almost brick-like and 4- wide. Conidia ellipsoidal, (n=48) (1.9-) 2.3-2.9 (-3.0) 6 × 3 µm, walls 1-2.5µm thick, yellow in lactic acid. × (1.5-) 1.6-2.2 (-2.4) µm, lacking a visible basal ab-Tissue immediately below the stromal surface of scission scar, smooth, green. Acremonium-like conidi­loosely intertwined, 2-3 µm wide, branching and sep- ophores arising from cottony, incipient stromata; tate, smooth-walled hyphae. Cells of the velvety re- conidiogenous cells 5-30 µm long, straight, smooth,

308 MYCOLOGIA

FIGS. 19-27. Hypocrea poronioidea. 19. Habit of two stromata. 20. Cells at stromal surface in the area of the ostiolar opening (arrow). 21. Median longitudinal section through a mature perithecium. 22. Detail of stromal surface between perithecia. 23. Internal tissue of stroma below perithecia. 24. Detail of perithecial apex. 25. Surface of stipe. 26, 27. Asci. Fig. 19 = BF; 20-22, 24-27 = DIC 23 = FL. Figs. 19, 26 from Rick 220 (S-Rehm), 20, 23-25 from “authentic” (FH-GEN), 21, 22, 27 from GJS 92-29. Scale bars: 19 = 10 mm; 20-25 = 50 µm, 26, 26 = 10 µm.

SAMUELS AND LODGE: HYPOCREA WITH STIPITATE STROMATA 309

FIGS. 28-35. Hypocrea poronioidea, synanamorphs. 28-32.Trichoderma synanamorph. 33-35. Acremonium-like synanamorph from incipient stromata. Figs. 28, 30 = FL; 29, 33, 34 = PC; 31, 32, 35 = DIC. Figs, 28-30from SNA, 31, 32 from PDA, 33­

=35 from CMD. Scale bars: 28-30, 33, 34 = 10 µm; 31, 32, 35

tip 1 µm wide, lacking periclinal thickening, not flared, base 2-3 µm wide. Conidia oblong, 7-13 × 3-4 µm, unicellular, lacking a basal abscission scar, hya­line. Chlamydospores not observed.

Habitat. On bark. Known distribution. Brazil, Dominica. Puerto Rico,

Uganda. TYPE. BRAZIL.. Sta. Catharina pr. Blumenau, in lig­

no putrido, leg. A. Möller (FH-GEN; specimen an­notated as “authentic”).

Additional specimens examined. BRAZIL. Estrella,

50 µm

1921, Rick s. n. (BPI-Lloyd 6106); São Leopoldo, in ligno frondoso, 1907, Rick Fungi Austro-Americani 220 (HBG-Magnus, HBG-Hamburgense, S-Rehm); São Leopoldo, ad ligno, Rick 109b (S-Bresadola). DOMINICA. Springfield Estate, on wood, 21 Jun. 1970, A. Y. Rossman 246 (BPI 630172, CUP-DO 42). PUERTO RICO. Mayaguez, on logs, 11 Dec. 7915, B. Fink 239 (BPI 630180, HOLOTYPE of P. orbiculare); on log, 12 Dec. 1915, B. Fink 922 (BPI 630181, as P. orbiculare); Luquillo Mts., Caribbean National Forest, El Verde Research Area, on log, 11 Jul. 1992, D. J.

310 MYCOLOGIA

FIGS. 36-40. Hypocrea poronioidea. 36, 37. Trichoderma synanamorph from PDA. 38. Acremonium-like synanamorph from incipient stroma on CMD. 39. Median longitudinal section through a whole stroma (diagramatic). 40 Ascus and ascospores. Figs. 36, 38 from GJS 93-29, 37 from GJS 92-29, 39 from Lloyd 6106; 40 from “authentic” (FH-GEN). Scale bars = 10 µm except 39 = 2 mm.

Lodge PR-913 (BPI 802499), El Verde Research Area, 1992, D. J. Lodge s.n. (Samuels culture 92-29, BPI); trail to the Radiation Center, on log, 23 Mar. 1993, on log, 22 Nov. 1992, S. M. Huhndorf 49 & D.J-D. J. Lodge PR-1026 (BPI); El Verde Research Area, Lodge PR-1081 (BPI, NY); El Verde Research Area, along La Prièta Trail, on fallen tree with bark, 1 Mar. trail to Río Sonadora, on right side just after leaving

SAMUELS AND LODGE: HYPOCREA WITH STIPITATE STROMATA 311

the El Verde Field Station, elev. 350 m, on log, 15 Dec. 1992, D. J. Lodge PR-961 (Samuels culture 93­29, ATCC 90748, CBS 37494; BPI). UGANDA. Kibale Natl. Park, Ngogo Station, on decorticated wood, 2 Aug. 1995, K.T. Hodge U0267 (BPI, Samuels culture 95-133).

Illustrations. Möller (1901, Taf. II, Fig. 37). Char­dón (1921, Pl. 13, Fig, 11). Lloyd (1923, Pl. 233, Fig. 2386).

Notes. Three recent collections of Hypoma poron­ioidea, two from Puerto Rico and one from Uganda, were brought into culture. The original isolation of Samuels 92-29 was made from tissue of the stipe. As­cospores were subsequently isolated from stromata that formed in culture.

This species was originally described by Möller (1901) from southern Brazil, and was redescribed by Chardón (1921) as Podostroma orbiculare from Puerto Rico. The species has apparently neither been re­ported nor described under other names from out­side of these areas (Rick, 1906; Theissen, 1911).

Hypocrea poronioidea is characteristic in that stro­mata comprise a nearly circular, brownish cap that is situated on a short stipe. This aspect is suggestive of stromata of the xylariaceous genus Poronia, hence the species epithet. Less frequently the stipe may be reduced and thus the cap is sessile; however, in these cases the stroma is attached at a central point and the margins are free.

Hypocrea poronioidea was included in Podocrea (Sacc.) Lindau, which is Podostroma (following Boe­dijn, 1938). While H. poronioidea, because of its flat­tened cap, does not fit in Podostroma in the sense of Boedijn, it is morphologically similar to P. brevipes. Stromata of P. brevipes are larger in stature, the cap is tuberculate from the perithecial elevations, and the ascospores are larger than those of H. poronioidea. On the basis of its stromal anatomy, H. poronioidea is re­ferable to Hypocrea sect. Hypocrea subsect. Hypocrea (Doi, 1972). Within that subsection, however, the per­ithecial apex of H. poronioidea that is formed of dense­ly interwoven, thick walled hyphae that are yellow in lactic acid is distinctive, as is the internal tissue of the stroma that is formed of thick walled hyaline hyphae.

Single part-ascospores were isolated from asci of H. poronioidea of the Puerto Rican and Ugandan collec­tions. The collections from both countries were het­erothallic. An analysis of mating-type segregation was undertaken with the Puerto Rican collections. Cul­tures derived from eight part-ascospores in each of several asci were self fertile, producing mature peri­thecia in turbinate stromata at the periphery of the colony. Colonies derived from the other eight part­ascospores produced conidia only. They did not form perithecia when mated among themselves or with the

self fertile colonies. Perithecia formed on CMD and PDA in unsealed petri dishes incubated at room tem­perature (ca. 21 C); they did not form in petri dishes incubated in an incubator at 20-21 C with alternating (21 h/12 h) darkness and cool white fluorescent light. Study of the genetics of sexuality in this species is continuing.

3. Hypocrea capitata Samuels, sp. nov. FIGS. 41-59

Stromata brunnea, breviter stipitata, capitata. Caputa convex vel hemisphaerica, tuberculata, 1.5-3 mm diam. Per­ithecia (190-) 197-219 (-241) µm alta, (127-) 132-162 (-182) µm lata. Asci cylindrici, (43-) 46-55 (-59) × (2.7-) 3.3-4.3 (-4.9) µm, ad apicem incrassati. Ascosporae bicellulares, ad sep­tum disarticulatae; parte distali globosa vel subglobosa, (2.0­) 2.1-2.7 (-2.9) × (1.8-) 1.9-2.6 (-2.9) µm parte proximi oblon­ga vel cuneiformi, (2.3-) 2.4-3.0 (-3.2) × (1.7-)1.8-2.2(-2.3) µm, hyalinae, minute verrucosae.

Anamorphe Trichoderma sp. Holotypus in cortice, Guyana, leg. A. Rossman 3298

(BPI).

Anamorph: Trichoderma sp. FIGS. 53-59

Stromata capitate-stipitate to pulvinate and then centrally attached with margins free; cap circular in outline, 1.5-3 mm diam, convex to nearly hemispher­ical, margin slightly inrolled, in colors of brown (6E F8), KOH-, surface glabrous, slightly tuberculate from perithecial elevations, ostiolar openings visible as darker brown dots against the lighter brown back­ground; undersurface of cap slightly lighter in color, KOH-, velvety with velvet continuing on stipe. Stipe 0.5-1.0 mm long, 0.5-10 mm diam, central, concol­orous with underside of cap, velvety. Cells of stromal surface, in surface view, lacking a definite outline or angular, walls to 3 µm thick. In section, stromal sur­face clearly differentiated from underlying tissue, crustose, 15-25 µm wide, cells angular and ca. 10 × 5-6 µm, walls 1.5 µm thick, yellow in lactic acid. Tis­sue immediately below the stromal surface of loosely intertwined, 2-3 µm wide, branching and septate, smooth-walled hyphae. Cells of the velvety region of the underside of the cap and of the stipe circular to angular, ca. 7 µm diam, thin-walled, producing in­conspicuous hyphal hairs < 25 µm long, septate, un­branched. Perithecia crowded below the stromal sur­face, oblong to elliptic in section, (n = 18) (190-) 197­219 (-241) µm high, (127-) 1.32-162 (-182) µm wide; cells at perithecial apex around ostiolar opening in­tertwined, short and conspicuously thick-walled, forming “shoulders” on the perithecium as seen in longitudinal section, yellow in lactic acid; ostiolar ca­nal 43-60 µm long, periphysate. Tissue of stromal interior below perithecia densely disposed 3 µm wide

312 MYCOLOGlA

SAMUELS AND LODGE: HYPOCREA WITH STIPITATE STROMATA 313

FIGS. 53-59. Hypocrea capitata, Trichoderma anamorph from CMD. Figs. 53, 54, 56, 57, 59 = PC; 55 = DIC; 58 = FL. All from Rossman 3298. Scale bars: 53, 54 = 50 µm, 55-59 = 10 µm.

FIGS. 41-52. Hypocrea capitata. 41, 42. Habit of stromata. 43. Median longitudinal section through a mature stroma. 44. Median longitudinal section through a mature perithecium. Detail of perithecial apex. 46. Stromal surface showing outlines of cells. 47. Detail of stromal surface. 48. Cells of stipe. 49. Internal tissue of stroma below perithecia. 50-52. Asci and ascospores. Figs. 41-43 = BF; 44-50, 52 = DIC; 51 = PC. All from Rossman 3298. Scale bars: 41 = 2 mm, 42 = 2 mm, 43 = 500 µm, 44-49 = 50 µm, 50-52 = 10 µm.

314 MYCOLOGIA

branched and septate hyphae with walls 1 µm thick; swollen or nodose elements lacking. Asci cylindrical, (n = 30) (43-) 46-55 (-59) × (2.7-) 3.3-4.3 (-4.9) µm, completely filled with ascospores; apex thickened and with an obscure ring; 8-spored, ascospores uni­seriate or partly biseriate. Part-ascospores dimorphic, distal part globose to subglobose, (n = 30) (2.0-) 2.1­2.7 (-2.9) × (1.8-) 1.9-2.6 (-2.9) µm; proximal part ob­long to narrowly wedge-shaped, (22%) 2.4-3.0(-3.2) × (1.7-) 1.8-2.2 (-2.3) µm; hyaline, ultimately becoming finely spinulose.

Ascospores germinating in low percentage within 24 h on CMD at 20 C. Colonies on CMD > 9 cm diam within 6 da, but growing slowly; aerial mycelium scant to lacking; a pale yellow pigment slowly devel­oping after 6 da and spreading through the agar; Conidia on CMD beginning to form in dense ‘balls’ or aggregates in the aerial myclium. Within 2 weeks conidia 29D-E8 (deep green). Colonies on PDA > 9 cm diam within 6 da; aerial mycelium scant, cottony, with a spreading yellow pigment (3B-C5-6: absinthe yellow, mustard yellow, grayish yellow) in colony re­verse; conidial production beginning within 6 da; co­nidia slowly turning green, after 14 da conidia 30E8 (parrot green) in the center to 30D6-8 (foliage green) at the margin. Colonies on ME > 9 cm within 6 da, much as on PDA but sterile; conidia developing slowly, eventually greenish yellow, after 14 da conidia 3C-D8 (olive yellow) to nearly white. Main axes of conidiophores formed on CMD 5-10 µm wide, fertile branches 2.5-4.5 µm wide, to 15 µm long, tending to radiate in groups of 3-4 from a central point, each rebranching and bearing a penicillus of 3-4 phiali­des. Phialides lageniform to ampulliform, (n = 30) (4.2) 5.1-6.9 (-7.9) µm long, (2.1-) 2.6-3.6 (-4.2) µm wide in the middle, (1.2-) 1.9-2.3 (-2.9) µm wide at the base. Hypha or branch immediately subtending each phialide (n = 30) (2.6-) 2.9-3.9(-4.4) µm. Conidia ob­long to ellipsoidal, (n = 35) (2.5-)2.6-3.2 (-3.7) × (1.5) 1.7-2.3 (-2.5) µm, lacking a visible basal abscis­sion scar, smooth, green. Chlamydospores not seen.

Habitat. Decorticated wood. Known distribution. French Guiana, known only

from the type. HOLOTYPE. FRENCH GUIANA. Route de Beli­

zon, 15 km from road N2, track to Montagne Tortue, 52°21'N, 04°25'W, on decorticated wood, 19 Feb. 1988, A. Y. Rossman 3298 (Samuels culture 88-11, ATCC 96281) (BPI).

Notes. Hypocrea capitata, H. poronioidea and H. brevipes differ most conspicuously in their ana­morphs. The stroma of the only known collection of H. capitata is considerably smaller than are the stro­mata of the other two species. The stromal surface of H. brevipes is slightly tuberculate from the perithe­

cial elevations, whereas the stromal surface of H. pa­ronioidea is plane. Ascospores and conidia of H. cap­itata and H. poronioidea are smaller than those of H. brevipes.

ACKNOWLEDGMENTS

James Plaskowitz processed film and prepared the photo­graphic prints. Anita Phillips provided technical assistance. Katleen van der Gucht, Kathie Hodge and Amy Rossman provided us with fresh collections of H. capitata and H. brevipes. Walter Gams corrected the Latin description. Ger­ald Bills and Amy Rossman provided helpful critical com­ment on the manuscript. We appreciate the loan of speci­mens from the following herbaria: B, FH, GENT, HBG, NY, PC, S.

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