The rise and fall of Sarawakus (Hypocreaceae, Ascomycota)

Abstract

Species of Sarawakus are rarely encountered. Their teleomorphs resemble sexual stages of Trichoderma, formerly called Hypocrea, but differ from that genus by unicellular ascospores. The two greenspored species S. britannicus and the type species of Sarawakus, S. lycogaloides, recently were collected, compared with their types and cultured. We redescribe and illustrate these species and transfer them to Trichoderma, based on phylogenetic analysis of the translation elongation factor 1-alpha encoding gene (tef1), containing the two last introns and exon, and a part of the rpb2 gene, encoding the second largest RNA polymerase subunit. Trichoderma lycogaloides, was found to cluster with Hypocrea sulawesensis, an unusual species of Trichoderma, while T. britannicum is closely related to T. aerugineum of the Spinulosa clade. The anamorphs of the two examined species are characterized by (odd) verticillium-like conidiophores, large cylindrical phialides and conidia, which belong to the largest of those species forming green conidia, oval to subglobose in T. lycogaloides and oblong in T. britannicum. All species currently recognized in Sarawakus are transferred to Trichoderma, introducing the new combinations T. fragile, T. hexasporum, T. izawae, T. sordidum, T. subtrachycarpum, T. succisum and T. trachycarpum and the new name T. rosellum. Trichoderma trachycarpum is redescribed and illustrated from an isotype.

Introduction

The family Hypocreaceae (Hypocreales), whose name is based on its type genus Hypocrea, currently contains 13 teleomorph and seven anamorph genera. Several segregates (Chromocrea, Creopus, Podocrea, Podostroma) of Hypocrea have been reunited with the genus in recent years (Chaverri and Samuels 2003; Chamberlain et al. 2004; Jaklitsch et al. 2008a; Jaklitsch 2009, 2011). Although many species of Hypocrea have not yet been combined in Trichoderma, Hypocrea is now abandoned in favor of its anamorph name Trichoderma in naming holomorphs, based mostly on opinion of non-taxonomists such as biotechnologists in a survey that was initiated by the first author of this paper on the ISTH webpage (ISTH crew pers comm); see also Rossman et al. (2013). As for other genera, little is known about Tilakidium Vaidya, C.D. Naik & Rathod, which might be a synonym of Hypocrea, while Aphysiostroma Barrasa, A.T. Martínez & G. Moreno, described as forming cleistothecial ascomata (see Rossman et al. 1999), resides in section Hypocreanum of Trichoderma. If the isolation was correct, it should be combined in Trichoderma. Also the genera Dialhypocrea Speg. and Pseudohypocrea Yoshim. Doi might prove to belong to this genus, but they have not yet been recollected for sequencing. All other genera so far have been recognized as distinct from Trichoderma. They include Arachnocrea Z. Moravec, Hypocreopsis P. Karst., Hypomyces (Fr.) Tul. & C. Tul., Protocrea Petch, Rogersonia Samuels & Lodge, Sarawakus Lloyd, Sphaerostilbella (Henn.) Sacc. & D. Sacc. and Sporophagomyces K. Põldmaa & Samuels. Except for Trichoderma, the now accepted holomorphic name for Hypocrea/Trichoderma and Gliocladium Corda sensu stricto, which belongs to Sphaerostilbella, all other named anamorphs (Cladobotryum, Mycogone, Sepedonium, Sibirina and Stephanoma) belong to Hypomyces.

Teleomorph genera have been distinguished mainly by differences in stroma anatomy and ascospore characteristics. Here we deal with the genus Sarawakus Lloyd. All species of this genus have distinctly warted, unicellular ascospores. Lloyd (1924) erected the genus based on two specimens labeled Hypoxylon lycogaloides Berk. & Broome (1873) and Hypocrea rhytidospora Ces. (1879). Boedijn (1934) redescribed Sarawakus lycogaloides from Java and thought that the genus is a transitional form between xylariaceous and hypocreaceous fungi. He, in line with other mycologists, however was not sure how to distinguish Sarawakus from the genus Thuemenella Penz. & Sacc.; he later (Boedijn 1964) described a similar fungus as Thuemenella hexaspora in a genus that he thought belonged to the Hypocreaceae. Von Arx and Müller (1954) listed Sarawakus in a broadly conceived Xylariaceae and Thuemenella as a synonym of Sarcoxylon. Rifai (1969) studied Sarawakus lycogaloides and observed that the ascospores in fresh material are dark green or olivaceous but turn brown after preservation. He (Rifai and Webster 1965) however did not recognize the affiliation of the fungus that he described as Thuemenella britannica to Sarawakus, although S. lycogaloides differs from the former fungus mainly by larger and more conspicuously tuberculate ascospores. Since then, Sarawakus lost focus and similar fungi were described either in Thuemenella (syn. Chromocreopsis Seaver fide Corlett 1985) or Hypocrea, although Rogerson (1970) kept Chromocreopsis, Sarawakus and Thuemenella separate. Doi (1972) described Thuemenella fragilis and T. sordida and at the same time two similar fungi as Hypocrea thuemenelloides and H. thuemenello-sulphurea; Doi (1975) added T. izawae and H. subtrachycarpa, which both fit in Sarawakus. Corlett (1985) regarded Thuemenella as a member of the Hypocreales. Samuels and Rossman (1992) finally clarified the concepts of the genera Thuemenella and Sarawakus. They recognized Thuemenella as a member of the Xylariaceae based on the Nodulisporium anamorph of the type species T. cubispora. They also found a heterogeneous mixture of species combined in Thuemenella, that several species have stromata and asci being indistinguishable from those of Hypocrea, detected that Trichoderma- and Gliocladium-like anamorphs had been described for several species (viz. T. britannica, T. fragilis, T. sordida) and therefore transferred several species to Sarawakus. This latter genus is identical to Hypocrea except for the unicellular, non-disarticulating, green to brown, rarely hyaline ascospores. The treatment by Samuels and Rossman (1992) has remained the standard for the taxonomy of the genus Sarawakus. G.J. Samuels (pers comm) anticipated that many species may belong to Hypocrea because of their Trichoderma anamorphs, but phylogenetic affiliations of Sarawakus remained unclear because Samuels and Rossman (1992) failed to culture S. lycogaloides and thus its anamorph has been unknown.

Species of Sarawakus are rare and scattered mostly in tropical regions; therefore it is unlikely that a larger number of species will be found soon. We therefore report here the phylogenetic relationships of two species including the generic type S. lycogaloides and redescribe the teleomorphs and describe for the first time the anamorph of S. lycogaloides. Both species belong to Trichoderma. In addition we transfer all residual species recognized in Sarawakus by Samuels and Rossman (1992) to Trichoderma.

Materials and Methods

Isolates and specimens

Cultures were prepared from ascospores and maintained as described in Jaklitsch (2009), except that for S. lycogaloides PDA (Difco, Detroit, Michigan) without antibiotics was used instead of CMD. Specimen information is given for each species after its description. Representative isolates have been deposited at the Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands (CBS). Specimens were deposited in the Herbarium of the Institute of Botany, University of Vienna (WU).

Morphology

Teleomorph and anamorph morphology was determined as described by Jaklitsch (2009). Cultures used for study of anamorph micromorphology were grown on malt extract agar (MEA; 2% malt extract, 2% agar-agar, both from Merck, Darmstadt, Germany), PDA or SNA at 25 C under alternating 12 h cool white fluorescent light and 12 h darkness. Determination of culture characteristics and morphological analyses of microscopic characters were carried out as described in Jaklitsch (2009); MEA was included in addition. Microscopic observations were made in 3% KOH, except for microtome sections that were examined in lactic acid. Data were gathered with a Nikon Coolpix 4500 or a Nikon DS-U2 digital camera and measured by NIS-Elements D 3.0 software. Methods of microscopy included stereo and Nomarski differential interference contrast (DIC). Kornerup and Wanscher (1981) was used as the color standard.

DNA extraction and sequencing

Extraction of genomic DNA was performed as reported in Voglmayr and Jaklitsch (2011) and Jaklitsch et al. (2012) with the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, Germany). A part of the translation elongation factor 1 alpha (tef1) was amplified with primers EF1-728F (Carbone and Kohn 1999) and TEF1LLErev (Jaklitsch et al. 2005) with these PCR conditions: 1 min at 95 C, followed by 35 or 40 cycles of 95 C for 20 s, 55 or 57 C for 35 s and 72 C for 1 min 30 s. An approximately 1 kb fragment of RNA polymerase II subunit B (rpb2) was amplified with the primer pair fRPB2-5f and fRPB2-7cr (Liu et al. 1999) with these PCR conditions: 1 min at 95 C, followed by 35 or 40 cycles of 95 C for 20 s, 55 C for 35 s and 72 C for 1 min 30 s. PCR products were purified with an enzymatic PCR cleanup (Werle et al. 1994) as described in Voglmayr and Jaklitsch (2008). DNA was cycle-sequenced with the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit 3.1 (Applied Biosystems, Warrington, UK) and the PCR primers.

Analysis of sequence data

An initial NCBI nucleotide BLAST query of rpb2 sequences of Sarawakus britannicus and S. lycogaloides retrieved numerous Trichoderma species with green ascospores as closest match. To infer the phylogenetic affiliations of the two species, a rough phylogenetic analysis was performed on an extensive matrix of rpb2 containing 223 sequences covering all lineages of Trichoderma for which rpb2 sequences were available. Alignments were produced with Muscle 3.6 (Edgar 2004). After exclusion of excessive leading/trailing gap regions, the matrix contained 1055 characters. Maximum likelihood (ML) analysis was performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 0.95 (Silvestro and Michalak 2012), with 100 rounds of random addition of sequences applying the gtrcatsubstitution model. According to this analysis, both Sarawakus britannicus and S. lycogaloides were confirmed to be affiliated with clades of species having green ascospores (data not shown; see Jaklitsch 2009).

Based on this tree, rpb2 sequences of Trichoderma species with teleomorphs having green ascospores and anamorphs belonging to the same clades were retrieved from GenBank and combined with newly generated sequences. The same was done for tef1 sequences, but those sequences, which are deposited in GenBank as separate parts (tef1 introns/tef1 exon), were united into single sequences (Table I).

Table I.

Strain numbers and GenBank accession numbers of sequences used for phylogenetic analyses

		GenBank accession no
Name	Straina	tef1	rpb2
Trichoderma aerugineum	Hypo 414 = CBS 120541	FJ860608	FJ860516
Trichoderma aggressivum f. europaeum	CBS 100526, CBS 100525; CBS 100525	AF348096, AF534614	AF545541
Trichoderma alni	Hypo 254 = CBS 120633	EU498312	EU498349
Trichoderma amazonicum	IB 95	HM142377	HM142368
Trichoderma aureoviride	Hypo 473 = C.P.K. 2848; Hypo 260 = CBS 120536	FJ860615	FJ179602
Trichoderma britannicum	SB	KF134795	KF134786
	SB1 = CBS 253.62	KF134796	KF134787
Trichoderma brunneoviride	Hypo 170 = CBS 121130; Hypo 442 = CBS 120928	EU498316	EU498358
Trichoderma candidum	P.C. 59	AY737742, AY391962	AY391899
Trichoderma catoptron	G.J.S. 02-76	AY737726, AY391963	AY391900
Trichoderma ceraceum	G.J.S. 95-159, G.J.S. 88-28; G.J.S. 95-159	AY937437, AY391964	AF545508b
Trichoderma ceramicum	CBS 114576	FJ860628	FJ860531
Trichoderma cerinum	S357	KF134797	KF134788
Trichoderma chlorosporum	P.C. 4, G.J.S. 88-33; G.J.S. 88-33	AF328561, AY391966	AY391903
Trichoderma chromospermum	G.J.S. 94-67; G.J.S. 94-68	AY737728, AY391973	AY391913
Trichoderma cinnamomeum	G.J.S. 97-237; G.J.S. 97-230	AY737732, AY391979	AY391918
Trichoderma compactum	CBS 121218	KF134798	KF134789
Hypocrea costaricensis	P.C. 21	AY737741, AY391980	AY391921
Trichoderma crassum	DAOM 164916; G.J.S. 01-227	EU280048, AF534615	AY481587
Trichoderma cremeum	G.J.S. 91-125; S112	AY737736, AF534598	KF134790
Trichoderma cuneisporum	G.J.S. 91-93	AY737727, AF534600	AF545512
Trichoderma dacrymycellum	Hypo 233 = WU 29044	FJ860633	FJ860533
Hypocrea danica	Hypo 402 = CBS 121273	FJ860634	FJ860534
Trichoderma epimyces	Hypo 194 = CBS 120534; Hypo 175 = C.P.K. 1980	EU498320	EU498359
Trichoderma estonicum	Hypo 501 = CBS 121556	FJ860637	FJ860536
Trichoderma gelatinosum	Hypo 139 = C.P.K. 1618	FJ179569	FJ179604
Trichoderma guizhouense	S278	KF134799	KF134791
Hypocrea lixii	Hypo 148 = C.P.K. 1934	FJ179573	FJ179608
Trichoderma longipile	Hypo 80 = CBS 120953	FJ860643	FJ860542
Trichoderma lycogaloides	SL = CBS 123493	KF134800	KF134792
Trichoderma nigrovirens	G.J.S. 99-64	AY737744, AF534582	AF545518
Trichoderma parepimyces	Hypo 357 = CBS 122769	FJ860664	FJ860562
Trichoderma parestonicum	Hypo 437 = CBS 120636	FJ860667	FJ860565
Trichoderma phyllostachydis	CBS 114071	FJ860673	FJ860570
Trichoderma pleuroti	CBS 124387	HM142382	HM142372
Trichoderma pleuroticola	CBS 124383	HM142381	HM142371
Trichoderma sinuosum	Hypo 13 = C.P.K. 1595	FJ860697	FJ179619
Hypocrea spinulosa	Hypo 424 = CBS 121280	FJ860699	FJ860589
Trichoderma spirale	DAOM 183974; DIS 311D	EU280049, AF534626	FJ442694
Trichoderma stramineum	G.J.S. 02-84	AY737746, AY391999	AY391945
Trichoderma strictipile	Hypo 24 = C.P.K. 1601	FJ860704	FJ860594
Hypocrea sulawesensis	G.J.S. 85-228	AY737730, AY392002	AY391954
Trichoderma surrotundum	G.J.S. 88-73	AY737734, AF534594	AF545540
Trichoderma tawa	DAOM 232841; G.J.S. 97-174	EU279972, AY392004	AY391956
Trichoderma thailandicum	G.J.S. 97-61	AY737748, AY392005	AY391957
Trichoderma thelephoricola	Hypo 344 = CBS 120925; Hypo 454 = CBS 121138	FJ860711	FJ860601
Trichoderma tomentosum	S33	KF134801	KF134793
Trichoderma velutinum	DAOM 230013; C.P.K. 298	AY937415	KF134794
Trichoderma virens	DAOM 167652, Gli39; Gli39	AY750891, AF534631	AF545558
Hypocrea virescentiflava	P.C. 278	AY737749, AY392007	AY391959

^atef1 introns, tef1 exon; rpb2.

^bAs Hypocrea atrogelatinosa in GenBank.

For combined analyses of tef1 and rpb2 sequences a single representative sequence was selected for each species, preferably from the types. Because several sequences of ex-type strains were short these were replaced by identical or highly similar sequences from other authentic sources where available or by those determined for newly isolated strains. For Sarawakus, all available, newly generated sequences were included in the matrix. Sequence alignments for final phylogenetic analyses were produced with MAFFT 6.847 (Katoh et al. 2002; Katoh and Toh 2008) implemented in UGENE 1.10.0 (http://ugene.unipro.ru), with a maximum of 100 iterative refinements and gap opening penalties of 1.53 for rpb2 and 0.80 for tef1 respectively. The resulting alignments were checked with BioEdit 7.0.9.0 (Hall 1999). After exclusion of excessive leading/trailing gap regions, the combined matrix contained respectively 1435 and 1072 characters from tef1 and rpb2. Before phylogenetic analyses, the approach of Wiens (1998) was applied to test for significant localized incongruence among the two gene partitions, using the level of bootstrap support (Sung et al. 2007). For this the 70% maximum likelihood (ML) bootstrap trees of the individual gene regions were compared; they were calculated with the same parameters as for the combined analysis below. No topological conflicts were observed between these bootstrap trees of tef1 and rpb2 except for an interchanged position of T. ceramicum and T. estonicum within the Ceramicum clade, indicating the absence of significant incongruence and combinability of both matrices (Wiens 1998).

Maximum likelihood (ML) and maximum parsimony (MP) analyses, including selection of models of sequence substitution for the former, were performed as described for tef1 and rpb2 in Jaklitsch and Voglmayr (2011). For ML analyses, 500 rounds of random addition of sequences as well as 1000 bootstrap replicates were computed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 0.95 (Silvestro and Michalak 2012) using respectively the gtrgammaand gtrcat substitution models. Maximum parsimony analyses were performed with PAUP* 4.0b10 (Swofford 2002) with 1000 replicates of heuristic search with random addition of sequences and subsequent tbr branch swapping (multrees option in effect, collapse = maxbrlen, steepest descent option not in effect). All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data. Bootstrap analysis with 1000 replicates was performed in the same way but with five rounds of random sequence addition and subsequent tbrbranch swapping during each bootstrap replicate. Sequence alignment files were deposited in TreeBASE and are available at http://purl.org/phylo/treebase/phylows/study/TB2:S14278.

Results

Molecular phylogenetic analyses

Of the 2507 characters included in the combined matrix, 707 were parsimony informative (413 in tef1, 294 in rpb2). MP analyses revealed eight MP trees with a score of 3854, which show minor topological differences in the position of T. brunneoviride relative to the clade of the T. harzianum complex and within the T. cremeum/sinuosum/surrotundum and the T. ceraceum/cerinum/tomentosum clades (data not shown). The best ML tree is lnL = −20815.2746 (Fig. 1) with ML and MP bootstrap support above 50% given at first and second positions respectively at each node. The ML tree is similar to the MP strict consensus tree except for topological differences in several basal nodes lacking significant MP bootstrap support (Fig. 1 asterisks). In the phylogenetic analyses, Sarawakus lycogaloides and S. britannicus were placed within Trichoderma with green ascospores but these species were not revealed as closely related (Fig. 1). Sarawakus britannicus was placed as sister taxon to T. aerugineum within the highly supported Spinulosa clade with medium (MP) to high (ML) support. The phylogenetic position of S. lycogaloides was less resolved in that it was not contained within a highly supported subclade, while it was revealed as sister taxon to Hypocrea sulawesensis with medium (MP) to high (ML) support.

Fig. 1.

Phylogram of the best ML tree (lnL = −220815.2746) revealed by RAxML from an analysis of the combined tef1-rpb2 alignment, showing the phylogenetic position of the two Sarawakus species (formatted in bold italics). ML and MP bootstrap support above 50% are given at first and second position respectively above or below the branches. Asterisks denote branches in conflict with the MP strict consensus tree.

Taxonomy

Trichoderma lycogaloides (Berk. & Broome) Jaklitsch, Lechat & Voglmayr, comb. nov. Fig. 2 MycoBank MB804561

• ≡Hypoxylon lycogaloides Berk. & Broome, J. Linn. Soc., Bot. 14:120 (1873) [1875].

• = Hypocrea rhytidospora Ces., Atti dell’Accademia delle scienze fisiche e matematiche Napoli 8:14 (1879).

• = Sarcoxylon lycogaloides (Berk. & Broome) Cooke, Grevillea 12:50 (1883).

• = Penzigia lycogaloides (Berk. & Broome) Sacc., Syll. fung. (Abellini) 9:569 (1891).

• = Clintoniella rhytidospora (Ces.) Sacc. & P. Syd., Syll. fung. (Abellini) 16:588 (1902).

• = Sarawakus lycogaloides (Berk. & Broome) Lloyd, Mycol. Notes 7:1258 (1924).

Fig. 2.

Trichoderma lycogaloides. a–o. Teleomorph. a. Fresh stromata of WU 32096. Bar = 2 mm. b, c. Dry stromata of WU 32096. Bars: b = 1 mm, c = 0.5 mm. d. Dry stroma of holotype K (M) 177253. Bar = 1 mm. e. Rehydrated stroma of WU 32096. Bar = 1 mm. f. Perithecium of WU 32096 in section. Bar = 0.1 mm. g. Cortical and subcortical tissue of WU 32096 in section. Bar = 15 μm. h. Subperithecial tissue of WU 32096 in section. Bar = 15 μm. i. Stroma base of WU 32096 in section. Bar =10 μm. j, k. Asci of WU 32096. j. In 3% KOH, immature. Bar = 10 μm. k. In water, mature. Bar = 15 μm. l–n. Ascospores of WU 32096. l, n. Face view. Bar = 10 μm. m. Section. Bar = 15 μm. o. Ascospores of holotype K (M) 177253 (section). Bar = 15 μm. p–w. Culture and anamorph, CBS 123493 (all at 25 C). p. Culture on PDA after 3 wk. q, r. Conidiophores from MEA after 11 d (q) and PDA after 3 wk (r). Bar = 30 μm. s, t. Phialides from MEA after 11 d. Bars: s = 10 μm, t = 15 μm. u–w. Conidia. from MEA after 11 d. Bar = 10 μm.

Stromata scattered or aggregated in groups of 2–3, pulvinate to discoid with depressed center, yellow, becoming green when fresh and mature; when dry 2.5–6(−8) × 2–5(−6.2) mm, (0.9−)1.2–2.2(−2.6) mm thick (n = 20); margin and fertile part for a large part free, often on a thick, short stipe; stipe bright yellow inside; sometimes surrounded by yellow mycelium. Stroma surface smooth, rugose or slightly tubercular, appearing gelatinous or waxy and translucent, yellow, yellow-brown or orange, with large distinct, convex, roundish or longish, orange to black ostiolar or perithecial protuberances (78−)92−235(−340) μm diam (n = 60); ostioles up to 160 μm diam, pale with dark margin; after rehydration dull orange-red in the stereo microscope, surface finely tubercular by slightly projecting, translucent dark green to black perithecial dots; not changing color in 3% KOH.

Stroma anatomy: Cortical layer (30−)38–58(−71) μm thick (n = 30), yellow, dense, opaque, comprising a textura angularis-epidermoidea of thick-walled (up to 4 μm) cells (5−)7–24(−41) × (4−)6–14(−20) μm (n = 35) in section, indistinct in upper part, more distinct with thinner wall downward, and some broad hyphae, covered by a thin amorphous surface layer of collapsed hyaline cells. Cells of lateral cortex tending to be larger. Subcortical tissue a textura epidermoidea of thin-walled, hyaline to yellowish cells (7−)9–32(−51) × (5−)7–16(−21) μm (n = 30) and some broad hyphae. Subperithecial tissue similar to subcortical tissue, comprising a textura epidermoidea of thin-walled, hyaline cells (5−)10–42(−75) × (5−)8–20(–30) μm (n = 35), smaller downward. Basal tissue yellow, comprising a textura epidermoidea-intricata, of (2.7−) 4.3–7.5(−11.2) μm wide hyphae (n = 30); where free similar to cortex but cells bright yellow and more distinct, with max. 1.5 μm thick walls; directly on bark partly a yellow textura angularis and cells thicker-walled close to the base, up to 24.5 μm long. Perithecia globose to flask-shaped, (330−)360–470(−530) μm high, (250−)300–380(−400) μm diam (n = 20); peridium (24−)26–35(−42) μm wide at the base, (14−)19–30(−33) μm at the sides (n = 20), hyaline. Ostioles (81−)108–162(−176) μm long, even with the surface or projecting to 40 μm, (46−)53–70(−77) μm wide at the apex inside, (103−)115–153(−177) μm including walls (n = 20), periphysate, with parallel, narrowly cylindrical, 2-4.5 μm wide apical cells; ostioles including surrounding cortex sometimes projecting up to 100 μm. Asci (112−)120–147(−170) × (9.8−) 10.7–13.0(−14.5) μm (n = 33), cylindrical, with eight uniseriate ascospores; stipe short; apex 1–2.5 μm thick, with a narrow central pore and sometimes a minute refractive ring. Ascospores narrowly ellipsoid to oblong, (13.0−)14.7–17.5(−19.5) × (5.0−)7.0–8.5(−9.3) μm, l/w = (1.6−)1.8–2.4(−3.1) (n = 98); including warts (15−) 17–20(−22) × (8.3−)9.0–10.8(−11.7) μm, l/w = (1.6−) 1.7–2.1(−2.3) ( n = 70), one-celled, in water and lactic acid green to olivaceous when fresh, in KOH and upon storage also in water brown, coarsely warted, warts in surface view appearing as (1.7−)2.5–6(−7) μm long plates, in section rounded, projecting to 1–2.5(−3) μm in water, warts sometimes (holotype) distinctly swelling in 3% KOH and projecting to for example 4.5 μm, becoming indistinct, then ascospores including warts up to ca. 23 × 14 μm.

Cultures and anamorph at 25 C under alternating 12 h darkness/12 h daylight: On CMD growth slow, conidiation effuse, in wet heads, dark green. On PDA centrally inoculated plate entirely covered within 3 wk, becoming hairy by aerial hyphae, turning green from the margin. Conidiation effuse, verticillium-like. On MEA covering a 90 mm Petri plate within 6–8 d, dense, aerial hyphae abundant, ascending up to the lid, colorless, center flat, turning yellow to greenish. Conidiation noticeable after 2 d, effuse, simple, verticillium-like. Conidiophores (3.5−)4–6(−8) μm wide, typically once branched near the base, with scarce unpaired side branches at higher levels, each terminating in a single, rarely two phialides. Phialides cylindrical or lageniform, (22−)26–38(−43) × (4.2−) 5.0–6.5(−7.5) μm, l/w = (3.5−)4.4–6.7(−7.6), (4.0−) 4.5–5.5(−6.0) μm wide at the base (n = 20), straight or curved to sigmoid, turning green with age. Conidia oval or subglobose, rarely oblong, (6.5−)9.5–13.5 (−15.0) × (5.8−)8.0–11.5(−12.5) μm, l/w = 1.1–1.2(−1.3) (n = 30), green, thick-walled, smooth, eguttulate; scar indistinct to truncate.

Distribution

pantropical (Costa Rica, French Guiana, Indonesia, Sri Lanka), also reported from Florida, USA (Lusk 1990); uncommon.

Habitat

on bark of broadleaf trees and probably fungi growing on it.

HOLOTYPE

SRI LANKA, CENTRAL PROVINCE, sine loc., on bark, soc. light corticiaceous fungus, Dec. 1868, G.H.K. Thwaites 1090 (K(M) 177253). Stromata are covered by a whitish layer of conserving agent and partly by cleistothecia.

Other material examined

SOUTH AMERICA, FRENCH GUIANA, Sinnamary, Parcelles Guyaflux, on bark of a broadleaf tree and an ascomycete, soc. white corticiaceous fungus, 30 Apr 2008, C. Lechat CLL 8032 (part: WU 32096; culture SL = CBS 123493).

Notes

Trichoderma lycogaloides is distinctive because of its ascospores that are covered by large, plate-like warts and by its large subglobose conidia formed on odd verticillium-like conidiophores. This species requires richer media (e.g. MEA) for growth than those typically used for Trichoderma, such as CMD or SNA. For additional descriptions and illustrations of this species see Boedijn (1934), Samuels and Rossman (1992) and Rossman et al. (1999).

Trichoderma britannicum (Rifai & J. Webster) Jaklitsch & Voglmayr, comb, nov. Fig. 3a-p MycoBank MB804562

• ≡ Thuemenella britannica Rifai & J. Webster, Trans. Br. mycol. Soc. 48:410 (1965).

• = Sarawakus britannicus (Rifai & J. Webster) Samuels & Rossman, Mycologia 84:34 (1992).

Fig. 3.

a–p. Trichoderma britannicum. a–f. Teleomorph, WU 32095. a. Fresh stromata. Bar = 1 mm. b. Subiculum. Bar = 0.3 mm. c. Dry stroma. Bar = 0.15 mm. d–f. Asci (e, f. in 3% KOH). Bar = 15 μm. g. Culture SB on MEA at 25 C after 35 d. h–p. Anamorph from SNA after 5–21 d. h. Conidiophore and phialides (SB). Bar = 30 μm. i. Conidiophore and phialides (CBS 253.62). Bar = 15 μm. j–l. Conidiophores and phialides (SB). Bars: j = 15 μm; k = 20 μm; l = 10 μm. m. Conidiophore and phialides (CBS 253.62). Bar 5 10 μm. n–p. Conidia. Bars = 10 μm. q–v. Trichoderma trachycarpum (isotype W 21407). q–s. Dry stromata. Bars: q = 2.5 mm; r, s = 0.5 mm. t–u. Asci (u. in cotton blue/lactic acid). Bars = 20 μm. v. Ascospores in cotton blue/lactic acid with warts. Bar = 5 μm. Images a, d by J. Kleine.

Stromata discoid with convex surface to turbinate or pulvinate, at the base surrounded by light brown radial mycelium; dark green when fresh; when dry (0.9−)1.2–2.2 × (0.8−)1.0–2.0 mm, 0.5–1.0 mm thick (n = 8), on a short, thick base, externally clothed with brown hyphae; margin free; surface finely rugose or tubercular, brownish between black perithecia; ostiolar dots absent, inconspicuous or convex to distinctly papillate, (27−)35–64(−90) μm diam (n = 30); stroma interior from the center whitish to green or olivaceous. Asci (108−)114–133(−137) × (5.8−)6.0–7.0(−7.2) μm (n = 30); stipe (15−)20–30(−34) μm long (n = 16); cylindrical, containing (6−)8 uniseriate ascospores; with slightly thickened apex, base thickened up to 8 μm, without croziers. Ascospores (10.0−)11.5–14.3(−16.0) × (4.5−)5.0–5.7(−6.2) μm, l/w = (1.9−)2.1–2.8(−3.2) (n = 70), oblong, one-celled, often attenuated toward the lower end and subtruncate, olive-green, brown in KOH, distinctly warted, warts flat, projecting to 1 μm, not swelling in KOH.

Cultures and anamorph

On CMD and PDA growth slow, colony whitish, finely farinose by scant effuse conidiation; on PDA reverse brown, surface turning yellow-brown. On MEA at 25 C after 5 d colony radius 4–6 mm; colony circular, dense, thick, first whitish yellowish, becoming zonate after a few weeks, turning olive-green to brown with yellow greenish, farinose center; conidiation effuse, on short odd verticilliumlike conidiophores. On SNA colony radius at 25 C after 2 wk 6–10 mm; colony dense, hyaline, turning greenish or olivaceous from conidia. Conidiation following growth, effuse, on aerial hyphae and short odd verticillium-like conidiophores, spreading from the plug. Conidiophores simple, comprising a stipe (main axis) 5–6 μm wide and swelling in KOH in basal regions, attenuated upward to 2–4 μm wide, bearing no or few asymmetric side branches (each with a whorl of phialides or few steep branches) and an apical whorl of few phialides or a broom-like verticil of up to four steeply ascending branches, terminated by solitary phialides or whorls of 2–3 divergent phialides, partly on an intercalary cell. Aphanophialides common, up to 4 μm long. Conidia formed in small numbers in wet heads. Phialides (10−)13–21(−30) × (2.5−)3.0–4.2(−5.0) μm, l/w = (2.2−)3.5–6.4(−10.1), (2.0−)2.5–3.5(−4.8) μm wide at the base (n = 50), cylindrical, less commonly lageniform, often thickest near the base. Conidia (4.7−)6.8–13.5(−19.3) × (4.0−) 4.5–5.5(−6.2) μm, l/w = (1.2−)1.5–2.6(−3.7) (n = 70), variable shape and size, typically oblong and pale olivegreen when fully mature, subglobose, oval or ellipsoid and hyaline when immature, straight or slightly curved, sides sometimes pinched, smooth, finely multiguttulate when fresh; base often truncate.

Distribution

Europe, known from Denmark, Germany and UK.

Habitat

on well decayed wood and bark of broadleaf trees and conifers, also pine needles.

HOLOTYPE

UNITED KINGDOM, ENGLAND, Chatsworth Park, Stand Wood, on dead bark of Pinus sp., 15 Oct. 1962 (according to the label), J.E. Harper, (K(M) 177252; ex herb. Sheffield 2543, ex IMI 90311; ex-holotype culture CBS 253.62). Rifai and Webster (1965) give wood of Quercus as the substrate and as collection date 15 Oct 1961. The material received from K contains two pieces of pine bark.

Other material examined

GERMANY, Sachsen, Leipzig, southern riparian forest on the western bank of the river Pleiße, MTB 4740,121, ca. 100 m, on rotten wood of Quercus robur, 7 Jul 2011, J. Kleine (WU 32095; culture SB).

Several additional records are listed in the database of the British Mycological Society, others were reported from Hessen, Germany, by Maas Geesteranus (1968) and by Læssøe and Olsen (2004) from Denmark (Sjælland, Vestskoven, Trippendalsvej, lat. 55.693022, long. 12.3751986, on conifer wood, 28 Sep 2003, S. Olsen s.n. (C)).

Notes

The studied specimens of T. britannicum contain only few stromata, not allowing microtome sections without destroying the material. Trichoderma britannicum is a distinctive, uncommon species and the only one known from Europe that has a Hypocrea morphology with one-celled, non-disarticulating ascospores. For additional descriptions and illustrations of this species see Rifai and Webster (1965) and Maas Geesteranus (1968).

Ten species have been established and keyed out in Sarawakus (Samuels and Rossman 1992). We see no reason to think that they do not belong to Trichoderma and therefore combine them in this genus. All have green ascospores except T. subtrachycarpum and T. trachycarpum. Trichoderma rosellum, T. sordidum, T. succisum and T. trachycarpum are known only from the type collections. Trichoderma succisum is possibly a synonym of T. hexasporum. The name Sarawakus frustulosus (Berk. & M.A. Curtis) Lar. N. Vasiljeva is not a hypocrealean fungus but a penzigioid Xylaria, recognized as Xylaria frustulosa (Berk. & M.A. Curtis) Cooke (see Ju et al. 2012).

Trichoderma fragile (Yoshim. Doi) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804563

• ≡Thuemenella fragilis Yoshim. Doi, Bull. natn. Sci. Mus., Tokyo 15:745 (1972).

• = Sarawakus fragilis (Yoshim. Doi) Samuels & Rossman, Mycologia 84:34 (1992).

• Japan, on wood, ascospores green, dimorphic, 8–11 × 5–7 μm and 6–9 × 5–6 μm.

Trichoderma hexasporum (Boedijn) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804564

• ≡Thuemenella hexaspora Boedijn, Persoonia 3:3 (1964)

• = Sarawakus hexasporus (Boedijn) Samuels & Rossman, Mycologia 84:34 (1992).

• Indonesia (Jawa), on wood, ascospores green, 12–26 × 7–12 μm; mostly six per ascus.

Trichoderma izawae (Yoshim. Doi) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804565

• ≡Thuemenella izawae Yoshim. Doi, Bull. natn. Sci. Mus., Tokyo, B 1:28 (1975).

• = Sarawakus izawae (Yoshim. Doi) Samuels & Rossman, Mycologia 84:35 (1992).

• Colombia, on wood, ascospores green, 9–14 × 5–6 μm.

Trichoderma rosellum Jaklitsch & Voglmayr, nom. nov. MycoBank MB804566

Replaced synonym: Sarawakus roseus Samuels & Rossman, Mycologia 84:35 (1992), non Trichoderma roseum Pers., Neues Mag. Bot. 1: 92 (1794).

Venezuela, on herbaceous stem, ascospores green, 9–16 × 6–8 μm.

Trichoderma sordidum (Yoshim. Doi) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804567

• ≡ Thuemenella sordida Yoshim. Doi, Bull. natn. Sci. Mus., Tokyo 15:747 (1972).

• = Sarawakus sordidus (Yoshim. Doi) Samuels & Rossman, Mycologia 84:35 (1992).

• Japan, on wood, ascospores green, 13–22 × 6–8 μm.

Trichoderma subtrachycarpum (Yoshim. Doi) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804568

• ≡ Hypocrea subtrachycarpa Yoshim. Doi, Bull. natn. Sci. Mus., Tokyo, B 1:14 (1975).

• = Sarawakus subtrachycarpus (Yoshim. Doi) Samuels & Rossman, Mycologia 84:35 (1992).

Colombia, on wood, ascospores hyaline to pale yellow, 13–18 × 7–8 μm.

Trichoderma succisum (Rifai) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804569

• ≡ Sarawakus succisus Rifai, Reinwardtia 7:567 (1969). Indonesia (Jawa), on bamboos, ascospores green, 15–23 × 8–10 μm.

Trichoderma trachycarpum (Syd.) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804570 Fig. 3q-v

• ≡ Hypocrea trachycarpa Syd., Ann. mycol. 28:126 (1930).

• = Sarawakus trachycarpus (Syd.) Samuels & Rossman, Mycologia 84:37 (1992).

Lectotype in S, selected by Samuels and Rossman (1992). We redescribe and illustrate this species here, based on an isotype in W: Venezuela, Los Rastrojos pr. Puerto La Cruz. Ad truncum subputridum Ingae spectabilis, on blackened wood surface, 31 Dec. 1927, H. Sydow (isotype W 21407, Sydow, Fungi exotici exsiccati 841; as Hypocrea trachycarpa; part separated as Petrak’s Pilzherbarium No. 36207).

Stromata when dry 0.5–3.5 mm diam, 0.3–1 mm thick (n = 8), gregarious or aggregated in small numbers, pulvinate, sometimes discoid, with roundish or irregular outline, broadly attached, but margin free, often wavy; stroma surface smooth or slightly velutinous, with distinct, reddish brown ostiolar dots (40−)50–90(−120) μm diam (n = 30), orange-yellow, ochraceous 5B4-6, more rust 6CD7-8 or dull brownish 5CD5-6 when old. Spore deposits yellow. Asci (92−)96–110(−115) × (6.7−)7.2–8.3(−8.5) μm, stipe (12−)14–26(−30) μm long (n = 10), cylindrical, with eight unicellular spores, apex 1–1.5(−3) μm thick, croziers absent. Ascospores oblong or ellipsoid, (7.5−)7.8–10.0(−12.0) × (4.0−)4.8–6.5(−7.5) μm, l/w = (1.1−)1.3–1.9(−2.3) (n = 30), hyaline to yellowish, distinctly warted; warts to ca. 0.75 μm wide and long.

Discussion

After a long taxonomic history we synonymize the genus Sarawakus, based on the phylogenetic position of its type species S. lycogaloides, with Trichoderma. Unicellular ascospores in teleomorphs of Trichoderma are unusual and to our knowledge described only for the species mentioned above. We show here that ascospore septation is not a generic diagnostic character in the Hypocreaceae. Ascospore septation, which has been used as a primary character to distinguish among species and genera, has been shown to be insignificant in the delimitation of genera in several other groups of the Ascomycota; Crous et al. (2003) for example showed that some species of the phragmosporous genus Sphaerulina Sacc. belong to Mycosphaerella Johanson. As another example, teleomorphs of Fusarium Link were recognized, among others, as Gibberella Sacc. with 2–4-celled ascospores, Haematonectria Samuels & Nirenberg with two-celled ascospores or Neocosmospora E.F. Sm. with aseptate ascospores. These genera currently are amalgamated in the single anamorphic genus Fusarium (Schroers et al. 2011, Rossman et al. 2013). As far as we are aware, no cytological studies on ascospores of the species presented above has been undertaken, therefore the reason for the unicellular ascospores in these species is not clear, although the ascospore shape in T. britannicum may suggest a developmental modification, where either an additional mitosis is lacking or a twocelled “Hypocrea-spore” is designed, but the genetic mechanism of septum formation is suppressed or lost. On the other hand the one-celled ascospores of these species might represent a reversal to an ancestral character state. Whatever the reason, the formation of eight ascospore cells evolved at least twice independently within Trichoderma (Fig. 1) and, considering the morphology of the species for which no sequence data are yet available, it is likely that the former genus Sarawakus is highly polyphyletic within Trichoderma. Also, the number of spores or spore cells in an ascus is not a generic phylogenetically informative character because asci of Protocrea (Jaklitsch et al. 2008b) contain eight ascospores, which disarticulate into 16 cells, but the genus is unrelated to Trichoderma.

Trichoderma lycogaloides clusters with Hypocrea sulawesensis (Fig. 1), which also has green ascospores but differs significantly by white stromata and a unique, peculiar anamorph that forms elongate phragmoconidia in addition to minute unicellular conidia unlike any other species of Trichoderma (Samuels et al. 1990). Trichoderma britannicum on the other hand fits nicely in the Spinulosa clade (Fig. 1) because of its green stromata. It is the second species in this clade in addition to its direct neighbor T. aerugineum, which forms an anamorph.