Mycologia, 106(1), 2014, pp. 133–144. DOI: 10.3852/13-117 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

The rise and fall of Sarawakus (Hypocreaceae, Ascomycota) Walter M. Jaklitsch1

INTRODUCTION

Department of Systematic and Evolutionary Botany, Faculty Centre of Biodiversity, University of Vienna, Rennweg 14, A-1030 Vienna, Austria

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. Martı´nez & 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. Po˜ldmaa & 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

Christian Lechat Ascofrance, 64 route de Chize´, 79360 Villiers en Bois, France

Hermann Voglmayr Department of Systematic and Evolutionary Botany, Faculty Centre of Biodiversity, University of Vienna, Rennweg 14, A-1030 Vienna, Austria

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. Key words: Ascomycota, Hypocrea, Hypocreaceae, Hypocreales, pyrenomycetes, rpb2, tef1, Thuemenella, Trichoderma

Submitted 15 Apr 2013; accepted for publication 30 May 2013. 1 Corresponding author. E-mail: [email protected]

133

134

MYCOLOGIA

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 Mu¨ller (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 cyclesequenced 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

JAKLITSCH ET AL.: SARAWAKUS 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 GTRCAT substitution 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). 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 GTRGAMMA and GTRCAT substitution models. Maximum parsimony analyses were performed with PAUP* 4.0b10 (Swofford 2002) with 1000 replicates of heuristic search

135

with random addition of sequences and subsequent TBR branch swapping (MULTREES option in effect, COLLAPSE 5 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 TBR branch 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 5 220815.2746 (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. 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]. 5 Hypocrea rhytidospora Ces., Atti dell’Accademia delle scienze fisiche e matematiche Napoli 8:14 (1879). 5 Sarcoxylon lycogaloides (Berk. & Broome) Cooke, Grevillea 12:50 (1883). 5 Penzigia lycogaloides (Berk. & Broome) Sacc., Syll. fung. (Abellini) 9:569 (1891). 5 Clintoniella rhytidospora (Ces.) Sacc. & P. Syd., Syll. fung. (Abellini) 16:588 (1902). 5 Sarawakus lycogaloides (Berk. & Broome) Lloyd, Mycol. Notes 7:1258 (1924).

136 TABLE I.

MYCOLOGIA Strain numbers and GenBank accession numbers of sequences used for phylogenetic analyses GenBank accession no. Name

Trichoderma aerugineum Trichoderma aggressivum f. europaeum Trichoderma alni Trichoderma amazonicum Trichoderma aureoviride Trichoderma britannicum Trichoderma brunneoviride Trichoderma candidum Trichoderma catoptron Trichoderma ceraceum Trichoderma ceramicum Trichoderma cerinum Trichoderma chlorosporum Trichoderma chromospermum Trichoderma cinnamomeum Trichoderma compactum Hypocrea costaricensis Trichoderma crassum Trichoderma cremeum Trichoderma cuneisporum Trichoderma dacrymycellum Hypocrea danica Trichoderma epimyces Trichoderma estonicum Trichoderma gelatinosum Trichoderma guizhouense Hypocrea lixii Trichoderma longipile Trichoderma lycogaloides Trichoderma nigrovirens Trichoderma parepimyces Trichoderma parestonicum Trichoderma phyllostachydis Trichoderma pleuroti Trichoderma pleuroticola Trichoderma sinuosum Hypocrea spinulosa Trichoderma spirale Trichoderma stramineum Trichoderma strictipile Hypocrea sulawesensis Trichoderma surrotundum Trichoderma tawa Trichoderma thailandicum Trichoderma thelephoricola Trichoderma tomentosum Trichoderma velutinum Trichoderma virens Hypocrea virescentiflava a b

Strain

a

tef1

rpb2

Hypo 414 5 CBS 120541

FJ860608

FJ860516

CBS 100526, CBS 100525; CBS 100525 Hypo 254 5 CBS 120633 IB 95 Hypo 473 5 C.P.K. 2848; Hypo 260 5 CBS 120536 SB SB1 5 CBS 253.62 Hypo 170 5 CBS 121130; Hypo 442 5 CBS 120928 P.C. 59 G.J.S. 02-76 G.J.S. 95-159, G.J.S. 88-28; G.J.S. 95-159 CBS 114576 S357 P.C. 4, G.J.S. 88-33; G.J.S. 88-33 G.J.S. 94-67; G.J.S. 94-68 G.J.S. 97-237; G.J.S. 97-230 CBS 121218 P.C. 21 DAOM 164916; G.J.S. 01-227 G.J.S. 91-125; S112 G.J.S. 91-93 Hypo 233 5 WU 29044 Hypo 402 5 CBS 121273 Hypo 194 5 CBS 120534; Hypo 175 5 C.P.K. 1980 Hypo 501 5 CBS 121556 Hypo 139 5 C.P.K. 1618 S278 Hypo 148 5 C.P.K. 1934 Hypo 80 5 CBS 120953 SL 5 CBS 123493 G.J.S. 99-64 Hypo 357 5 CBS 122769 Hypo 437 5 CBS 120636 CBS 114071 CBS 124387 CBS 124383 Hypo 13 5 C.P.K. 1595 Hypo 424 5 CBS 121280 DAOM 183974; DIS 311D G.J.S. 02-84 Hypo 24 5 C.P.K. 1601 G.J.S. 85-228 G.J.S. 88-73 DAOM 232841; G.J.S. 97-174 G.J.S. 97-61 Hypo 344 5 CBS 120925; Hypo 454 5 CBS 121138 S33 DAOM 230013; C.P.K. 298 DAOM 167652, Gli39; Gli39 P.C. 278

AF348096, AF534614 EU498312 HM142377 FJ860615 KF134795 KF134796 EU498316 AY737742, AY391962 AY737726, AY391963 AY937437, AY391964 FJ860628 KF134797 AF328561, AY391966 AY737728, AY391973 AY737732, AY391979 KF134798 AY737741, AY391980 EU280048, AF534615 AY737736, AF534598 AY737727, AF534600 FJ860633 FJ860634 EU498320 FJ860637 FJ179569 KF134799 FJ179573 FJ860643 KF134800 AY737744, AF534582 FJ860664 FJ860667 FJ860673 HM142382 HM142381 FJ860697 FJ860699 EU280049, AF534626 AY737746, AY391999 FJ860704 AY737730, AY392002 AY737734, AF534594 EU279972, AY392004 AY737748, AY392005 FJ860711 KF134801 AY937415 AY750891, AF534631 AY737749, AY392007

AF545541 EU498349 HM142368 FJ179602 KF134786 KF134787 EU498358 AY391899 AY391900 AF545508b FJ860531 KF134788 AY391903 AY391913 AY391918 KF134789 AY391921 AY481587 KF134790 AF545512 FJ860533 FJ860534 EU498359 FJ860536 FJ179604 KF134791 FJ179608 FJ860542 KF134792 AF545518 FJ860562 FJ860565 FJ860570 HM142372 HM142371 FJ179619 FJ860589 FJ442694 AY391945 FJ860594 AY391954 AF545540 AY391956 AY391957 FJ860601 KF134793 KF134794 AF545558 AY391959

tef1 introns, tef1 exon; rpb2. As Hypocrea atrogelatinosa in GenBank.

JAKLITSCH ET AL.: SARAWAKUS

137

FIG. 1. Phylogram of the best ML tree (lnL 5 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.

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) 3 2–5(–6.2) mm, (0.9–)1.2–2.2(–2.6) mm thick (n 5 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) mm diam (n 5 60); ostioles up to 160 mm diam, pale with dark margin; after rehydration dull orange-red in the

138

MYCOLOGIA

FIG. 2. Trichoderma lycogaloides. a–o. Teleomorph. a. Fresh stromata of WU 32096. Bar 5 2 mm. b, c. Dry stromata of WU 32096. Bars: b 5 1 mm, c 5 0.5 mm. d. Dry stroma of holotype K (M) 177253. Bar 5 1 mm. e. Rehydrated stroma of WU 32096. Bar 5 1 mm. f. Perithecium of WU 32096 in section. Bar 5 0.1 mm. g. Cortical and subcortical tissue of WU 32096 in section. Bar 5 15 mm. h. Subperithecial tissue of WU 32096 in section. Bar 5 15 mm. i. Stroma base of WU 32096 in section.

JAKLITSCH ET AL.: SARAWAKUS 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) mm thick (n 5 30), yellow, dense, opaque, comprising a textura angularis-epidermoidea of thick-walled (up to 4 mm) cells (5–)7–24(–41) 3 (4–)6–14(–20) mm (n 5 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) 3 (5–)7–16(–21) mm (n 5 30) and some broad hyphae. Subperithecial tissue similar to subcortical tissue, comprising a textura epidermoidea of thin-walled, hyaline cells (5–)10–42(–75) 3 (5–)8–20(–30) mm (n 5 35), smaller downward. Basal tissue yellow, comprising a textura epidermoidea-intricata, of (2.7–) 4.3–7.5(–11.2) mm wide hyphae (n 5 30); where free similar to cortex but cells bright yellow and more distinct, with max. 1.5 mm thick walls; directly on bark partly a yellow textura angularis and cells thickerwalled close to the base, up to 24.5 mm long. Perithecia globose to flask-shaped, (330–)360–470(–530) mm high, (250–)300–380(–400) mm diam (n 5 20); peridium (24–)26–35(–42) mm wide at the base, (14–)19–30(–33) mm at the sides (n 5 20), hyaline. Ostioles (81–)108–162(–176) mm long, even with the surface or projecting to 40 mm, (46–)53–70(–77) mm wide at the apex inside, (103–)115–153(–177) mm including walls (n 5 20), periphysate, with parallel, narrowly cylindrical, 2–4.5 mm wide apical cells; ostioles including surrounding cortex sometimes projecting up to 100 mm. Asci (112–)120–147(–170) 3 (9.8–) 10.7–13.0(–14.5) mm (n 5 33), cylindrical, with eight uniseriate ascospores; stipe short; apex 1–2.5 mm 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) 3 (5.0–)7.0–8.5(–9.3) mm, l/w 5 (1.6–)1.8–2.4(–3.1) (n 5 98); including warts (15–) 17–20(–22) 3 (8.3–)9.0–10.8(–11.7) mm, l/w 5 (1.6–) 1.7–2.1(–2.3) ( n 5 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) mm long plates, in section rounded, projecting to 1–2.5(–3) mm

139

in water, warts sometimes (holotype) distinctly swelling in 3% KOH and projecting to for example 4.5 mm, becoming indistinct, then ascospores including warts up to ca. 23 3 14 mm. 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) mm 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) 3 (4.2–) 5.0–6.5(–7.5) mm, l/w 5 (3.5–)4.4–6.7(–7.6), (4.0–) 4.5–5.5(–6.0) mm wide at the base (n 5 20), straight or curved to sigmoid, turning green with age. Conidia oval or subglobose, rarely oblong, (6.5–)9.5–13.5 (–15.0) 3 (5.8–)8.0–11.5(–12.5) mm, l/w 5 1.1– 1.2(–1.3) (n 5 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 5 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

r Bar 510 mm. j, k. Asci of WU 32096. j. In 3% KOH, immature. Bar 5 10 mm. k. In water, mature. Bar 5 15 mm. l–n. Ascospores of WU 32096. l, n. Face view. Bar 5 10 mm. m. Section. Bar 5 15 mm. o. Ascospores of holotype K (M) 177253 (section). Bar 5 15 mm. 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 5 30 mm. s, t. Phialides from MEA after 11 d. Bars: s 5 10 mm, t 5 15 mm. u–w. Conidia. from MEA after 11 d. Bar 5 10 mm.

140

MYCOLOGIA

FIG. 3. a–p. Trichoderma britannicum. a–f. Teleomorph, WU 32095. a. Fresh stromata. Bar 5 1 mm. b. Subiculum. Bar 5 0.3 mm. c. Dry stroma. Bar 5 0.15 mm. d–f. Asci (e, f. in 3% KOH). Bar 5 15 mm. 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 5 30 mm. i. Conidiophore and phialides (CBS 253.62). Bar 5 15 mm. j–l. Conidiophores and phialides (SB). Bars: j 5 15 mm; k 5 20 mm; l 5 10 mm.

JAKLITSCH ET AL.: SARAWAKUS 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). 5 Sarawakus britannicus (Rifai & J. Webster) Samuels & Rossman, Mycologia 84:34 (1992).

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 3 (0.8–)1.0–2.0 mm, 0.5–1.0 mm thick (n 5 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) mm diam (n 5 30); stroma interior from the center whitish to green or olivaceous. Asci (108–)114–133(–137) 3 (5.8–)6.0–7.0(–7.2) mm (n 5 30); stipe (15–)20–30(–34) mm long (n 5 16); cylindrical, containing (6–)8 uniseriate ascospores; with slightly thickened apex, base thickened up to 8 mm, without croziers. Ascospores (10.0–)11.5–14.3(–16.0) 3 (4.5–)5.0–5.7(–6.2) mm, l/w 5 (1.9–)2.1–2.8(–3.2) (n 5 70), oblong, one-celled, often attenuated toward the lower end and subtruncate, olive-green, brown in KOH, distinctly warted, warts flat, projecting to 1 mm, 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 mm wide and swelling in KOH in basal regions, attenuated upward to 2–4 mm wide, bearing no or few asymmetric side branches (each with a

141

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 mm long. Conidia formed in small numbers in wet heads. Phialides (10–)13–21(–30) 3 (2.5–)3.0–4.2(–5.0) mm, l/w 5 (2.2–)3.5–6.4(–10.1), (2.0–)2.5–3.5(–4.8) mm wide at the base (n 5 50), cylindrical, less commonly lageniform, often thickest near the base. Conidia (4.7–)6.8–13.5(–19.3) 3 (4.0–) 4.5–5.5(–6.2) mm, l/w 5 (1.2–)1.5–2.6(–3.7) (n 5 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).

r m. Conidiophore and phialides (CBS 253.62). Bar 5 10 mm. n–p. Conidia. Bars 5 10 mm. q–v. Trichoderma trachycarpum (isotype W 21407). q–s. Dry stromata. Bars: q 5 2.5 mm; r, s 5 0.5 mm. t–u. Asci (u. in cotton blue/lactic acid). Bars 5 20 mm. v. Ascospores in cotton blue/lactic acid with warts. Bar 5 5 mm. Images a, d by J. Kleine.

142

MYCOLOGIA

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

; Thuemenella sordida Yoshim. Doi, Bull. natn. Sci. Mus., Tokyo 15:747 (1972). 5 Sarawakus sordidus (Yoshim. Doi) Samuels & Rossman, Mycologia 84:35 (1992).

Japan, on wood, ascospores green, 13–22 3 6–8 mm. Trichoderma subtrachycarpum (Yoshim. Doi) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804568 ; Hypocrea subtrachycarpa Yoshim. Doi, Bull. natn. Sci. Mus., Tokyo, B 1:14 (1975). 5 Sarawakus subtrachycarpus (Yoshim. Doi) Samuels & Rossman, Mycologia 84:35 (1992).

Colombia, on wood, ascospores hyaline to pale yellow, 13–18 3 7–8 mm. Trichoderma succisum (Rifai) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804569 ; Sarawakus succisus Rifai, Reinwardtia 7:567 (1969).

Japan, on wood, ascospores green, dimorphic, 8–11 3 5–7 mm and 6–9 3 5–6 mm.

Indonesia (Jawa), on bamboos, ascospores green, 15–23 3 8–10 mm.

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

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

; Thuemenella hexaspora Boedijn, Persoonia 3:3 (1964) 5 Sarawakus hexasporus (Boedijn) Samuels & Rossman, Mycologia 84:34 (1992).

; Hypocrea trachycarpa Syd., Ann. mycol. 28:126 (1930). 5 Sarawakus trachycarpus (Syd.) Samuels & Rossman, Mycologia 84:37 (1992).

Indonesia (Jawa), on wood, ascospores green, 12– 26 3 7–12 mm; mostly six per ascus.

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 5 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) mm diam (n 5 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) 3 (6.7–)7.2–8.3(–8.5) mm, stipe (12–)14–26(–30) mm long (n 5 10), cylindrical, with eight unicellular spores, apex 1–1.5(–3) mm thick, croziers absent. Ascospores oblong or ellipsoid, (7.5–)7.8–10.0(–12.0) 3 (4.0–)4.8–6.5(–7.5) mm, l/w 5 (1.1–)1.3–1.9(–2.3) (n 5 30), hyaline to yellowish, distinctly warted; warts to ca. 0.75 mm wide and long.

Trichoderma izawae (Yoshim. Doi) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804565 ; Thuemenella izawae Yoshim. Doi, Bull. natn. Sci. Mus., Tokyo, B 1:28 (1975). 5 Sarawakus izawae (Yoshim. Doi) Samuels & Rossman, Mycologia 84:35 (1992).

Colombia, on wood, ascospores green, 9–14 3 5–6 mm. 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 3 6–8 mm. Trichoderma sordidum (Yoshim. Doi) Jaklitsch & Voglmayr, comb. nov. MycoBank MB804567

JAKLITSCH ET AL.: SARAWAKUS

143

DISCUSSION

ACKNOWLEDGMENTS

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.

We thank Begon ˜ a Aguirre-Hudson of the fungarium Kew and Walter Till at WU for sending and managing collections, Jesko Kleine for sending a fresh collection and images of Sarawakus britannicus, Trix Merkx and Gerard Verkley (CBS) for patience with our cultures, Anton Hausknecht for insertion of specimens into WU and Thomas Læssøe for specimen information. The financial support by the Austrian Science Fund (FWF; project P22081-B17) is gratefully acknowledged.

LITERATURE CITED Boedijn KB. 1934. The genus Sarawakus in the Netherlands Indies. Bull Jard Bot Buitenzorg, se´r. 3, 13:263–265. ———. 1964. The genus Thuemenella with remarks on Hypocreaceae and Nectriaceae. Persoonia 3:1–7. Carbone I, Kohn LM. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553–556, doi:10.2307/3761358 Chamberlain HL, Rossman AY, Stewart EL, Ulvinen T, Samuels GJ. 2004. The stipitate species of Hypocrea (Hypocreales, Hypocreaceae) including Podostroma. Karstenia 44:1–24. Chaverri P, Samuels GJ. 2003. Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): species with green ascospores. Stud Mycol 48:1–116. Corlett M. 1985. Taxonomy of Thuemenella (Chromocreopsis) cubispora. Mycologia 77:272–277, doi:10.2307/3793079 Crous PW, Groenewald JZ, Wingfield MJ, Aptroot A. 2003. The value of ascospore septation in separating Mycosphaerella from Sphaerulina in the Dothideales: a Saccardoan myth? Sydowia 55:136–152. Doi Y. 1972. Revision of the Hypocreales with cultural observations IV. The genus Hypocrea and its allies in Japan (2). Enumeration of the species. Bull Nat Sci Mus (Tokyo) 15:649–751. ———. 1975. Revision of Hypocreales with cultural observations VII. The genus Hypocrea and its allied genera in South America (1). Bull Nat Sci Mus Ser B 1:1–33. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797, doi:10.1093/nar/gkh340 Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis. program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98. Jaklitsch WM. 2009. European species of Hypocrea I. Stud Mycol 63:1–91, doi:10.3114/sim.2009.63.01 ———. 2011. European species of Hypocrea II. Fungal Divers 48:1–250, doi:10.1007/s13225-011-0088-y ———, Gruber S, Voglmayr H. 2008a. Hypocrea seppoi, a new stipitate species from Finland. Karstenia 48:1–11. ———, Komon M, Kubicek CP, Druzhinina IS. 2005. Hypocrea voglmayrii sp. nov. from the Austrian Alps represents a new phylogenetic clade in Hypocrea/Trichoderma. Mycologia 97:1365–1378, doi:10.3852/mycologia.97.6.1365 ———, Po˜ldmaa K, Samuels GJ. 2008b. Reconsideration of Protocrea (Hypocreales, Hypocreaceae). Mycologia 100: 962–984, doi:10.3852/08-101

144

MYCOLOGIA

———, Stadler M, Voglmayr H. 2012. Blue pigment in Hypocrea caerulescens sp. nov. and two additional new species in sect. Trichoderma. Mycologia 104:925–941, doi:10.3852/11-327 ———, Voglmayr H. 2011. Nectria eustromatica sp. nov., an exceptional species with a hypocreaceous stroma. Mycologia 103:209–218, doi:10.3852/10-178 Ju Y-M, Hsieh H-M, Rogers JD, Fournier J, Jaklitsch WM, Courtecuisse R. 2012. New and interesting penzigioid Xylaria species with small, soft stromata. Mycologia 104: 766–776, doi:10.3852/11-313 Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30: 3059–3066, doi:10.1093/nar/gkf436 ———, Toh H. 2008. Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinf 9: 286–298, doi:10.1093/bib/bbn013 Kornerup A, Wanscher JH. 1981. Taschenlexikon der Farben. Muster-Schmidt Verlag, Zu¨rich-Go¨ttingen. Læssøe T, Olsen S. 2004. Sarawakus britannicus—en ny og spændende dansk kernesvamp. Svampe 49:39–40. Liu YL, Whelen S, Hall BD. 1999. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Mol Biol Evol 16:1799–1808, doi:10.1093/ oxfordjournals.molbev.a026092 Lloyd CG. 1924. Miscellaneous Hypocreaceae. Mycol Notes Lloyd Lib Mus 7:1258–1262. Lusk DE. 1990. A new pyrenomycete record for the New World. Mycotaxon 37:273. Maas Geesteranus RA. 1968. Ein zweiter Fundort von Thuemenella britannica. Westfa¨l Pilzbr 7:1–3. Rifai MA. 1969. Sarawakus Lloyd, a genus of the Pyrenomycete family Hypocreaceae. Reinwardtia 7:561–578. ———, Webster J. 1965. An undescribed British species of Thuemenella. Trans Br Mycol Soc 48:409–413, doi:10.1016/S0007-1536(65)80061-4 Rogerson CT. 1970. The Hypocrealean Fungi (Ascomycetes, Hypocreales). Mycologia 62:865–910, doi:10.2307/ 3757604 Rossman AY, Samuels GJ, Rogerson CT, Lowen R. 1999. Genera of Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales, Ascomycetes). Stud Mycol 42:1–248. ———, Seifert KA, Samuels GJ, Minnis AM, Schroers H-J, Lombard L, Crous PW, Po˜ldmaa K, Cannon PF, Summerbell RC, Geiser DM, Zhuang W-Y, Hirooka Y,

Herrera C, Salgado-Salazar C, Chaverri P. 2013. Genera in Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales) proposed for acceptance or rejection. IMA Fungus 4:41–51, doi:10.5598/imafungus.2013. 04.01.05 Samuels GJ, Doi Y, Rogerson CT. 1990. Contributions toward a mycobiota of Indonesia: Hypocreales. Mem New York Bot Gard 59:6–108. ———, Rossman AY. 1992. Thuemenella and Sarawakus. Mycologia 84:26–40, doi:10.2307/3760399 Schroers HJ, Gra¨fenhan T, Nirenberg HI, Seifert KA. 2011. A revision of Cyanonectria and Geejayessia gen. nov. and related species with Fusarium-like anamorphs. Stud Mycol 68:115–138, doi:10.3114/sim.2011.68.05 Silvestro D, Michalak I. 2012. raxmlGUI: a graphical front end for RAxML. Org Divers Evol 12:335–337, doi:10.1007/ s13127-011-0056-0 Stamatakis E. 2006. RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690, doi:10. 1093/bioinformatics/btl446 Sung GH, Sung JM, Hywel-Jones NL, Spatafora JW. 2007. A multigene phylogeny of Clavicipitaceae (Ascomycota, Fungi): identification of localized incongruence using a combinational bootstrap approach. Mol Phylogenet Evol 44:1204–1223, doi:10.1016/j.ympev.2007.03.011 Swofford DL. 2002. PAUP* 4.0b10: phylogenetic analysis using parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates. Voglmayr H, Jaklitsch WM. 2008. Prosthecium species with Stegonsporium anamorphs on Acer. Mycol Res 112:885– 905, doi:10.1016/j.mycres.2008.01.020 ———, ———. 2011. Molecular data reveal high host specificity in the phylogenetically isolated genus Massaria (Ascomycota, Massariaceae). Fungal Divers 46:133–170, doi:10.1007/s13225-010-0078-5 von Arx JA, Mu¨ller E. 1954. Die Gattungen der amerosporen Pyrenomyceten. Beitr Krypt Schweiz 11:1–434. Werle E, Schneider C, Renner M, Vo¨lker M, Fiehn W. 1994. Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Res 22: 4354–4355, doi:10.1093/nar/22.20.4354 Wiens JJ. 1998. Combining datasets with different phylogenetic histories. Syst Biol 47:568–581, doi:10.1080/ 106351598260581