Recognition of hypoxyloid and xylarioid Entonaema species and allied Xylaria species from a comparison of holomorphic morphology, HPLC profiles, and ribosomal DNA sequences

Mycol Progress (2008) 7:53–73 DOI 10.1007/s11557-008-0553-5

ORIGINAL ARTICLE

Recognition of hypoxyloid and xylarioid Entonaema species and allied Xylaria species from a comparison of holomorphic morphology, HPLC profiles, and ribosomal DNA sequences Marc Stadler & Jacques Fournier & Thomas Læssøe & Christian Lechat & Hans-Volker Tichy & Meike Piepenbring

Received: 22 September 2007 / Revised: 7 January 2008 / Accepted: 15 January 2008 / Published online: 21 February 2008 # German Mycological Society and Springer-Verlag 2008

Abstract The genus Entonaema comprises Xylariaceae with hollow, gelatinous stromata that accumulate liquid. Some of its species, including the type species, appear related to Daldinia from a polyphasic approach, comprising morphological studies, comparisons of ribosomal DNA sequences, and high performance liquid chromatography (HPLC) profiles with diode array and mass spectrometric detection (HPLC-DAD-MS). This methodology was used to study Entonaema pallidum. Its major stromatal constituent was identified as xylaral, a secondary metabolite known from Xylaria polymorpha. This compound was detected in

several Xylaria spp., including the tropical X. telfairii and morphologically similar taxa, whose stromata may also become hollow and filled with liquid. Cultures of E. pallidum resembled those of Xylaria, substantially differing from other Entonaema spp., in their morphology, 5.8S/ITS nrDNA sequences, and HPLC profiles. The type specimen of E. mesentericum was located in the spirit collection of the herbarium B and found to agree morphologically with the nomenclatorily younger E. pallidum. Traces of xylaral were even detected by HPLC-DAD-MS in the spirit in which the fungus had been preserved. Entonaema pallidum is

Taxonomic novelty Xylaria mesenterica (Möller) M. Stadler, Læssøe & J. Fournier.

C. Lechat AscoFrance, 64 route de Chizé, 79360 Villiers-en-Bois, France e-mail: [email protected]

This paper is dedicated to the memory of our colleague Jean-F. Magni. M. Stadler (*) InterMed Discovery GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany e-mail: [email protected] M. Stadler Lehrstuhl für Pflanzensystematik, Department Mycology, University of Bayreuth, Universitätstrasse 30, NW1, 95440 Bayreuth, Germany J. Fournier Las Muros, 09420 Rimont, France e-mail: [email protected] T. Læssøe Institute of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark e-mail: [email protected]

H.-V. Tichy LUFA-ITL-GmbH, Dr.-Hell-Str. 6, 24107 Kiel, Germany e-mail: [email protected] M. Piepenbring Institut für Ökologie, Evolution und Diversität, J.W. Goethe-Universität Frankfurt am Main, Siesmayerstr. 71-73, 60323 Frankfurt am Main, Germany e-mail: [email protected]

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thus regarded as a later synonym of E. mesentericum. Therefore, the latter name is transferred to Xylaria. A key to entonaemoid Xylariaceae is provided. Colour reactions (NH3, KOH) of the ectostroma were applied to a limited number of Xylaria spp., but metabolite profiles of cultures appear more promising as chemotaxonomic traits to segregate this genus. As xylaral was also found in Nemania and Stilbohypoxylon spp., while being apparently absent in Hypoxylon and allied genera, it may be a chemotaxonomic marker for Xylariaceae with Geniculosporium-like anamorphs.

Introduction The Xylariaceae are a large cosmopolitan family of stromatic pyrenomycetes, displaying their highest apparent diversity in the tropics (Rogers 2000). Ju and Rogers (1996) divided them into two major lineages, according to the types of conidiogenous structures (“Geniculosporiumlike” in Xylaria Hill ex Schrank and allies; “Nodulisporiumlike” in Hypoxylon Bull. and relatives). We further refer to these groups as “xylarioid” and “hypoxyloid” Xylariaceae1. Some small genera (e.g., Creosphaeria Theiss.) deviate from the above lineages, since they have diatrypaceous anamorphs. Those and other non-stromatic and coprophilous Xylariaceae genera are not considered further here. The extraordinary morphological diversity of tropical Xylariaceae was already explored by mycologists in the nineteenth and early twentieth centuries, when generic concepts relied exclusively on their teleomorphic morphology, including stromatal anatomy. Most of the presently accepted genera were erected during that time, or even earlier. Even anamorphs were described for some Xylariaceae quite a long time ago (Tulasne and Tulasne 1863; Nitschke 1867), but the conidiogenous structures were at first not regarded taxonomically informative. Only much later, Greenhalgh and Chesters (1968), Jong and Rogers (1972), Petrini and Müller (1986), and others cultured and compared a considerable number of species. This provided a prerequisite to the “modern” generic concepts, which are based on holomorphic morphology. Cultures of Xylariaceae have also been shown to produce various unique or chemotaxonomically significant secondary metabolites (Whalley and Edwards 1995; Stadler and Hellwig 2005; Bitzer et al. 2008). However, several tropical genera and species have never been cultured. They are still only known from the original descriptions and, if not lost, from ancient type material. One of these “problem genera” is Entonaema A. Möller, whose conspicuous, azonate stromata become 1 These terms correspond to “subfamilies”, Hypoxyloideae and Xylarioideae, which were used in previous publications but are not valid taxa; see Bitzer et al. (2008).

Mycol Progress (2008) 7:53–73 Fig. 1 Stromata of Entonaema spp. ss. Rogers (1981) and Xylaria„ telfairii. a,b E. mesentericum (= Xylaria mesenterica), type material from spirit collection (B); a stroma, showing characteristic wrinkled surface which gave rise to the epithet (Möller 1901); b stroma cut open (diam. 4.5 cm). c Fresh stromata, cut open, of X. telfairii (MP 3730). d Fresh stroma of X. telfairii MP3730, cut open so the gelatinous interior becomes evident. e,f E. liquescens: e fresh stromata (MP 3669), diam. of largest stroma ca. 3.5 cm; f type material from spirit collection (B), which has been removed from the spirit and left to dry (diam. 3 cm). g,h E. cinnabarinum (holotype of Sarcoxylon aurantiacum (FH): g stromatal habit (dried stroma ca. 5 cm diam.); h augmented perithecial section, showing thick stromatal context (100 μm)

hollow and filled with liquid. Möller (1901) erected this genus to accommodate E. liquescens A. Möller and E. mesentericum A. Möller, which he collected during his stay in southeastern Brazil. Möller’s descriptions were only based in part on field work and studies of fresh material. He also relied on material that was preserved in alcohol to complete his studies. Most of his specimens were deposited in the herbarium B, whose collections were unfortunately destroyed to a great extent during WWII2. A “complete” inventory of specimens (Friederichsen 1977) only included basidiomycetes, indicating that none of Möller’s ascomycete type specimens had survived. Accordingly, only duplicates of the original material located in S and FH, which frequently constitute only fragmentary, immature, and/or depauperate stromata, have been available for postwar revisions. The taxonomic history of Entonaema was reviewed by Rogers (1981), shortly thereafter followed by the description of the anamorph of E. liquescens (Rogers 1982). The latter study confirmed Möller’s hypothesis that this fungus has close affinities to Daldinia Ces. & de Not. and Hypoxylon. Meanwhile, chemotaxonomic studies (Stadler et al. 2001, 2004a; Hellwig et al. 2005; Quang et al. 2005) using high performance liquid chromatography (HPLC) with diode array detection (DAD) and electrospray ionisationbased mass spectrometric detection (ESI-MS) allowed for a survey of characteristic chemical traits in the aforementioned genera, even providing conclusive results based on ancient specimens. This study revealed that several specific marker molecules of Xylariaceae remained stable for centuries in their herbarium specimens, and that cultures of two Entonaema spp. contain the same specific metabolites as those of Daldinia. The close relationship of the above genera was also corroborated by molecular phylogenetic data on 5.8S/ITS rDNA (Triebel et al. 2005). Nevertheless, the above described polyphasic evaluation of Entonaema and allies only appears sound as far as E. liquescens, E. cinnabarinum (Cooke & Massee) Lloyd, and E. globosum Heim are concerned. Those species yield 2

See http://www.bgbm.org/BGBM/research/colls/herb.

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py were provided by Merck (Darmstadt, Germany). Xylaral (1 in Fig. 2) was kindly provided by Norbert Arnold. All other standard compounds (including some metabolites depicted in Fig. 2, which are referred to in the text by bold numbers as defined there) and a database of HPLC profiles were available from previous work (Stadler and Hellwig 2005; Stadler and Fournier 2006; and references therein). HPLC and other spectral techniques were carried out as described in Hellwig et al. (2005). Non-invasive HPLC studies on irreplaceable type specimens were carried out, using the extraction protocol described in Stadler et al. (2004b), and quantification of secondary metabolites was performed as described by Stadler et al. (2007b). The stromata of the type specimen of E. mesentericum from the alcohol collection in B, which had possibly been stored in the spirit for over 100 years, did not any longer contain any methanol-extractable material. Circa 10 ml of the spirit (presumably ethanol) were withdrawn and an aliquot analysed as described above. However, analyses of this sample turned out to be inconclusive. It was thus evaporated in vacuo to yield an amorphous residue (26 mg),

orange stromatal pigments in KOH, due to the presence of mitorubrin and other azaphilones (Stadler et al. 2004b; Quang et al. 2004), which are typical of particular species of hypoxyloid Xylariaceae. The spectra and chromatograms from previous chemotaxonomic work are stored in a HPLC library that allows us to obtain conclusive results on further specimens by comparing Rt and spectra. We have recently located Möller’s type specimens of Entonaema. Here, we will report their characteristics in comparison with fresh material from the neotropics and discuss these findings with respect to the generic organisation of the Xylariaceae (Figs 1 and 2).

Materials and methods General If not indicated otherwise, all chemicals were obtained in analytical grade from Sigma-Aldrich (Deisenhofen, Germany), while solvents for chromatography and spectroscoFig. 2 Chemical structures of metabolites reported from stromata (1–3; 5,6) and cultures (4,7) of Xylaria spp.: 1 Xylaral; 2 Coloratin A; 3 Coloratin B; 4 Globoscinic acid; 5 Xylactam; 6 Phlegmacin A 8,8′-di-O-methyl ether; 7 2,3-Didehydrotelfairic anhydride

CH3

O

OH

OH

CH3 HO

O

OH

O

H3C

O

O

O

OH

1 O

HO

OH

O

HO

O

O

O 2

O CH3

O

H3C O

O 3

CH3

CH3 OH

O

O

CH3 OH

OH

O

4

CH3 H3C O

CH3

NH

H3C

O

HO H3C

OH O H3C

O

O

CH3

O O

O

O 7

OH 6

5

O

HO

H3C

OH

O

CH3

O

O

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Table 1 Xylaria spp. and other xylarioid Xylariaceae studied for comparison Nemania diffusa France, Ariège, Rimont, Peyrau, on bark of Hedera helix, 12 Jun 2002 (JF 02106). La Réunion (France), Notre Dame de la Paix, Plaine des Câpres, western slope of Volcan de la Fournaise, 9 Mar 2000, leg. G. Gilles, comm. by F. Candoussau (in herb. J.F.). Stilbohypoxylon quisquiliarum (Mont.) J.D. Rogers & Y.M. Ju Guadeloupe (French West Indies), Petit Bourg, Montebello, ravine Nicolas, 1 Apr 1989, leg. J. Vivant & F. Candoussau in personal herb. JF (see also Stadler et al. 2007b). Xylaria aenea Mont. Panama, Chiriqui, highlands of Boquete, Bajo Mono, 14 Sep 2005, leg. J. Gossmann in herb. R. Mangelsdorff RMP 322 (MP, PMA) X. badia Pat. Thailand, Chiang Mai Prov., Bahn Pha Deng, Mushroom Research Centre, 07 Jun 2005, on bamboo, JF TH 0701 Xylaria curta Fr. Panama, Chiriqui, Estero de Bdo. San José, on angiosperm wood, 7 Nov 2005, Olmedo Frago s/n (culture MUCL 47604), det. by J.F. ss. Callan and Rogers (1993) USA, Hawaii, Big Island, Puna, Lava Tree State Park, MSA/MSJ Pre-Congress Foray, 29 Jul 2005, on wood, leg. S. Peterson (STMA 05214, culture MUCL 49354). (Culture studied for comparison: USA, New York, Hamilton Co. Fagus sylvatica, leg. Samuels & Rodrigues, det. J. D. Rogers, ATCC 66841) Xylaria carpophila (Pers. : Fr.) Fr. France, Ariége, Prat Communal, Loumet, 100m, on buried fruits of Fagus sylvatica, 25 Aug 2004, JF-04188. X. corniformis (Fr.) Fr. var macrospora Bres. apud Theissen French West Indies, Martinique, Case Pilote, Bois La Roche, 22 Aug 2005, leg. C. Lechat CLL 5131, conf. Y.-M. Ju. X. cubensis Mont. French West Indies : Martinique, Saint Esprit, Bois La Charles, 29 Aug 2005, leg. C. Lechat CLL 5261 X. cf. dealbata Berk. & M.A. Curtis ss auct. (=X. fockei (Miq.) Cooke) French Guiana, Saul, Monts La fumée, 23 Mar 2006, leg. J.L. Cheype, JLC3, conf. Y.-M. Ju. (hardly mature stromata, morphology similar to the description of X. dealbata, but with smaller ascospores and apical apparatus. X. enterogena (Mont.) Fr. French Guiana, Cayenne, Paracou, Cirad, 26 Feb 2007, leg. C. Lechat CLL 7043 (culture CBS 121674, MUCL 49335, GenBank Acc. No. AM900589); vicinity of Cayenne, Feb 2002, leg. Y. Bellanger, JF 02038, comm. & det. F. Candoussau. French West Indies, Guadeloupe, Trace de Sofaia, Nov 2005, leg. C. Lechat CLL 5427 X. guyanensis (Mont.) Mont. French Guiana, Saul, Monts La Fumée, 21 Mar 2005, leg. J.L. Cheype G8 in herb JF X. hyperythra (Mont.) Fr. French West Indies, Martinique, Crête Jean Louis, 27 Aug 2004, leg. C. Lechat CLL 2156 X. hypoxylon (L. : Fr.) Greville Austria, Lower Austria, Mauerbach, nature reserve “Kartause”, 17 Jul 2005, on Betula pendula, M. S. (STMA05172); Lower Austria, near St. Aegyd, nature reserve “Lahnsattel, 17 Jul 2005, on decorticated wood of Fagus sylvatica, leg. M. S. (STMA05166 and STMA05171). France, Ariége, Rimont, Las Muros, 460m, on log of Fraxinus excelsior, 16 Dec 2004, JF04258, (culture CBS 21679, MUCL 49353). Germany, North Rhine Westphalia, Gruiten, cf. Corylus avellana, 1 Jan 2006, M.& B. Stadler STMA 05047; Rheinland-Pfalz, Wachenheim, near Kurpfalz-Park, in beech forest on old trunk of Fagus sylvatica, 23 Jul 2005, STMA 05143, culture CBS 121680) X. laevis C.G. Lloyd French West Indies, Martinique, Le Prêcheur, Anse Couleuvre, 3 Sep 2003, leg. C. Lechat CLL 0785 X. longipes Nitschke Austria, Lower Austria, St. Aegyd, Ahornhof, 17 Jul 2005, on Acer pseudoplatanus, leg. H. Voglmayr, STMA 05159. France, Ariége, Prat Communal, Loumet, 100 m, Acer pseudoplatanus, 25 Aug 2004, JF 04178. Germany, North Rhine Westphalia, Solingen, Ohligser Heide, mixed deciduous forest near Engelsberger Hof, on highly putrified angiosperm wood, 3 Jul 2005, det. M. S., STMA 05139, culture in MUCL; Wuppertal, Tescher Busch, on trunk of Acer campestre, 25 Jul 2005, STMA 05144. Cultures studied concurrently: CBS 580.88 (Germany, GenBank Acc. No. AY909015); CBS 148.73 (Germany, GenBank Acc. No. AF163038) X. moelleroclavus J.D. Rogers, Y.M. Ju & Hemmes French West Indies: Guadeloupe, Marie Galante, Ravine de Saint Louis, 2 Nov 1993, leg. J. Vivant 7, associated with X. telfairii, comm. Candoussau. Panama: Chiriqui, highlands of Boquete, Bajo Mono, 14 Sep 2005, leg. R. Mangelsdorff RMP 323 (PMA, MP) X. obovata ((Berk.) Fr. sensu Rogers et al. 1988 and Dennis 1956) French West Indies Martinique, La Pirogue, 26 Aug 2004, CLL 2146 X. olobapha Penz. & Sacc. French Guiana, Saul, Monts La Fumée, Mar 2005, leg. J. L. Cheype G4, conf. Y.-M. Ju X. poiteana (Lév.) Fr.

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Table 1 (continued) Panama, Chiriqui, Dolega, Los Algarrobos, close to Casa de la Alemana, alt. ca. 150 m, on angiosperm wood, 5 Nov 2005, leg. M. Piepenbring (& students of UNACHI) MP 3622 (PMA, culture MUCL 47968). X. polymorpha (Pers.) Grev. Germany, Badenia-Württemberg, Freiburg, Aug 1997, leg. H.-V. Tichy, det. M. S. & H. Wollweber (STMA 97160) France, Ariege, vicinity of Rimont, wood of Quercus, 19 Jul 2006 (immature, JF-07142); same collection site and substrate, mature 9 Sep 2007 (JF-07152). Portugal, Isla de Madeira, vicinity of Funchal, Nov 2004, leg. W. Jäger (STMA 05059). X. scruposa (Fr.) Berk. Japan, Honshu Island, Mt. Fuji area, Oct 2004, leg. T. Hashimoto, STMA-04-FJ1. Panama, Chiriqui, Boquete, Bajo Mono, alt. ca. 1,600 m, 14 Sep 2005, M. Piepenbring et al. MP 3548 (PMA, 2 packets; culture MUCL 47967). X. telfairii (Berk.) Fr. French Guiana, Cayenne, Crique Macouria 1 Feb 2007, leg. CLL 7107 ( culture CBS 121673, MUCL 49334; GenBank Acc. No. AM900590); vicinity of Cayenne, Feb 2002, leg. Y. Bellanger, FC5376-1, comm. and det. F. Candoussau; Saul, Monts La Fumée, 10 Mar 2006, leg. J.-L. Cheype JLC2. French West Indies, Martinique, Rivière Rouge, Pierre Dennis, 29 Aug 2004, leg. C. Lechat CLL 2224; Guadeloupe, Marie Galante, Ravine de Saint Louis, 2 Nov 1993, leg. J. Vivant 7, comm. F. Candoussau. Panama, Alto Chiquero, 24 Mar 2006, leg. M. Piepenbring MP 3730 (PMA). Materials originating from Austria were collected during the mycological excursion of the International Botanical Congress, Vienna 2005, and materials originating from Hawaii were collected during the foray of the Joint MSA/MSJ meeting in Hilo, 2005. Unless stated otherwise, the specimens were collected and identified by J. Fournier.

which was redissolved in 10 ml water/ethyl acetate (1:1) in an ultrasonic bath for 30 min. The organic phase was then dried over anhydrous Na2SO4, filtered and evaporated in vacuo (40°C) to yield a residue (4.5 mg). This material was separated by preparative HPLC (for method, see Hellwig et al. 2005), and the resulting fractions studied by HPLC-MS for occurrence of known metabolites of the Xylariaceae. Morphological characters of the stromata and teleomorphs were evaluated as described by Stadler et al. (2004a), except that critical examination of ascospore germ slit was carried out from slides mounted for 48 h in PVA-lactophenol (van Brummelen 1967; polyvinylic alcohol solution in water 15 g for 100 ml: 56 ml; lactic acid: 22 ml; phenol: 22 ml). Cultures were obtained and studied as described by Stadler et al. (2004b) and for secondary metabolite production as described by Bitzer et al. (2008). The strains were grown in batches of several shake flasks and monitored during a period of up to 268 h. Daily samples were taken for determination of pH and glucose. Ethyl acetate extracts of the culture broth were analysed by HPLC and compared with those of other Xylariaceae studied previously (Stadler et al. 2001, 2004b; Bitzer et al. 2008). Chemical pigment reactions were assessed as described in Wollweber and Stadler (2001). Colour codes follow Rayner (1970) and, therefore, also Ju and Rogers (1996). Specimens examined of Entonaema ss. Rogers (1981) are listed in the taxonomic part, whereas the Xylaria spp. studied for comparison are listed in Table 1. Most of the materials studied and all cultures obtained are deposited in public collections; some specimens are also available from the herbaria of the authors or were kindly provided by our colleagues. Specimens designated STMA and Ww are kept at the Fuhlrott-Museum, Wuppertal,

Germany. CLL specimens were collected by C. L. and will be deposited in LIP. 5.8S/ITS nrDNA sequences were obtained in a similar manner as described by Triebel et al. (2005) and compared with entries in the EMBL database of fungal DNA sequences http://www.ebi.ac.uk/embl) by performing FASTA searches (Bitzer et al. 2008) and alignments.

Results Chemotaxonomy The search for E. mesentericum and xylaral HPLC profiles of all newly examined specimens listed in the taxonomic part of E. cinnabarinum and E. liquescens agreed with previously reported data, confirming that the secondary metabolism is consistent in these species. Azaphilones of the mitorubrin/rubiginosin type were always fairly detectable in very young, mature and even overmature and depauperate specimens of these species. We obtained several fresh collections of E. pallidum and type material of this fungus from FH. Our previous HPLC profiling work revealed several unknown metabolites with characteristic chromophores (Stadler et al. 2004b, Fig. 12) as major stromatal metabolites, while azaphilone pigments and orsellinic acid (see Fig. 6) are apparently lacking in E. pallidum. In fact, all specimens of this species listed in “Taxonomy” showed similar HPLC profiles. We compared them with entries in our spectral database. Surprisingly, the characteristic metabolites of E. pallidum were observed in

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Xylaria enterogena, X. telfairii, and some other Xylaria taxa listed in Table 1 (see chromatograms and spectra in Fig. 3), but they were not detected in over 2,500 specimens of hypoxyloid Xylariaceae studied in the past decade. Using an original standard, one of these common prominent metabolites was identified as xylaral (1 in Fig. 2), reported from stromata of X. polymorpha (Gunawan et al. 1990). Putative xylaral derivatives (indicated as congeners “C” in Fig. 3), showing UV-visual spectra similar to those of (1), but deviating in their retention times (Rt) and molecular masses, were also detected in the above fungi. Noting the similarities of mature stromata of E. pallidum to Möller’s description and illustrations of E. mesentericum, we continued our search for authentic material of the latter name. This was finally located in B with kind assistance by the curators, in a cabinet which contained various other containers with ascomycetes stored in spirit (basidiomycetes listed in 1977 by Friederichsen were stored in different cabinets). Several further specimens, including type material of E. liquescens, were meanwhile already deposited in the regular herbarium, after the spirit evaporated from the containers. After enrichment by preparative HPLC, we

detected traces of xylaral or a closely related compound in fractions of the concentrated spirit in which E. mesentericum was preserved. Xylaral (1), which actually corresponds to the peak “E4” in Stadler et al. (2004b, Fig. 12), was therefore probably present in the stromata, but had been extracted due to the “preservation” procedure.

Fig. 3 HPLC-UV chromatograms of MeOH extracts (210 nm) from stromata of Entonaema pallidum TL-9701 (a) and Xylaria telfairii MP3730 (b), including HPLC-UV-Vis spectra of xylaral (1) and some

unidentified congeners (C) that are presumably chemically related to 1 because they show similar, characteristic UV-Vis spectra

Pigment reactions for characterisation of Xylaria In contrast to Hypoxylon (Ju and Rogers 1996), colour reactions of stromatal extracts are not regarded as taxonomically informative in Xylaria. To the best of our knowledge, their significance has never been proven by systematic studies on a representative number of taxa. Since the isolation of xylaral was initially attempted following an observation that stromatal extracts of X. polymorpha showed a purple colour in 25% NH3 (Gunawan et al. 1990), we used this agent in addition to 10% KOH to evaluate the stromatal metabolites in Xylaria. As shown in Table 2, the colours of pigments coincided well with the presence of xylaral or related compounds. Greenish Yellow (16) pigments in KOH and Vinaceous (57) to Livid Red

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Table 2 KOH pigment reaction and characteristic secondary metabolites of various Xylaria and Entonaema spp Species

25% NH3

10% KOH

HPLC profile

Xylaria aenea X. badia

NAP A (slow)

NAP A (immediately)

X. carpophila X. corniformis var. macrospora

NAP NAP

NAP NAP

X. cubensis X. curta (Hawaii)/X. cf. curta (Panama) X. cf. dealbata ss. auct. X. enterogena 02/2002 X. guyanensis X. hyperythra X. hypoxylon X. laevis X. longipes X. moelleroclavus X. obovata X. olobapha var. camptospora X. poiteana X. polymorpha (Germany) X. polymorpha (France, JF-07142) X. polymorpha (France, JF-07152) X. cf. polymorpha (Madeira) X. scruposa RMP317 X .cf. scruposa MP3548 X. telfairii CLL 2224 X. telfairii 02/2002 X. telfairii FC5376-1 X. telfairii JLC2 X. telfairii 11/1993 X. telfairii MP 3730 E. pallidum (= X. mesenterica) For comparison Entonaema cinnabarinum, E. globosum, E. liquescens

NAP NAP NAP V (++) none V (+) NAP NAP NAP NAP NAP V (++) NAP NAP NAP NAP V (+) NAP V (+) V (+) V (++) V (++) V (++) V (++) V (+) V (+)

NAP NAP Faint yellow GY (+) Faint yellow GY (++) NAP NAP NAP NAP NAP GY (++) NAP NAP NAP NAP GY (+) GY (+) GY (+*) A (+++) GY (++) GY (++) GY (++) GY (++) GY (+) GY (+)

Inconclusive No xylarals detected; different unknown hydrophilic components Inconclusive Metabolites with similar UV spectra to chaetoglobosins Inconclusive Inconclusive Small amounts of unknown compounds xylarals (+) Inconclusive xylarals (++) Inconclusive Series of uv-inactive lipophilic components Inconclusive Inconclusive Inconclusive Xylarals (+++) Inconclusive Inconclusive Xylarals (+) Xylarals (+) Xylarals (+) Xylarals (+) Xylarals (+) Xylarals (++) and unknown co-metabolites Xylarals (+++) Xylarals (+++) Xylarals (+++) Xylarals (++) Xylarals (+) Xylarals (+/++)

Orange (7) (++/+++)

Fulvous (43) or Luteous (12) (++/+++)

Mitorubrins/rubiginosins

Legends (numbers refer to colour codes in Rayner 1970): GY Greenish Yellow (16), A Amber (47), V Vinaceous (57) to Livid Red (56), NAP no apparent pigments. Intensity: (+) weak, (++) moderate; (+++) strong. Unless individual collections are listed, all specimens of a particular taxon showed similar reactions.

(56) pigments in 25% NH3 indicated the presence of xylaral in many cases. The latter colour reactions are in accordance with the “violet” colour reported for xylaral and X. polymorpha by Gunawan et al. (1990). Only in the stromata of X. aenea, X. polymorpha 07142 and 07152 from France, and X. cf. polymorpha from Madeira, were no apparent pigments observed with the chemical reagents despite HPLC also revealing traces of xylaral derivatives in these specimens. Apparently, the pigment reaction with the alkaline chemicals is less sensitive than the concurrent HPLC-MS analyses, as the concentrations of xylaral detected in the KOH- and NH3-negative specimens by HPLC were in general lower than in those specimens that showed positive colour reactions. Xylaria badia showed

similar colours with both reagents as the xylaral-containing species, but HPLC data proved that different, yet unknown compounds are involved. Comparing different collections of X. telfairii, variations in the intensities of these pigment colours were observed. This was in part due to different pigment concentrations, and/or to the presence of mixtures of metabolites that showed the xylaral chromophore upon DAD (examples in Fig. 3 as “C”; congeners). Over 20 different compounds of this type were detected in the specimens examined. Interestingly, various other Xylaria spp. studied for comparison, were devoid of such putative xylaral derivatives, and their stromatal pigments were absent or faint. Most of these “NH3/KOH-negative” taxa contained little extractable material, discounting ubiquitous

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fatty acids and ergosterol. A range of further randomly selected xylarioid Xylariaceae were also studied for the occurrence of xylaral. Among those were specimens of Entoleuca Syd., Euepixylon Füisting, Kretzschmaria Fr., Nemania S.F. Gray, and Rosellinia Desmaz., previously reported (Stadler et al. 2001, 2007b) and additional ones from the personal herbarium of J. Fournier. A complete inventory is not given here, because a systematic evaluation of this phenomenon would be outside the scope of this paper. Out of more than 50 specimens studied, xylaral was only encountered in stromata of Stilbohypoxylon quisquiliarum, as well as in two specimens of Nemania diffusa. The concentrations of xylaral (1) estimated by HPLC in E. pallidum, Nemania, and Stilbohypoxylon were considerably lower as compared to some Xylaria spp. (e.g., 1.4 mg per gram dried stromatal material in X. telfairii MP 3730 vs 0.1 mg per gram in E. pallidum TL-9701). Accordingly, the stromatal pigment colours in NH3 are much more obvious in X. telfairii and allies (cf. Table 2) than in E. pallidum. Nemania, Stilbohypoxylon, and some Xylaria specimens in which xylaral was detected did not show apparent pigments at all. Secondary metabolism in cultures of Entonaema and Xylaria Two cultures of E. pallidum and some cultures of Xylaria spp. (Fig. 4) (including European X. hypoxylon, which we regard as representative) were studied for comparison on secondary metabolite production in different culture media. Fig. 4 Agar cultures (plates 9 cm diam.) of Xylaria mesenterica (“Entonaema pallidum”) (a,b,e) and X. telfairii (c) and microphotographs of ascospores of the type material of E. mesentericum (d). A Culture ex Rodriguez 2818 (Mexico), on OA, after 4 weeks. b Culture ex MP3685 (Panama), on YMG agar, after 3 weeks. c X. telfairii CLL-7107, on OA, after 3 weeks. e Microphotograph of conidiophore with conidium attached, from the culture depicted in b. d and e were recorded by phase contrast brightfield microscopy at 1,000×, and scale is indicated by bar)

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In cultures of E. pallidum and Xylaria, no chromone or naphthol derivatives were detected. Their metabolite patterns drastically differed from those of E. liquescens and E. cinnabarinum, both of which showed similar HPLC profiles as Daldinia. These results agree with previous studies (Whalley and Edwards 1995; Espada et al. 1997; Abate et al. 1997), where cytochalasins, succinic acid derivatives, and various other compound classes were reported from the genus Xylaria. Mellein, already reported in Xylaria by Whalley and Edwards (1995), however, was occasionally detected in the Xylaria spp. studied. Moreover, cytochalasins appeared as closest matches by a search in a comprehensive HPLC library containing spectral data of various metabolites from fungi and other sources (cf. Bitzer et al. 2007). The two cultures of E. pallidum studied agreed with one another with respect to the time course of secondary metabolite production as revealed by HPLC. Some of their major metabolites were also detected concurrently in the Xylaria strains. Interestingly, X. hypoxylon and most of other Xylaria spp. examined tended to produce metabolites with cytochalasin-like HPLC-MS characteristics during early stages of fermentation, turning to the production of different compounds later on. In contrast to most species of hypoxyloid Xylariaceae (except for H. fragiforme and allies, which, however, produce different metabolites as Xylaria; see Bitzer et al. 2008), the putative taxonomically significant metabolites of the Xylaria spp. and E. pallidum did not always accumulate towards the end of the fermentation process. A comparison

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Fig. 5 HPLC-UV chromatograms of ethyl extract acetates redissolved in methanol after drying of E. pallidum ex MP 3685 and X. longipes ST;A 05139 (YMG cultures, 168 h) and above HPLC-DAD spectra of

unknown compounds, one of which shows cytochalasin-like (Cy) HPLC-MS data, whereas the other two (U1, U2) have not yet been identified

of the extracts from cultures of E. pallidum and X. longipes (Fig. 5) by HPLC revealed some common metabolites in the medium polar range (U1, U2), whose identification is pending. Various further, yet unknown hydrophilic compounds, eluting at retention times (Rt) lower than 6 min were detected in both extracts, as well as in other Xylaria strains. A search in our HPLC library revealed that most of these peaks did not occur as major component in any HPLC profile derived from the numerous cultures studied of Daldinia, Entonaema, and Hypoxylon.

Entonaema, Daldinia, and Hypoxylon were not even found on the lists including the closest 50 matches. The most similar sequences were derived from unidentified “Xylaria spp.”, such as P106 (GenBank Acc. No. EF423545, see Gilbert and Webb 2007), MS 106 (1066) 86 (Acc No. AF153724, see Guo et al. 2000), and NR-2006-A59 (Acc. No. DQ480344, see Promputtha et al. 2007). Among the most similar sequences derived from material with designated species names were several sequences of X. longipes, but not even those strains were all available in public collections. Among them were strains CBS 146.73 and CBS 580.88, whose taxonomy was verified using the key by Callan and Rogers (1993), and by comparison with our own cultures of X. longipes. Our cultures of X. enterogena and X. telfairii (Fig. 4c) showed similar morphologies to those reported by Callan and Rogers (1990) for the same species. Ribosomal DNA sequences, obtained from these taxa for the first time,

Comparison of ribosomal DNA sequences of E. pallidum and Xylaria species The 5.8S/ITS nrDNA sequences of two E. pallidum strains were compared with those in public databases using a FASTA search. The results revealed sequences of Xylaria spp. to have the highest similarity, while those of

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proved somewhat different from other Xylaria spp., but still the greatest homologies among published sequences were found within the genus Xylaria. Deviations to other Xylaria spp. (and thus to E. pallidum) were noted, especially in the ITS1 region of their rDNA gene, presumably due to specific introns. Hence, only about 55% similarity with E. pallidum was found as inferred from a clustal alignment using DNAStar (Stadler et al. 2007a).

Taxonomy Entonaema A. Möller, Bot. Mitt. Trop. 9: 306. (1901) emend. M. Stadler, Læssøe & J. Fournier Typus (Lectotype, selected by Clements and Shear (1931) as “lignescens”): Entonaema liquescens A. Möller. Generic description, modified from Rogers (1981) to exclude E. mesentericum: Stromata pulvinate, semiglobose or globose, often irregular, solitary or aggregated, attaining a cavity filled with liquid when fresh. Internal concentric zones absent. Flesh of fully developed stromata gelatinous when wet, but horny and very hard when dry, carbonaceous to some extent due to the incorporation of melanin pigments. Stromatal surface smooth when fresh, becoming wrinkled in dry state. Ostioles obsolete, umbilicate, punctiform, or slightly papillate. Perithecia monostichous, developing beneath a bright-coloured, thin outer pellicle, frequently with a dark thin carbonised zone just underneath the coloured outer part of the mature stromata. Asci tubular, stipitate, with amyloid apical ring. Ascospores unicellular, also when immature, brown to dark brown, ellipsoidinequilateral to almost rectangular, or sometimes almost fusiform-inequilateral, with longitudinal ventral germ slit. Anamorph, where known, showing a Nodulisporium-like branching pattern as defined in Ju and Rogers (1996). Stromatal pigments of the mitorubrin/rubiginosin type (in three accepted species including the type) or unknown. So far, no binaphthalenes, which are ubiquitous in Daldinia, and widespread in Annulohypoxylon (Y.M. Ju, J.D. Rogers & H.M. Hsieh) and Hypoxylon were observed. Prevailing secondary metabolites of cultures: naphthol derivatives or chromones. Mellein-like dihydroisocoumarins and cytochalasins were so far not detected. Notes Clements and Shear (1931) lectotypified Entonaema with E. liquescens, an action accepted by Læssøe (1994). Although attempts have been made to typify with the other element introduced by Möller (1901), we can see no valid arguments for rejecting the original lectotypification. Furthermore, the original choice clearly stabilises the concept of a fungus related to the pigmented Hypoxylon

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species rather than to a poorly understood group of Xylaria species, despite material of E. mesentericum (i.e. the species described first by Möller) has now been located. Besides microscopic details drawn from fresh material, Möller (1901) even included a photograph of E. liquescens. From his description and discussion of evolutionary lineages in the Xylariaceae, Möller evidently intended to describe a genus that has close affinities to the hypoxyloid Xylariaceae, and especially the genus Daldinia, rather than an aberrant form of Xylaria. The rationale of the taxonomic treatment provided below is in accordance with the revision of Hypoxylon s. lat. in Miller (1961) and in Ju and Rogers (1996) and many other taxonomic changes concerning xylariaceous taxa that were recently proposed (see recent revisions of genera like Entoleuca, Euepixylon, and Nemania, discussed by Ju and Rogers 1996): All species showing anamorphic features, secondary metabolites and/or molecular data different from the type species (i.e. a member of the hypoxyloid Xylariaceae) should be expelled from Entonaema, even if they have hollow stromata. Anamorphs and other characters of some Entonaema spp. are still unknown. E. dengii and E. moluccanum (of which only teleomorphic features are known) are therefore retained for the time being in Entonaema. The morphologically similar genus Sarcoxylon Cooke, whose type species may also have closer affinities to Xylaria, and which at one time included several taxa that are now accommodated in Entonaema (cf. Rogers 1981), is retained for the time being until anamorphic characters and molecular data can be studied.

Species descriptions Our results from type studies and authentic material, of all taxa regarded as members of Entonaema by Rogers (1981), are summarised below. Details are only provided for the most well known species, or if new evidence was obtained in the course of this study. We add some data on chemotaxonomic features including pigment reactions of the stromata, which have not been evaluated for Entonaema. Entonaema liquescens A. Möller, Phycom. Ascom. Bras.: 306, Figs. p. 248 and plate (Tafel) VIII, Fig. 108 (1901); types: Brazil (Südbrasilien), Santa Catharina, Blumenau, “an morschen Baumstämmen” (decayed tree trunks), A. Möller (B, ex Alkoholsammlung No. P144aholotype; FH (fide Rogers 1981) and S (F-38124)—isotypes (fragments of dried specimens that were not preserved in alcohol). Synonyms Xylaria splendens Berk. & M.A. Curtis, J. Linn. Soc. 10: 382 (1869).

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Glaziella splendens (Berk. & M.A. Curtis) Berk., Vidensk. Medd. Nat. For. Kjobenh. 31/32: 31 (1879–80). Entonaema splendens (Berk. & M.A. Curtis) Lloyd, Mycol. Notes 7(4): 1203 (1923); types: Cuba, immature (Kholotype; – BPI-isotype). Selected illustrations Rogers (1981), Figs. 24–26 and 47– 49 (teleomorph); Rogers (1982), Figs. 1–8 (anamorph). Geographic distribution/habitats/host range On wood and bark of various broadleaved trees; Africa (Uganda), Americas (temperate, subtropical and tropical zones ranging from Kansas and Georgia, USA in the north to Argentina in the south), Asia (China fide Rogers 1981; Thailand fide Sihanonth et al. 1998). Not yet recorded from Europe and Australia. Teleomorph Stromata globose to cerebriform, solitary or confluent, sometimes highly irregular. Solitary stromata often attached by narrow basal connective to the substrate, hollow, deliquescent in age, but frequently never becoming fertile (Rogers 1981), 1–13 cm wide × 1–6 cm high. (fide Möller 1901, cf. p. 238 confluent stromata may attain a diameter of up to 40 cm). Surface bright sulphur yellow in young specimens to dull yellow or olive yellow with orange tinges in mature ones, discolouring to greenish when handled fresh, surface layer easy to remove, exposing a dark thin stromatic layer below. Ostioles sparse, owing to the presence of rather few perithecia, punctiform to papillate. Flesh of the mature entostroma gelatinous, up to 1 cm thick when wet, but hardly 2 mm thick in dry stage. Perithecia tubular to obovoid, 0.5–0.6 cm high × 0.3– 0.6 mm wide. Asci tubular, eight-spored, early deliquescent, total length 110–140 μm, p. sp. 60–80×6–9 μm, stipe 35– 55 μm. Ascal apical apparatus discoid, 1.5–2.5 μm high × 2– 2.5 μm wide, amyloid. Ascospores light brown to brown, ellipsoid-inequilateral with narrowly rounded ends to equilateral and almost cylindrical, mostly biguttulate, with perispore indehiscent in 10% KOH, (8)9.5–13.5(15) × (4.5) 5–6.5(7.5) μm (M=11.3–5.4 μm, n=70), with germ slit faint, dorsal (in inequilateral spores), straight, almost spore length. Cultures and anamorph See Rogers (1982); culture deposited as ATCC 46302, showing a Nodulisporium-like anamorph with holoblastic conidiogenesis (GenBank Acc. No: AY616686; molecular data see Triebel et al. 2005). Secondary metabolites Stromata contain mitorubrin, rubiginosin A and orsellinic acid as major components. The ratio of these compounds is fairly consistent and mostly equivalent to the HPLC diagram in Stadler et al. (2004b, Fig. 10) in all developmental stages we studied so far. Stromatal pigments are Orange (7) to Luteous (12) in 10%

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KOH as well as in 25% NH3. Cultures contain the same compounds as those of E. cinnabarinum (see below). Notes The above description constitutes a synopsis of the descriptions by Möller (1901) and Rogers (1981), complemented by additional data on type material in B, now removed from the spirit (Fig. 1f). The glass container (including the dried “extract”) was not located for HPLC studies. The specimen consists of a single, lobed stroma of ca. 5 cm diam., which is in excellent shape, aside from the lack of pigments. Only some parts of the stromatal surface still contained remnants of a yellowish pigment. Rubiginosin A and orsellinic acid (structures see Fig. 6) were tentatively detected by HPLC-MS in a sample taken from such regions, after pre-concentration. Conclusive HPLC profiles, however, were obtained from portions of the material derived from Möller’s collection in S, which were very similar to the profiles of recently collected specimens. The material in B has slightly larger asci and ascospores than reported by Rogers (1981). However, we agree with his measurements from our own examination of the mature specimen listed below. We did not search for ascospores in the isotype in S because not much material is left. Entonaema splendens is in our mind a synonym of E. liquescens (rather than E. mesentericum as assumed by Rogers 1981). The type specimen of X. splendens is immature but shows the specific HPLC profile of E. liquescens (Stadler et al. 2004b), whereas other species with similar secondary metabolism (E. cinnabarinum and E. globosum) contain different azaphilone patterns. Because various other specimens listed below contained immature as well as mature stromata showing essentially the same HPLC profile, identification of this species appears feasible based on chemotaxonomic methodology in conjunction with morphological traits of the immature stromata. Further specimens examined Brazil, Sta. Catharina, São Canisio de Porto Novo, Uruguay River, 1928, J. Rick (BPI 715365); ibid., Blumenau, J. Rick (BPI 715362); Rio Grande do Sul, São Leopoldo, no date, J. Rick 272 ex herb. Bresadola (M-0055871); ibid., Sta. Cruz, Thaxter 4209, det. J. Rick (FH 42769; BPI 715363); J. Rick s/n (FH 79765, det. J. D. Rogers (immature); J. Rick 246, det. J. D. Rogers as “Sarcoxylon sp., immature” (FH 79766 (immature)); J. Rick s/n (FH 79767, immature); Thaxter 4209, det. J. Rick,; J. Rick 246 as Sarcoxylon sp. (FH 79670); J. Rick 762 as Sarcoxylon, rev. J. D. Rogers (as E. cf. cinnabarinum) (FH 79779). Panama, G. W. Martin 3280, rev. J. D. Rogers as “immature E. liquescens” (FH 79761, immature) ; Soberana, MP 3669 (specimen currently lost or misplaced but depicted in Fig. 1). USA: Florida, Gainesville, Celtis laevigata, 11 Sep 1952, A. S. Rhoads, as E. aurantiacum (rev. Rogers 1981) (BPI 629857); Florida,

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Fig. 6 HPLC-UV chromatogram (210 nm) of the stromatal methanol extract of Entonaema mollucanum sensu Sanchez-Jacome and GuzmánDavalos (2005), specimen GD 7359 (C), showing rubiginosin A and orsellinic acid as major components. This profile drastically differs from that of the holotype of E. moluccanum (cf. Stadler et al. 2004b). Above

left micrograph of ascospore (15.5×7 μm) of this specimen in 10% KOH, recorded using phase contrast brightfield microscopy (1,000×), showing dehiscent perispore, which indicates that this specimen represents an undescribed species

Gainesville, rotten log, 3 Jul 1939, E. West (det. Rogers 1981) (BPI 630383); Maryland; Calvert Co., Flag Ponds County Park, Jul 1988, D. & E. Farr (BPI 802219); Kansas, Leavenworth Co., Leavenworth Co. State Lake, 27 Sep 1979, R. W. Lichtwardt & students, (Rogers 1981, 1982; culture ATCC 46302) (pers. herb. J. D. Rogers and pers. herb. M. S.).

Mexico), Bulgaria (Benkert 1993, Læssøe 1997), and France (Stadler et al. 2004b). Also occurs in Iran (current study) and, doubtfully fide Rogers (1981), in Estonia (see note below). Known host spectrum: on bark or decorticated, wood. Recorded from Europe on Acer, Fraxinus, and Platanus.

Entonaema cinnabarinum (Cooke & Massee) Lloyd, Mycol. Notes 7 (4): 1203 (1923) [as “cinnabarina”]. Basionym Xylaria cinnabarina Cooke & Massee, Grevillea 15: 101 (1887), types: Queensland, Daintree River, on wood, no date, F. Mueller (K - holotype-; BPI 715361 isotype). Synonyms Sarcoxylon aurantiacum Pat., Bull. Soc. Mycol. France 27: 331 (1911). Entonaema aurantiacum (Pat.) Lloyd, Mycol. Notes 7 (4): 1203(1923) [as “aurantiaca”], type: New Caledonia, A.J. Le Rat 192, ex herb Patouillard (FH 79774 – holotype). Selected illustrations: Læssøe (1997), Rogers (1981) Figs. 10–13, 46, 54–55; Stadler et al. (2004b) Figs. 1–5 (see also http://pyrenomycetes.free.fr/entonaema/index.htm). Geographic distribution/habitats/host range Widespread in warmer climates. Reported from Africa (“Portuguese Congo”), Australia, New Caledonia, Asia (Japan, Philippines, Sri Lanka fide Rogers 1981); America (Costa Rica,

Teleomorph Stromata superficial, pulvinate, folded (when overmature or when drying up), irregular to subglobose, somewhat constricted at the base, soft, shrivelling upon drying, 18–42 mm diam × 15–32 mm high; surface Sienna (8), Rust (39), Bay (6) to Dark Brick (60), eventually blackish, when immature coated with a pale Luteous (11) pruina readily rubbed off, smooth when fresh, deeply folded when dry. Outer layer a continuous crust 60– 80 μm thick, of orange to orange red granules with KOHextractable pigments Orange (7); the tissue enclosing the perithecia 0.45–0.6 mm thick, blackish, replaced by a thin whitish layer just beneath the outer crust; a gelatinous layer is present beneath the perithecial layer, Luteous (12), greenish toward the base, soft, 6–1.3 mm thick on fresh material, blackish, horny, 1–1.5 mm thick on dried material. Mature stromata hollow, filled with a luteous liquid with a striking curry-like odour (reminiscent of fenugreek) that still prevails in dried material. Perithecia spherical to obovoid, 0.4–0.55 mm high × 0.3–0.35 mm wide, outlines slightly visible. Ostioles lower than the stromatal surface, slightly papillate or obsolete. Asci tubular, long-stipitate, 110– 125 μm total length × 6.5–9 μm broad, p. sp. 59–73 μm long, stipes 55–70 μm long, with a discoid amyloid apical

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apparatus 0.6 μm high × 2–2.4 μm broad. Ascospores brown, broadly ellipsoid, nearly equilateral, with broadly rounded ends, conspicuously biguttulate, 9.5–11.5 × 4.8– 6 μm (M ¼ 10:6
5:4 2m), with faint straight germ slit 2/3 spore-length; perispore indehiscent in 10% KOH. Owing to the presence of oil drops, the faint germ slit is not seen in water, barely visible in KOH or Melzer’s reagent, but clearly seen in PVA-lactophenol.

Cultures and anamorph Nodulisporium-like (see Stadler et al. 2004b) with holoblastic conidiogenesis. Culture: CBS 113034 (GenBank Acc. No for rDNA sequence: AY616685; Triebel et al. 2005). Secondary metabolites see Stadler et al. (2004b). Stromata contain orsellinic acid and azaphilones. Cultures contain the same major constituents as those of Daldinia, namely chromones, naphthols, as well as phytotoxins of the “AB5046” type that were recently identified by Bitzer et al. (2008), as major components. The stromatal HPLC profiles of this species proved less consistent than those of E. liquescens when a range of specimens from different geographic regions were compared. Specimens from the tropics showed a smaller number of azaphilones than the recently collected European (Stadler et al. 2004b) and Iranian (this study) material. Stromatal pigments in KOH are Orange (7) to Luteous (12), as in E. liquescens. Further specimens examined Bulgaria, Black Sea Coast, Varna, Kamtschija Reserve, Fraxinus angustifolia, 30 Jul 1996, TL-4197 (Læssøe 1997) (C 35468, K(M) 53712). Costa Rica, San José Province, Nov 1929, C.W. Dodge & W.S. Thomas (as E. mesenterica), rev. Rogers (1981), (M0055872 ex herb. F. Petrak, BPI 630384, FH 79768). France, Pyrénées Atlantiques, Auterrive, Ile du Gave d’Oloron, Fraxinus excelsior, 6 Aug 1999, J. Vivant (pers. herb. F. Candoussau 665, BPI 747841, FH 79777, pers. herb. J.D. Rogers and J. Fournier), culture: CBS 113034; same locality, Fraxinus excelsior and Platanus sp., 5 Sep 1999, J.F., J.-F. Magni & C. Girard (JF-99200); same locality, F. excelsior, 30 Jun 2004, J.F. & M. S (pers. herb. JF-04112, immature). Iran, road from Asalem to KhalkhalGilan, on rotten wood, 17 Jun 1991, Saber et al., det. B. M. Spooner (K(M) 39857). Japan, Iyo Province, 15 Aug 1916, A. Yasuda as Sarcoxylon splendens, rev. Stadler et al. (2004b) (BPI 714489). México, Jalisco Prov., San Sebastian del Oeste (1400 m), in garden, 30 Aug 1994, O. Rodríguez 1088, det. L. Guzmán-Davalos (GUAD n.v., C). New Caledonia, A. J. Le Rat 151, as S. aurantiacum (FH 79774). Philippines, Laguna Province, 30 Jul 1919, A. Manza as Sarcoxylon splendens, rev. Stadler et al. (2004b) (BPI 714490). Sri Lanka, T. Petch 4205 (FH 79776 (immature)).

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Uganda, R.A. Dümmer 1443 (FH 79775 (immature); duplicate in Kew treated in Rogers 1981 n.v., said to be mature). Notes The above description is mainly based on fresh material collected in France. Nonetheless, the type specimens of S. cinnabarinum and S. aurantiacum (Fig. 1g-h) revealed essentially the same morphological characters. Interestingly, anamorphic Xylariaceae, whose ITS nrDNA sequences matched closely that reported by Triebel et al. (2005) from the cultured specimen BPI 747841, were recently reported by Šrutka et al. (2007) to be associated with woodwasps in the Czech Republic. Rogers (1981) doubted whether a record in K of E. cinnabarinum from Estonia was genuine (no other Entonaema sp. was at that time reported from Europe). If hymenopteran insects do really act as vectors for the fungus, it could in principle turn up anywhere in temperate central Europe. However, 5.8S/ ITS nrDNA sequences do not even appear to be specific enough to tell the genera Daldinia and Entonaema from one another (cf. phylogenies in Triebel et al. 2005). The anamorphic E. cinnabarinum described by Šrutka et al. (2007) also produced secondary conidia from attenuated, elongate conidia, i.e., a feature that was hitherto not observed in Daldinia or Entonaema. Additional studies are needed on this interesting phenomenon in any case. Entonaema dengii, J.D. Rogers Mycologia 73: 55 and Figs. 20, 21, 50 (1981). Type P. R. China, Hainan, 11 Dec. 1934, S.Q. Deng 7478 (BPI 586728-holotype). Notes According to Rogers (1981) and our own observations, this monotypic species is similar to E. cinnabarinum but has larger ascospores (13–15×6.5–8 μm) with acute ends. In addition, it differs from the latter species in lacking stromatal pigments in 10% KOH and 25% NH3, and accordingly it does not contain orange granules surrounding perithecia. Cultures and anamorphs are unknown. HPLC analyses did not reveal any known Xylariaceae metabolites (Stadler et al. 2004b). It remains unclear whether this species is really related to E. liquescens and other mitorubrin-containing members of the genus.

Entonaema globosum Heim, Bull. Mycol. Soc. France 76: 121, 129 (1960). [as “globosa”] Type Mexico, Oaxaca, near Rio Santiago, alt. 14–1,500 m, 10–12 Aug 1959, R. Heim 524, and loc. cit., 11 Jul 1959, R. Heim 5350, “type” of E. globosum f. aurantiaca (nom. nud.- no latin diagnosis) (PC - holotype, not seen; both forms immature fide Rogers 1981).

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Epitype (selected here) Mexico, Campeche, Escárcega Municipality, Ing. Eduardo Sangri Serrano Forestry Experimental Station, on wood of Nectandra salicifolia in median subevergreen rainforest, 9 Nov 1988, F. San Martín 1171 (WSP 69647). Notes Since type material, albeit immature, appears to be extant at PC fide Rogers (1981), the “neotype” erected by Rogers et al. (1996) is here selected as epitype. Significant morphological traits of this species are the long-stipitate asci (p. sp. 48–50 μm; total length up to 200 μm), and the ascospores, 8–10 × (5)6–6.5 μm, ellipsoid–inequilateral with narrowly rounded ends, and a ventral, conspicuous, broad germ slit, with perispore indehiscent in 10% KOH. The spores are smaller and their size range is narrower than in E. liquescens and E. cinnabarinum. Rogers (1981) stated that the “type material” of Heim’s invalid “E. globosa forma aurantiaca” is immature as well, and pigments of both specimens have been removed upon storage. The epitype specimen was studied by Stadler et al. (2004b) and found to contain mitorubrin, orsellinic acid, and what we meanwhile identified as rubiginosin A and entonaemin A, respectively (see Quang et al. 2004). The pattern differed from those of both E cinnabarinum and E. liquescens in that dimeric azaphilones similar to rutilins (Stadler and Fournier 2006), probably corresponding with the blood red granules, were present. Cultures and anamorphs of this species are still unknown. Entonaema moluccanum J.D. Rogers Mycologia 73: 56, and Figs. 22, 23, 52, 53 (1981). Type Indonesia, North Moluccas, Halmahera, upper slopes of peak of Djaibolo, 13 Aug 1954, A.H.G. Alston 16826 as “Xylaria gigantea” (K(M) 110675-holotype). Notes This monotypic Indonesian species is characterised by ellipsoid-inequilateral, somewhat lemon-shaped ascospores that are larger than in any other member of the genus, measuring (13)14–17 × 7–8 μm. The ascospore perispore of the type material is indehiscent in KOH, and no pigments were extracted with either 25% NH3 or 10% KOH, which agrees with the inconclusive HPLC profile reported in Stadler et al. (2004b). We have studied two specimens referred to as E. moluccanum by Sánchez-Jácome and Guzmán-Dávalos (2005). The duplicates in C were largely immature, and no asci were seen. The material left in GUAD may be more mature. Aside from the common metabolite, orsellinic acid, the immature GD 7285 contained mitorubrin and rubiginosin A, whereas GD 7359 contained only the latter compound and some derivatives thereof in small quantities (see Fig. 6). Its ascospores were smaller in average than in

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the type of E. moluccanum (13.5–15.5×6.5–7.5 μm), ellipsoid with narrowly rounded ends, but not actually lemon-shaped, with straight obscure germ slits almost spore length. Perispores were dehiscent in 10% KOH (Fig. 6), a feature that was not observed before in an Entonaema. Even the type of E. moluccanum features ascospores with an indehiscent perispore (M. S., unpubl.) and its stromata are devoid of mitorubrin and any other pigments. As even very old specimens of pigmented Entonaema spp. always gave very intense pigments (Stadler et al. 2004b), we exclude the identity of the GD specimens with the Asian E. moluccanum. An additional species in Entonaema should eventually be erected for the Mexican fungus. Specimens examined (of Entonaema sp. nov. ined., aff. moluccanum) México, Jalisco Prov., San Sebastian del Oeste, Arroyo El Viborón, in tropical semi-deciduous forest, 15 Aug 1998, L. Guzmán-Dávalos 7359 (GUAD, not seen; duplicate in C, containing a few ascospores); same locality, Huerta de Javier Curiel, 14 Aug 1998, L. GuzmánDávalos 7285 (GUAD, not studied; duplicate in C, immature)-both det. M. R. Sánchez-Jácome as E. moluccanum.

Excluded taxa (Xylaria) Xylaria mesenterica (A. Möller) M. Stadler, Læssøe & Fournier, comb. nov. Basionym Entonaema mesentericum A. Möller, Phycom. Ascom. Bras. p. 306, Figs. p. 248 and Tafel VIII, Fig. 108 (1901). Type Brazil, Sta. Catherina, Seeseite der (sea side of) Serra Geral, 400 m, on decayed wood (B (ex spirit collection No. 58, P115, label reading “Südbrasilien, auf faulendem Holz”) - holotype). Synonym Entonaema pallidum G.W. Martin, Mycologia 30: 431 (1938) [as “pallida”]. Type in FH (see below), isotypes in NY Selected Illustrations Martin (1938), Figs. 1–4; Rogers (1981), Figs. 17–19, 51.3 Geographic distribution/host range Neotropics, on angiosperm wood.

3 A luxurious collection (TL-8380, unfortunately lost in a fire) can be viewed on http://www.mycokey.com/Ecuador.htmls.

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Teleomorph Stromata globose to foliate-lobed, mostly with narrow base, hollow and filled with liquid when fresh, 1– 5 cm diam, with Greenish Yellow (16) pigments in 10% KOH (57) to Livid Red (56) pigments in 25%NH3. Surface grayish white to gray, dotted with black punctiform perithecia. Flesh surrounding ostioles and bordering cavity gelatinous, 5–7 μm thick when fresh, but blackish and only 1 mm diam when dry, a mature specimen almost explodes when picked. Perithecia spherical to tubular, 0.3–0.6 mm wide × 0.5–0.6 mm high. Asci eight-spored, early deliquescent, stipe not observed, p. sp. tubular, 75–85×7–10 μm, with amyloid apical ring, 1.25–1.75 high × 2–2.25 μm wide. Ascospores (Fig. 4d) brown, ellipsoid-inequilateral to equilateral, variable in size and shape, with both ends broadly rounded or with one end attenuate, (9.5)11–14(14.5) × (4.5) 5–6.5 μm (M ¼ 12:3
5:2, n=50), with perispore indehiscent in KOH, germ slit dorsal (in inequilateral spores), straight, almost spore length. Cultures and anamorph Colonies on Difco Oatmeal agar (Fig. 4a) covering a 9-cm plate in 2–3 weeks, at first white, velvety, zonate, with finely lobed margins, sparsely developing brown patches. Aerial mycelium later attaining a pinkish colour. Reverse mostly remaining uncoloured for up to four weeks, finally turning pale greyish brown. Sometimes, pulvinate, primordia-like structures were observed in the centre of the colonies, but those never differentiated into stromata. Conidiogenous structures not observed on this medium. Colonies on YMG agar (Fig. 4b) covering a 9-cm agar plate in 9–11 days, at first white, velvety, darkening from centre outwards with a pale olivaceous velvety layer of hyphae. Reverse remaining uncoloured for the first two weeks of incubation, thereafter becoming olive brown in patches, finally melanising to some extent after 3 weeks of incubation, especially in the centre of colonies. Formation of stromata or primordia not observed even at prolonged incubation times (>2 months). Conidiophores (Fig. 4e) arising from olivaceous or fuscous regions after 3–4 weeks of incubation, sparse, 30–75×2.5–3.5 μm, unbranched, sometimes forming a palisade-like layer like in some species of Xylaria. Conidiogenous cells inflated 8–14×3– 4(4.5) μm, producing one or more conidia from the apical region in a holoblastic or, less frequently, an enteroblastic manner, becoming geniculate when several conidia are formed. Conidia smooth, hyaline, globose to ellipsoid, 4– 5.5×3–4.5 μm. Secondary metabolites Xylaral and derivatives (in stromata); cytochalasins in solid YMG and OA, as well as in liquid submerged YMG cultures. Binaphthalenes, azaphilones (in stromata), melleins, naphthols, chromones (in cultures) and other metabolites of hypoxyloid Xylariaceae not detected.

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Notes Our teleomorph descriptions largely agree with those by Martin (1938) and Rogers (1981), based on the type material of E. pallidum, and with the description by Möller (1901), aside from the stromatal surface. Möller (1901, p. 246) explicitly stated that some of the characteristic features he described for E. mesentericum, such as the mesenteric surface structure (see Fig. 1a), may have become prominent only after storage in alcohol. He also “did not recall whether the stromata were pigmented when collected fresh”. Our microscopic studies on the type in B revealed only a few ascospores (10–13×5–6 μm), which were slightly larger than reported by Möller (1901) and well inside the range observed in E. pallidum. Martin (1938) did not check the type of E. mesentericum in B for comparison when describing E. pallidum as new. Further specimens examined Ecuador, Sucumbios, La Selva, opposite Añangu, canopy tower N of lodge at Garzacocha, alt. 300 m., on rotten branch attached to living Ceiba at ca. 40 m height, 15 Jun 2002, T. Læssøe TL-9701 (C-58426, QCA); Manabi, Río Ayampe near the village, alt. 45 m, on rotten dicot wood at ground level, 31 Jul 2004, T. Læssøe, K. Hansen, J.H. Petersen & A. Alsgård Jensen, TL-11725 (QCNE, C). Mexico, Jalisco, Mpio. de Cabo Corrientes, 9.8 km on road from Brecha El Tuito to Aquiles Serdán (alt. 570 m), in tropical semi-deciduous forest, Sep 13, 2004, O. Rodríguez 2818 (GUAD, C), culture in MUCL and CBS 121671, GenBank Acc.No. AM900591. Panama, Canal Zone, Barro Colorado Island, on stump near termite nesting ground, back of laboratory, 10 Aug 1937, G. W. Martin 4003 (FH 79771, FH 79772), co-types of E. pallidum (isotype extant in NY fide Rogers 1981, n. v.) Chiriqui Prov., Parque Nacional Soberania, Sendero el Charco, on liana, 19 Nov. 2005, M. Piepenbring & students MP 3685 (PMA, culture CBS 121678, MUCL 49332 and, GenBank Acc. No. AM900592). Peru, Dep. Madre de Dios, Rio Tambopata, Tambopata Nature reserve, Explorer’s Inn, 17 Apr 1987, T. Læssøe TL-P152 and TL-P64 (C). Trinidad & Tobago, Port of Spain, R. Thaxter, Mar 1913, labelled ‘E. grisea” (BPI 715364, FH 79763); same collection data but labelled Thaxter 4211 (FH 79762, see Rogers 1981). Venezuela, Amazonas, Neblina Base Camp on Rio Baria (Mawarinuma), 28 Jan 1985, A. Rossman (BPI 1107645).

Xylaria mesenterica and the X. telfairii complex As discussed above, X. mesenterica has chemotaxonomic affinities to X. telfairii and X. enterogena. These species and their presumed relatives (for synonyms, see Dennis 1956, 1957, 1961), are characterised by a rather similar stromatal morphology. They have medium sized to large, internally hollow stromata up to 15 cm high, which are

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frequently covered in youth by a fawn to pale orange or yellowish crust. Stromata of the above Xylaria spp. are more or less rubbery to internally somewhat gelatinous when fresh. At maturity, the pruina disappears to some extent, slightly papillate ostioles become visible, and the stromata are in overmature specimens often split longitudinally with broken edges curled inwards (i.e., appearing involute) or becoming so when dried. Our field observations have revealed that the stromata are filled with liquid, when collected fresh, and this liquid is even used as medical remedy by Indians in lowland Ecuador (T.L., unpublished), but we failed to find a reference to this phenomenon in the literature (presumably because most authors have been dealing with herbarium specimens that were actually collected previously by others). As shown by Callan and Rogers (1990), anamorphic morphology for the above species is also highly similar. In Entonaema spp. and in X. mesenterica, the horny black layer is readily rehydrated, turning somewhat gelatinous. In contrast, rehydration can not usually be accomplished with stromata of the X. telfairii group. In these species a well-defined hollow cavity exists but the texture and colour of the inner stromatal layer are different. We do not think they are among the closest relatives of X. mesenterica, but their hollow stromata have probably evolved in an analogous manner to the cavities of the latter species. Key to Entonaema and other Xylariaceae with hollow, liquid-filled stromata 1 Stromatal pigments greenish yellow in 10% KOH, red in NH3 (Xylaria) 2 1* Stromatal pigments absent or orange in KOH, absent or orange in NH3 (Entonaema) 3 2 Stromata clavate, erect, stipitate, texture carbonaceous Xylaria telfairii and allies (not keyed further out here ) 2* Stromata entonaemoid; non-stipitate, semiglobose, constricted at base or lobed, texture soft to horny X. mesenterica. 3 Stromata with orange or red granules beneath surface layer 4 3* Stromata with green granules beneath surface layer or granules absent 5 4 Subsurface granules orange or orange brown; ascospores (9)10–13×5–7 μm4 E. cinnabarinum 4* Subsurface granules blood-red; ascospores 8–10× 5–5.5(6) μm E. globosum 5 Subsurface granules green; ascospores 8–10.5×3.5– 5 μm E. siamensis 5* Subsurface granules absent; ascospores 8–18×4.5– 8.5 μm 6 4 (for specimens with larger ascospores cf. E. moluccanum ss. Sánchez-Jacomé & Guzmán-Davalos 2005).

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6 Stromatal pigments orange (7) in 10% KOH; ascospores (8)9.5–13.5(15)×(4.5)5–6.5(7.5) μm E. liquescens 6* Stromata without apparent pigments in 10% KOH; ascospores 13–18×6.6–8.5 μm 7 7 Ascospores 13–15×6.5–8 μm E. dengii 7* Ascospores 14–18×6.5–7.5(8.5) μm E. moluccanum Notes The above key was adapted from Rogers (1981) and emended including data on KOH-extractable pigments. The Asian species, E. siamensis Sihan., Thienh. & Whalley has not yet been studied for KOH-extractable pigments and secondary metabolites, but keys out (from E. globosum) because of the greenish subsurface colour and granules reported by Sihanonth et al. (1998). We were so far unable to locate authentic material. Unfortunately, no type material has been deposited in BKF and E, according to the information provided by the curators of these herbaria. Notably, a greenish subsurface colour was also reported by Rogers (1981) upon studies of fresh E. liquescens. The neotype of E. globosum was described from dried, rather than fresh material. Hence, the erection of E. siamensis was mainly based on chemotaxonomic evidence, on the other hand it might as well be a Sarcoxylon from the morphological descriptions provided. The HPLC profile of this species should be checked for comparison with E. globosum. It might actually be revealed to contain reduced mitorubrin derivatives such as hypomiltin (Hellwig et al. (2005), which yields greenish yellow colours.

Discussion Chemotaxonomy As stated in the Introduction, we have included pigment reactions following the detection of xylaral (1). Gunawan et al. (1990) reported that stromata of X. polymorpha gave a violet colour in 25% NH3, which they reported to be due to this compound. No voucher specimen was deposited, and only a standard of the pure compound was available. The yields of xylaral (1) from X. polymorpha, however, were reported to be extremely low (240 mg from 479 g of dried stromata; cf. Gunawan et al. 1990), which may explain that we were unable to find this compound by HPLC profiling in some of our collections of X. polymorpha from the European mainland, and only found xylaral in two specimens from France (JF-07142 and JF-07152). The concentration of xylaral derivatives in these fungi (Table 2) might be correlated with the physiological stage under which the stromata were initially collected. A comparison of different developmental stages by HPLC profiling, using a similar

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approach as that recently reported by Stadler et al. (2006) might provide further evidence. Aside from xylaral, not many metabolites have been reported from stromata of Xylaria. Quang et al. (2006) reported coloratins A and B (2, 3) from stromata of X. intracolorata (J.D. Rogers, Callan & Samuels) J.D. Rogers & Y.M. Ju, and Wang et al. (2005a) reported phlegmacin and emodin derivatives (e.g., 6 in Fig. 2) from a Chinese “X. euglossa Fr.”. Wang et al. (2005b) also published xylactam (5 in Fig. 2), which appears chemically similar to xylaral, from the same fungus. Curiously, X. euglossa is a synonym of X. telfairii according to Dennis (1956, 1961), but no taxonomic literature was cited by Wang et al. (2005a, b). The taxonomy of the material studied by Wang et al. (2005a, b) should be revised. Especially in Xylaria, it is difficult to assess the correspondence of taxa from which unique metabolites have been isolated, as these compounds may have been obtained from immature material, which can usually easily be cultured from tissue but not determined at species level (cf. Stadler and Hellwig 2005). Even at the mature teleomorphic level, there are considerable identification problems in this non-monographed “mega-genus”. There is also little knowledge on the correlation of secondary metabolite profiles in stromata and cultures of Xylaria and other xylarioid Xylariaceae. In general, the secondary metabolism in Xylaria cultures seems to be more diverse than in the hypoxyloid Xylariaceae, as was already illustrated by numerous reports on apparently specific compounds by Whalley, Edwards, and co-workers (cf. Whalley and Edwards 1995). Cultures of X. telfairii have been reported to yield telfairic acids, including the corresponding anhydride (7 in Fig. 2; Adeboya et al. 1996), but these compounds were not available as standards for our HPLC study. Aside from globoscinic acid (4 in Fig. 2) and globoscin from cultures of X. obovata (Adeboya et al. 1995), which are not phthalides, not even remotely similar aromatic compounds were reported in the literature from cultures of this genus (cf. Stadler and Hellwig 2005). Our results on diverging HPLC profiles in cultures of Xylaria imply that it might be necessary to monitor a large number of strains for a certain period of time, using various fermentation conditions, before conclusive results on the specificity of individual components may be obtained. We can so far only vouch for the absence of certain marker compounds that are ubiquitously distributed in what we regard as the hypoxyloid clade of the Xylariaceae. While this study provided the first hint as to the relevance of stromatal metabolites in the xylarioid Xylariaceae, there is little information on the specificity and chemotaxonomic relevance of the various compounds produced by their cultures in relation to morphological concepts. Not even the previous reports on Xylaria metabolites by Whalley and Edwards (1995, and refs. therein) included a comparison of

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individual morphological characters and growth characteristics of the strains examined. In these publications, it was not stated whether and where the stromata and corresponding cultures studied have been deposited. A study on their chemotaxonomic significance will only become feasible, once these compounds have been reisolated and tested for occurrence in a large panel of wellcharacterised cultures. Taxonomy and affinities of Entonaema At present molecular data do allow for safe discrimination between xylarioid and hypoxyloid Xylariaceae, but they do not provide anything new beyond the classical concepts that have been established for Xylaria by traditional microscopic methods. The relevance of a study by Lee et al. (2000) is highly questionable because these authors did not concurrently report any morphological features of the strains they studied. They used several cultures, mostly of Xylaria spp., deposited in public collection whose taxonomy remains to be confirmed and clarified, but it can safely be said that some of these identifications are more than dubious. A molecular phylogeny inferred from α-actin and β-tubulin sequences (Hsieh et al. 2005), using wellcharacterised strains, agreed fairly well with the gross segregation of hypoxyloid and xylarioid Xylariaceae. The Xylaria spp. included there for comparison appeared as sister group to the clade comprising all the above genera. Daldinia appeared to be derived from within Hypoxylon and far apart from Xylaria taxa. While Hsieh et al. (2005) did not study Entonaema, two representatives of the genus were included by Triebel et al. (2005). In their phylogeny, based on 5.8S/ITS nrDNA sequences, E. cinnabarinum and E. liquescens both clustered within a branch of a monophyletic clade within Hypoxylon that otherwise comprised Daldinia. In conformity with other molecular phylogenies (Sánchez-Ballesteros et al. 2000; Polishook et al. 2001), the study by Triebel et al. (2005) suggested a distinction of Xylariaceae into several clades that agreed with the modern concepts based on anamorphic morphology and chemical data. However, in their phylogenetic trees the xylarioid Xylariaceae, the hypoxyloid clade, the Diatrypaceae, and some minor groups were only poorly resolved. A concise molecular phylogeny of the genus Xylaria is not likely to become realistic until further sequences of cultures obtained from well-characterised teleomorphic material become available. As exemplified above, many rDNA sequences recorded from the genus at present, have been derived from supposedly endotrophic fungi. Many of these data are unpublished, or no morphological evaluations were presented concurrently, and corresponding cultures are frequently not available from public collections. We therefore

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decided not to provide an extensive molecular phylogeny including sequences of other Xylaria spp. from GenBank. Chemotaxonomically significant characters in xylarioid Xylariaceae So far, stromatal secondary metabolites have mainly proven useful for hypoxyloid taxa (Stadler and Fournier 2006). However, our preliminary results on the detection of stromatal metabolites in Xylaria provided evidence on the potential utility of the KOH and NH3 pigment reactions for taxonomic purposes in this genus, too. We propose that this feature be applied on additional specimen contingents and species. As exemplified here by the lack of apparent pigments in some Xylaria stromata, which were shown concurrently to contain xylaral derivatives by HPLC, it should be kept in mind that the classical method for determination of pigments is sometimes less sensitive than modern analytical techniques. The concentrations of pigments, and thus the intensity of colour reactions, may at times vary with the age of the examined specimens, as well as during stromatal ontogeny (Stadler et al. 2006, 2007b). Similar phenomena have been encountered in Hypoxylon, where, e.g., overmature type material of H. carneum Petch lacked stromatal pigments altogether, while fresh young specimens of the same species readily yielded purple pigments in KOH (Stadler et al. 2004a). Correlations between the state of development and the pigment colours need to be established. Pigments should at best be assessed and recorded in fresh material immediately after collection. The stromatal HPLC profiles of some groups in Xylaria, such as the X. hypoxylon complex, were entirely inconclusive. We expect that screening of stromatal extracts by HPLC will only give limited informative data. Nevertheless, such information may turn out to be valuable at least in some species groups. Differences in the production of xylaral derivatives might eventually prove to be taxonomically informative in certain groups of Xylaria. However, before this hypothesis can be verified, a broad range of additional specimens and taxa should be studied. Xylaral was reported by Gunawan et al. (1990) to lack antibiotic activities, albeit no detailed results were provided. The possible natural function of these molecules is therefore still unclear, and they might well be revealed in future to possess a more specific function. It should also be interesting to further pursue the use of the fluid from stromata of the X. telfairii group to see whether xylaral or other metabolites accumulate there and whether the therapeutic value can be deduced from the traditional usage as ethnomycological drug. The apparent lack of informative chemical traits in stromata of various Xylaria spp., however, implies that cultures will ultimately need to be studied to attain a better

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understanding of their chemotaxonomic affinities. For this, a modification of the approach outlined by Whalley and Edwards (1995, and references therein) might be feasible to establish a sound taxonomy of this large and poorly understood genus. However, most of the original cultures studied by Whalley and Edwards (1995) and their coworkers appear not to be extant in public collections. No details were reported on the number of specimens cultured, and thus the chemotaxonomic significance of the metabolites. It was apparently not attempted to detect the specific compounds of certain Xylaria spp. in related genera and species, and in most cases not even the literature used for identification was cited. Another complication we observed in our study relates to the higher apparent diversity of secondary metabolism in Xylaria as compared to hypoxyloid Xylariaceae, and to the fact that not all chemotaxonomic marker compounds tend to accumulate at prolonged fermentation times in their cultures. It will therefore be a great challenge to undertake a comprehensive study of this kind in Xylaria and its allies, because they appear more diverse with regard to their secondary metabolism, especially when their entire “secondary metabolome” is concerned. It remains to be seen whether this indicates that they are evolutionary derived. Acknowledgements Our warmest thanks go to Burghard Hein and Harrie Sipman (B), Genevieve Lewis-Gentry (FH), Anna-Lena Anderberg (S), Begoña Aguirre-Hudson (K), Ellen Bloch (NY), Erin McCray (BPI), Laura Guzmán-Dávalos (GUAD) and all other curators of public herbaria, for kindly sending us specimens, and to Dagmar Triebel (M), also for help with international loan transactions. We acknowledge the input by Régis Courtecuisse, Lille, for facilitating collection work in the French West Indies. Furthermore, we thank Jack D. Rogers, Francoise Candoussau, Jean Louis Cheype and Ralph Mangelsdorff, for providing us with specimens, and Wolfgang Steglich (Munich, Germany) and Norbert Arnold (Leibniz Institute for Plant Biochemistry, Halle/Saale, Germany) for providing us with a standard of xylaral. Expert technical assistance by Dirk Müller (InterMed) for recording HPLC-MS spectra, and by Beata Schmieschek and Michael Benfer (dto.) for assistance in handling the cultures and performing HPLC-DAD analyses, is also gratefully acknowledged. Finally, we thank two anonymous referees for their valuable comments, which substantially improved our manuscript.

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