Current Research in Environmental & Applied Doi 10.5943/cream/1/2/1 Importance of secondary metabolites in the as parameters for assessment of their , phylogeny, and functional biodiversity

Stadler M1, 2

1 InterMed Discovery GmbH, Dortmund, Germany 2 Department Mycology, University of Bayreuth, Bayreuth, Germany

Stadler M 2011 – Importance of secondary metabolites in the Xylariaceae as parameters for assessment of their taxonomy, phylogeny, and functional biodiversity. Current Research in Environmental & Applied Mycology 1(2), 75-133, Doi 10.5943/cream/1/2/1

This paper constitutes a synopsis of our polythetic studies of the Xylariaceae, which was originally compiled in the course of a Habilitation thesis. Based on several thousands of specimens and several hundreds of cultures, morphological studies of the teleomorphs and anamorphs of these fungi were combined with chemotaxonomic studies based on HPLC-DAD/MS profiling, as well as PCR fingerprinting and molecular phylogenetic analyses. Numerous novel pigments and other natural products, many of which were shown to have biological activities were also isolated and identified, and their production in the course of the life cycle of their producer organisms was followed by HPLC profiling and biological assays. Numerous new species and even new genera were recognised in the course of this work. Finally, secondary metabolite production in cultures of Xylariaceae was correlated with molecular data and the production of certain chemotaxonomic marker compounds was found to be strongly correlated with a phylogeny based on ITS nrDNA, demonstrating that secondary metabolite profiles are not only important species-specific characters but even have phylogenetic significance. The work on Xylariaceae is proposed as a model how interdisciplinary, international collaborations can help to increase our understanding of the phylogenetic and evolutionary relationships, as well as the biology of fungal organisms. Similar work on other groups of the and Basidiomycota would certainly be rewarding.

Key words – chemosystematics – – extrolites – metabolomics – bioprospecting

Article Information Received 19 August 2011 Accepted 22 August 2011 Published online 29 October 2011 Corresponding author: Marc Stadler – e-mail – [email protected]

Introduction ples to be included in High-Throughput This study originates from a pilot project Screening libraries, was outlined elsewhere in at Bayer AG, when I had been responsible to detail (Stadler & Hellwig 2004). Briefly, we build a culture collection of secondary meta- aimed to find predictive parameters that could bolite producing fungi for pharmaceutical and be effectively used to characterise the in-house agrochemical screening, which meanwhile fungal culture collection and screening libra- comprises over 10.000 strains. The Xylariaceae ries. Chemotaxonomic information was com- were taken as model organisms to evaluate cor- bined with classical taxonomy and PCR-based relations between the diversity of morphotype- methods, to improve the efficacy of the screen- based taxonomy, secondary metabolite produc- ing process. The Xylariaceae were chosen as tion and PCR-based molecular data. This ap- model organisms for the following reasons. proach, relating to the need for implementation This family of Ascomycota represents of rigorous pre-selection procedures for sam- one of the predominant groups of fungal 75 endophytes, which became of great interest as fuelled by the inestimable help of numerous sources for novel lead compounds, but are par- other mycologists and natural product chemists ticularly difficult to classify by morphological from around the world. This thesis is intended methods. As will be outlined below, - to present the results in a historical and ceae are “model endophytes”. methodical context and give an outlook on Monographs of (Ju et al. 1997) what can be done in the future. and (Ju & Rogers 1996), relying in The model character of this study for part on chemotaxonomic characters had just interdisciplinary mycology and an integrative been published. Stromatal pigments were re- approach to biodiversity assessments in fungi garded as taxonomically significant, but their will also be briefly addressed. chemical structures remained widely unknown, leaving options for discovery of new bioactive The Xylariaceae represent one of the molecules. On the other hand, Whalley & largest and most important families of the As- Edwards (1995) had demonstrated the chemo- comycota and the Fungi. The exact number of taxonomic value of metabolites from cultures. currently accepted taxa varies between authors. The ubiquitous wood-decomposing Xyla- For instance, sixty genera that potentially be- riaceae are also of interest in biodegradation of long to the Xylariaceae were listed by xenobiotics. Together with Heinz-Georg Lumbsch & Huhndorf (2007). The Dictionary Wetzstein and Hans-Volker Tichy, we studied of the Fungi (Kirk et al. 2008) listed 85 accep- them to evaluate their capabilities for biodegra- ted genera (including 71 synonyms) and 1343 dability of quinolone antibiotics, along with ba- accepted species. The vast majority of those are sidiomycetes (cf. Wetzstein et al. 1996). These associated with plants, but some genera and studies included the characterisation of the fun- species are known from herbivore dung or ter- gi based on molecular methods. The Xylaria- mite nests. As discussed below, further associa- ceae were at first studied by PCR fingerprin- tions of Xylariaceae with other insects have ting; but DNA sequences were also generated recently been discovered. later on. The hypothesis by Hawksworth (1991), Sophisticated analytical methods such as who postulated 1.5 million of fungal species to High performance liquid chromatography, hy- be extant (of which only 5% are hitherto phenated with diode array detection (HPLC- known to Science), is largely based on an as- DAD) and mass spectrometry (HPLC-MS) had sumed ratio of five plant-associated fungal spe- just become available in our laboratory in the cies per plant species, which can be assumed to search for novel drug candidates (Stadler et al. be realistic from our current knowledge on the 1998). HPLC-DAD and direct inlet mass spec- biodiversity in the well-investigated geographic trometry were already used in the chemotaxo- areas of the world. Accordingly, as many as nomy of Penicillium (Smedsgaard & Frisvad 10.000 additional Xylariaceae species may re- 1996), and HPLC-DAD was employed by main to be described. As outlined by Rogers Lumbsch (1998) for chemotaxonomy of liche- (2000), their morphological diversity is particu- nised ascomycetes. Inspired by these studies, larly high in the tropics, and indeed, several we extended such techniques to the Xylaria- genera have an exclusively tropical distribu- ceae (albeit HPLC-MS was to my knowledge tion. On the other hand, most taxonomic and used in fungal chemotaxonomy by us for the chorological studies were carried out in the first time). temperate regions of the Northern hemisphere, In retrospective, the project, baptised and biogeographic information on the global “Molecular chemotaxonomy of the Xylaria- distribution of the Xylariaceae is still widely ceae” (Stadler et al. 2001d), proved extremely amiss. helpful to increase the diversity of the screening libraries as the Xylariaceae are not Higher classification and teleomorphic the only group of filamentous fungi in which stages of the Xylariaceae chemotaxonomic information is well-correlated The Xylariaceae belong to tribe Ascomy- with molecular and morphological data. The cota, class , subclass Xylario- project soon developed its own dynamics, mycetidae, Xylariales. In previous classi-

76 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 fications of ascomycetes they were included in designed for anemochoric dispersal. class Pyrenomycetes, as their ascocarps are pe- The anamorphic structures are most often rithecia. In the Xylariaceae, the perithecia are referred to the genus Nodulisporium Preuss, frequently embedded in a conspicuous stroma. which was already linked by Tulasne & Morphological and anatomical features of the Tulasne (1863) to xylariaceous teleomorphs. stroma have been traditionally used in generic Nevertheless, it took another century until the classification of the Xylariaceae. The typical significance of anamorphic morphology was xylariaceous octosporous asci are tubular and fully appreciated in their classification and ta- stipitate, featuring an amyloid apical apparatus, xonomy. Greenhalgh & Chesters (1968), Mar- which may be reduced in some genera and tin (1969a, b, and further papers of his series), species. The spores are actively released from and Jong & Rogers (1972) gave extensive refe- the perithecial ostioles in those species that rence of anamorphic traits in several species. have an apical apparatus. The ascospores (Fig. Petrini & Müller (1986) studied numerous 2A) are arranged uniseriately in the asci, one- European Xylariaceae for anamorphic and celled, mostly ellipsoid to ellipsoid-inequilate- teleomorphic features. Today, the microscopic ral and dark brown, and have a germ slit. They characters of the conidiophores and the macro- are designed to survive in the environment for morphology of mycelial cultures are regarded a long time after discharge, and the germ slit highly important at various taxonomic levels. allows a rapid germination under favourable New species are now often recognised, based conditions. The ecophysiological features of on new combinations of teleomorphic and the Xylariaceae indicate a xerophilous lifestyle anamorphic characters, applying a holomorphic of their ancestors (Rogers 2000). morphological species concept. Jack D. Rogers, Y.-M. Ju and their co- Infrageneric affinities and anamorphic workers provided numerous important mono- stages graphs based on holomorphic morphology and While stromata of many plant-associated published several papers on cultural and ana- Xylariaceae (Fig. 1) are frequently and ubiqui- morphic characteristics of the known species. tously encountered, others form stromata and The term “Nodulisporium-like anamorph” was sexual stages on the hosts only sparsely and restricted by Ju & Rogers (1996) to the asexual occasionally. Their life cycle is apparently stages of Hypoxylon Bull., and related genera. dominated by asexual propagation, and their This definition now also embraces morpho- mycelia remain dormant in the host plants for a types such as Xylocladium P. Syd. ex Lindau, a long time. As in other groups of Ascomycota characteristic anamorph of Fr., as (e.g., in Aspergillus P. Micheli ex Haller and well as the Virgariella-like, Sporothrix-like and Penicillium Link), many Xylariaceae may have Periconiella-like conidial stages that are found abandoned the production of the sexual stages instead of or besides the common Nodulispo- altogether. In contrast to the aforementioned rium type (Fig. 2B) in several species of Hypo- “moulds”, the Xylariaceae, however, are not xylon and its immediate allies (e.g., Daldinia often encountered in soil (see Ecological as- Ces. & De Not.). These anamorph types mainly pects). Their asexual propagation is generally differ in the complexity of their conidio- accomplished via the production of conidia. phores, whereas the morphology of conidio- These propagules are borne on conidiophores genous cells appears rather uniform. The coni- of rather characteristic structures, which may diogenesis occurs from apical regions of the arise either from the stromata or from the my- terminal (or rarely, intercalary) swollen, denti- celia on the substrate. Many species will rea- culate, cylindrical to clavate conidiogenous dily produce anamorphic structures in laborato- cells. Most species produce conidia from sym- ry culture. Their production is not as dependent podially proliferating conidiogenous cells in a on the season as that of the sexual stages and holoblastic manner, albeit exceptions are may occur several times throughout the vege- known. For instance, some Daldinia spp. asso- tation period under favourable conditions. They ciated with Betulaceae, including D. petriniae are relatively short-lived in contrast to the Y.M. Ju, J.D. Rogers & San Martín and D. ascospores and do not appear to be ideally decipiens Wollw. & M. Stadler, predominantly

77

Fig. 1 – Stromata of Xylariaceae. A . B Hypoxylon fragiforme. C Hypoxylon rubiginosum. D . Images by Jacques Fournier

Fig. 2 – Characteristic microscopic features of the Xylariaceae, as exemplified by Daldinia eschscholtzii (Ehrenb.) Rehm. A Ascospores (SEM, 5000×). B Nodulisporium conidiophore (phase contrast micrograph, 400×). show an enteroblastic conidiogenesis and pro- described in serpens (Chesters & duce conidia from percurrently proliferating Greenhalgh 1964, as Hypoxylon serpens). conidiogenous cells (cf. Petrini & Müller 1986, Aside from the genus Nemania S.F. Gray, this sub D. occidentalis Child; Ju et al. 1997, sub group comprises Sydow, Euepixylon D. petriniae; Stadler et al. 2001c). Füisting, Fr., de Not., The second, large group within the fami- Xylaria Hill ex Schrank, and many other gene- ly is characterised by having “Geniculospo- ra of the Xylariaceae. In contrast to the Noduli- rium-like” anamorphic stages sensu Ju & sporium type, their conidiogenous cells become Rogers (1996), referring to Geniculosporium geniculate after repetitive production of conidia Greenh. & Chesters, the asexual stage first from different areas on a relatively long rachis

78 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 as the production of conidiogenesis is not 1993). These examples show the difficulties to restricted to the apical portion of the conidio- draw clear boundaries between the families of genous cell. The Geniculosporium type of the Xylariales. The evolution of this fungal conidiophores (including the associated mor- order obviously resulted in numerous conver- phological structures) is more variable than the gent developmental lines, which remain to be Nodulisporium type. Geniculate conidiophores evaluated by modern taxonomic techniques. may simply arise from vegetative hyphae, but Nonetheless, the higher taxonomic classi- they may also be arranged in palisades (as in fication of Xylariomycetidae is outside the ma- the Dematophora R. Hartig stages of some jor scope of this study, which concentrated on Rosellinia spp.; cf. Petrini 1992, 2003. 2005). infrageneric and intergeneric relationships of In Xylaria hypoxylon (L.) Greville, they can the Xylariaceae. Only a few representatives of even be borne on coremia-like, conspicuous other families were included for comparison. stromata. The genus Collodiscula I. Hino & This topic is therefore not further addressed Katum., which is now included in the Xylaria- here. A current overview on the organisation of ceae despite its two-celled ascospores, has an the higher taxa in the Sordariomycetes and Acanthodochium Samuels et al. anamorph. their phylogenetic relationships has been pub- This was also categorised by Ju & Rogers lished by Zhang et al. (2006). (1996) as Geniculosporium-like. The ana- morphs of the known coprophilous Xylariaceae Taxonomic history of the Xylariaceae genera also belong to this group, but, as The basionym of the type species of the exemplified by the Lindquistia Subram. & family, Clavaria hypoxylon L. (syn. Xylaria Chandrash. stage of Willd., they show hypoxylon) goes back to Linnaeus (1753). specific morphological adaptations to the diffe- Persoon (1799) and Fries (1823) still preferred rent habitat. The conidia of most of the genera to use the generic name Sphaeria Persoon. with Geniculosporium-like and Nodulisporium- Tulasne & Tulasne (1863) are often referred to like anamorphs are ellipsoid or ovoid. Some as the mycologists who erected the family Xylariaceae genera cannot be assigned to either name. However, they used the term “Xylariei”, of the above groups and differ from them in and it is not clear whether this was meant to re- producing anamorphic structures similar to lated to a family concept as we would under- those of the related family Diatrypaceae. They stand it today. Nevertheless, the Tulasne Bro- produce long, slender, scolecosporous conidia, thers and Nitschke (1867) provided remarkable similar to those of the coelomycete genus Li- and valuable early contributions to their taxo- bertella Desmaziéres, which is rather distantly nomy. Many other famous mycologists of the related to the Xylariaceae. Genera with 19th century, including M.J. Berkeley, M.C. Libertella-like anamorphs [e.g., Cooke, J.B. Ellis, J.F.C. Montagne, and L.D. Theiss., Lopadostoma (Nitschke) Traverso, von Schweinitz, also dealt with them and Jumillera J.D. Rogers et al., J.D. erected new taxa. In his Sylloge fungorum, P.A. Rogers et al.] are maintained in the Xylariaceae Sac-cardo provided an account of all fungal because of their characteristic ascospore species that were then known to Science (most morphology. In case of Jumillera, the of the Xylariaceae were treated in the first conidiophores are Geniculosporium like, while volume; Saccardo 1882) and listed several only the conidia differ in their morphology hundreds of Xylariaceae taxa. However, owing (Rogers et al. 1997). Both, Libertella-like and to the mo-numental task he had to accomplish, Nodulisporium-like anamorphs have been he mainly checked herbarium material and had reported from the genus to use rather broad taxonomic concepts, which Saccardo. must be kept in mind when interpreting his To further complicate the taxonomy of data. the Xylariales, there are also genera with dia- A brilliant early study was published by trypaceous teleomorphs and xylariaceous ana- Möller (1901) on neotropical Xylariaceae. He morphs, such as Graphostroma Pirozynski included meticulous field observations, micro- (Pirozynski 1974), for which an own family, scopic studies on their anamorphs and stroma- Graphostromataceae, was erected (Barr et al. tal development of the species he observed in

79 Brazil. In retrospective, his monograph appears were not validly erected by Dennis (1961), and visionary, as it relates to concepts that were they remain to be emended and officially pro- only established several decades later. Another posed in a taxonomic paper. Dennis did not yet mycologist who undoubtedly strongly consider anamorphic morphology to be signifi- influenced our modern concept of the Xylaria- cant. He could therefore not possibly have en- ceae was Julian H. Miller, who worked on visaged some major taxonomic changes rela- these fungi for several decades. He provided ting to the resolution of Hypoxylon sensu several important papers on their taxonomy, Miller (1961). Rogers (1994) pointed out some including the first world monograph on Hypo- problems that remain to be solved before final xylon (Miller 1961). Meanwhile, Dennis (1957, conclusions on higher classification of the Xy- 1959, 1961, 1963, 1964) revised Xylariaceae lariaceae at suprageneric ranks can be drawn, from around the tropics. His work heavily con- and some of these still persist even today. tributed to our present knowledge on the family Strikingly, there are not many features re- in warmer climates and constituted an impor- lating to the teleomorph that can be universally tant starting point for our own work on their ta- applied to segregate the Hypoxyloideae from xonomy. the Xylarioideae. Their ascospore and ascal However, Dennis and Miller both still morphology appear rather similar. The mor- used rather broad concepts. Only In the years phology of the ascal apical apparatus, which following Miller’s monograph, data on ana- differs between the xylarioid and the hypoxy- morphic morphology (see above), and details loid genera, may be taken as an additional on ascal and ascospore morphology and ultra- diagnostic tool for differentiation of xylarioid structure became increasingly available. The and hypoxyloid teleomorphs (Rogers 1979). work by Pouzar (1985a,b) was instrumental in Several genera and species are characterised by segregating Nemania S. F. Gray from Hypoxy- having an aberrant ascal morphology. Cultures lon sensu Miller (1961). In the following, this and anamorphs of numerous genera and species genus was segregated by applying a holomor- are as yet unknown. Many of our own studies phic concept. For instance, Læssøe et al. (1989) (see below) were aimed at gaining further in- treated Camillea Fr. and revised some other formation on this matter, and were designed to taxa in Hypoxylon that have light-coloured as- employ complementary methodology to eva- cospores. In the 1990s, various taxonomic revi- luate infrageneric and intergeneric affinities, sions by J.D. Rogers, Y.-M. Ju and co-workers above all in the Hypoxyloideae. resulted in the recognition of additional genera of predominantly tropical distribution that had Ecological aspects also been included in the “old” broad concept As pointed out by Whalley (1996), the of Hypoxylon. However, as with the aforemen- Xylariaceae have long been considered to be tioned Nemania, it was often possible to resur- wood-destroying or coprophilous saprobes, rect old generic names, such as Creosphaeria aside from a few facultative tree parasites. En- Theiss. (Ju et al. 1993), Entoleuca (Rogers & toleuca mammata (Wahlenberg) J.D. Rogers & Ju 1996), and Kretzschmariella Viegas (Ju & Y.M. Ju, the causative agent of “Hypoxylon Rogers 1994). The type species of these genera canker” of Populus spp. (Ostry & Anderson were all included as synonyms of Hypoxylon 2009) and (Hoffm.) by Miller (1961), but proved to have ana- P.M.D. Martin, a frequent parasite of beech, morphs other than Nodulisporium and were are among the few important xylariaceous plant therefore expelled from the large genus. parasites of the Northern Hemisphere. Certain In Table 1 the above mentioned concept species of Rosellinia are known to cause se- of “subfamilies” Hypoxyloideae and Xylarioi- rious damage as pathogens of trees or agricul- deae) is adopted. Those names were proposed tural plants (Edwards et al. 2003, ten Hoppen by Dennis (1961) in a different context, but & Krauss 2006), but aside from R. necatrix will here be used for the two major groups of Prillieux, those are mainly distributed in the Xylariaceae sensu Ju & Rogers (1996) with tropics. These parasites all belong to the Nodulisporium-like and Geniculosporium-like Xylarioideae as understood here. The only eco- anamorphs, respectively. These “subfamilies” nomically important pathogen among the

80 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 Table 1 Gross classification of the major Xylariaceae genera into “subfamilies”.

Hypoxyloideae Xylarioideae Others/Genera incertae sedis Anthostomella Sacc. p.p.* Astrocystis Berk. & Broome Anthostomella Sacc. p.p.* Areolospora S.C. Jong & E.E. Davis Berk. Ascovirgaria J. D. Rogers & Y.-M. Ju (= Phaeosporis Clem.) Kuntze Discoxylaria Lindquist & J. Wright Creosphaeria Theiss. Camillea Fr. Engleromyces Henn. Kretzschmariella Viégas Chlorostroma A.N. Miller et al. Entoleuca Syd. Jumillera J.D. Rogers et al. ** Lopadostoma (Nitschke) Traverso Daldinia Ces. & de Not. Euepixylon Füisting Whalleya J.D. Rogers et al. A. Möller Whalley et al. Hypoxylon Bull. (Fr.) J. Kickx fil. Induratia Samuels et al. Jumillera J.D. Rogers et al. Obolarina Pouzar Kretzschmaria Fr. Lév. Leprieuria Læssøe et al. Pyrenomyxa Morgan Nemania S.F. Gray D. Hawksworth Ellis & Holw. Ehrenb. Poronia Willd. Theissenia Maubl. Rosellinia de Not. Penz. & Sacc. Sarcoxylon Cooke Versiomyces Whalley & Watl. Stilbohypoxylon Henn. Vivantia J.D. Rogers et al. Xylaria Hill ex Schrank Largely based on the overview by Ju & Rogers (1996), except that genera with Libertella-like anamorphs are listed here as “genera incertae sedis”, and that recent taxonomic papers have been considered. The genera of which a significant number of representatives (in relation to the number of described species) were already included in our chemotaxonomic evaluation are printed in bold. Those taxa of which a significant number of representatives was also included in our molecular phylogenetic study are highlighted in grey. Some recently erected genera with unknown anamorphs, and non-stromatic genera that were placed in the Xylariaceae based on molecular phylogenetic evidence are not included. * Anthostomella species may have either Libertella-like or Nodulisporium-like anamorphs. **Several Jumillera species have Libertella-like anamorphs with Geniculosporium-like synanamorphs.

Hypoxyloideae in Europe is Biscogniauxia produce stromata on damp, already highly mediterranea (De Not.) Kuntze. This decomposed wood (Laessøe & Spooner 1994). seriously affects evergreen oaks in the The mechanisms of wood biodegradation Mediterranean (Collado et al. 2001, Nugent have been studied extensively in some tempe- 2005). It has therefore been studied more rate (Sutherland & Crawford 1981, Boddy et intensively than most other Xylariaceae by al. 1985, Boddy & Rayner 1993) as well as the plant pathologists. tropical Xylariaceae (Pointing et al. 2003, The vast majority of the stromatic Xyla- 2005), albeit most of the basic research with riaceae colonise dead and decaying wood of regard to this topic has been dedicated to their angiospermous plants. Saprotrophic Xylaria- basidiomycete counterparts. For instance, Pha- ceae are predominant in virtually all forested nerochaete chrysosporium Burds. has been habitats of the world. They are considered to be evaluated extensively as a “model wood-des- white-rot fungi, owing to their ability to de- troyer” for more than two decades, and even grade lignin, but they even can degrade cellu- the sequencing of its genome has been accom- lose very effectively (Merrill et al. 1964, Wei plished. On the other hand, it remains widely et al. 1992). Some species (including Hypoxy- obscure whether the biogenetic pathways that lon fragiforme and the “primary colonisers” of have evolved in the Xylariaceae for decompo- the genus Daldinia, which are discussed further sition of wood and other organic substrates are below) appear in the succession of wood- analogous or homologous to those of the basi- decomposing fungi quite early. Their activities diomycetes. Shary et al. (2007) have recently (or at least the formation of their stromata) demonstrated that wood-grown cultures of a apparently cease when the more competitive fungus they named “Daldinia concentrica” polyporaceous Basidiomycetes arrive. Other degraded 14C-labelled synthetic lignin in a Xylariaceae including Euepixylon udum (Pers.) similar manner as white-rot basidiomycetes. Füisting and many Nemania spp., exclusively However, their conclusions were drawn from

81 chemical analyses of the biodegradation able to form their massive stromata on such process, based on the end-products of the relatively small and fragile plants, even after degradation process. It still remains unclear decomposition. To my knowledge, anamorphic whether the molecular and biochemical stages of Xylariaceae growing as saprobes on prerequisites for this process have evolved in decaying liverwort thalli have likewise not homology or in convergence in Xylariaceae been encountered. It is not clear whether the and basidiomycetes. fungi are horizontally transmitted, and how the The Xylariaceae have also received con- liverwort thalli are infected by fungal propa- siderable attention because of their mutualistic gules. relationships with plants. Carroll (1988), c) A study in a national park of Thailand, Petrini & Petrini (1985) and others showed by using extensive culturing and similar methods classical microbiological methodology and for characterisation of genotypes (Okane et al. morphology-based identification techniques 2008), revealed over 270 endophytic Xylaria- that xylariaceous anamorphs constitute one of ceae in different seed plants. Over 100 strains the predominant groups of fungal endophytes obtained from representative teleomorphic ma- in various habitats and plant taxa. Recent mole- terials from the same site were examined for cular ecology studies (Arnold et al. 2001, 2003, comparison. The authors recognised 21 diffe- Guo et al. 2000, 2001) reinforced this view. rent genotype clades that may represent spe- Even fungal DNA extracted from the host cies. Several of those did not correspond to as- plants can now be assigned to the Xylariaceae cospore isolates from the same site. As inferred as inferred from similarity analyses of their from a comparison of DNA sequence data with rDNA genes. Recent reviews on fungal endo- those in public domain databases, some of phytes in general have been compiled by these “endophyte” taxa may well correspond Schulz & Boyle (2005) and Hyde & Soytong with the teleomorphic stages of fungi that have (2008). The role of the Xylariaceae as endo- been studied in other parts of the world, since a phytes was last reviewed by Petrini et al. high degree of homology to the corresponding (1995). Out of the vast amount of literature on published data in GenBank (http://www.ncbi this topic, only some recent studies dealing .nlm.nih.gov/Genbank) was observed. with Xylariaceae are treated here in detail. Despite the general availability of sophis- a) Orchids are very well known as sym- ticated molecular methods, a problem still per- bionts of mycorrhizal fungi, but Yuan et al. sists regarding the safe identification of the (2008) focused on their endophytic mycobiota, xylariaceous endophytes. While the keys by revealing that 14 out of 33 fungal “morphospe- Petrini & Petrini (1985) and other detailed cies” belonged to the Xylariaceae and verified morphological descriptions of Xylariaceae ana- their results by molecular data. The Xylaria- morphs will often help in narrowing down the ceae were found in all organs of the plants. identification to the level of genus or species b) Even liverworts are predominantly co- group, endophytes often remain entirely sterile lonised by “xylarialean” endophytes in diffe- in culture. In such cases, or when the identifi- rent regions of the world (Davis et al. 2003, cation is only based on “environmental” DNA Davis & Shaw 2008). Even though Davis & sequences, the fungi can at present hardly be Shaw (2008) referred to the order Xylariales, identified with certainty. An exception from most of the genera mentioned in the earlier this rule may be the fungal endophytes belon- paper apparently belong to the Xylarioideae. ging to the genus Nodulisporium, which pro- They comprised about two-thirds of the total duce the antiparasitic agent nodulisporic acid endophyte populations, as revealed from and occur all over the tropics (Polishook et al. 5.8S/ITS nrDNA sequences. Most of these 2001). Notably, the detection of the antiparasi- liverwort-associated Xylariaceae have not been tic secondary metabolite coincided with rather identified conclusively to species level for lack strong homologies of rDNA sequence data. Up of reliable data on teleomorphic material for to date, no xylariaceous teleomorph was asso- comparison, but in most cases, high similarities ciated to these endophytes. The DNA sequen- were observed to sequence data derived from ces derived from the producer strains are signi- stromatic taxa. Evidently, the fungi will not be ficantly distinct from those of all published

82 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 teleomorphic Xylariaceae. Possibly, the produ- vegetation periods, even living trees may be- cers of nodulisporic acid constitute a highly come seriously affected by “saprobic” or “en- specialised group within the Xylariaceae that dophytic” Xylariaceae. Nevertheless, as com- has abandoned production of the teleomorph. pared to other groups of fungi, the damages However, most xylariaceous endophytes caused in agriculture and forestry by parasitic appear to have close affinities to certain teleo- Xylariaceae appear rather moderate. morphic species or constitute the asexual stages Fungal endophytes were even postulated of those fungi themselves. Not only are the to have a positive effect on their host plants ascospores of this family perfectly designed to (Arnold et al. 2003). They might indeed act as withstand environmental hardships, but they antagonists to parasites, impeding colonisation may also have found a way to awake from dor- by parasitic fungi. Evidently, a plant that is al- mancy at the right time. Chapela et al. (1990, ready inhabited by mutualistic organisms will 1991) studied in detail the process of host not be as easily affected by parasites as an infection for H. fragiforme. For this species uncolonised plant. Possibly, the fungal endo- and Fagus, an intricate host/fungus recognition phytes dispose of their own armoury to defen- mechanism was assumed. Ascospore germina- ding their habitat, such as antifungal secondary tion is mediated by mannolignol glucosides, metabolites. Such a phenomenon was proven present in the host, acting as specific recogni- conclusively in case of the systemic clavicipita- tion messengers. Ascospore eclosion and ger- ceous endophytes of ryegrass (Schardl et al. mination is induced by the messenger com- 2004), which produce alkaloids with antifee- pound (Chapela et al. 1993), facilitating the dant and other biological activities. Those com- colonisation of the host. A similar mechanism pounds may substantially accumulate in the may be generally involved in the establishment host plants and thus protect them against herbi- of an endophytic relationship between other vore feeding. Xylariaceae and their host plants. This pheno- The general availability of molecular menon deserves further study, using a broad identification techniques would be very helpful range of host plants and xylariaceous model or- for studies of endophytic Xylariaceae. How- ganisms. ever, reliable reference sequence data in public Some other studies dealt with the role of databases are still amiss for numerous taxa of xylariaceous and other fungal endophytes in this family. During the last decade, several en- the process of wood decay, in which these fun- tries of new “unidentified” xylariaceous and gi turn from endophytes to saprotrophs (Boddy xylarialean DNA sequences were made in the & Griffith 1989, Griffith & Boddy 1990). They aforementioned databases from inventories of examined the ecophysiological processes and fungal endophytes. the fungal succession in detail, highlighting the Innovative methods, such as the design of role of certain Xylariaceae in the decay com- specific primers (Johannesson et al. 2000a) or munity. For instance, it was established that the approaches based on microarrays or real time anamorph of D. concentrica (Bolt.) Ces. & de PCR have recently been developed to detect Not., which preferentially forms stromata on and study fungi in the environment. With re- , is often present on other angiosper- gard to Xylariaceae, their application still re- mous hosts as an endophyte. This was later mains largely restricted to economically impor- confirmed by Nugent (2003) using molecular tant pathogens (see e.g., Schena et al. 2002 for identification methods. R. necatrix; Luchi et al. 2005 for Biscogniauxia The boundaries between the endophytic, mediterranea). It would be a great benefit for parasitic, and saprotrophic lifestyles are still studies aimed at elucidation of the ecology and obscure. They may just reflect an equilibrium the life cycle of the Xylariaceae if such in the fungus-host relationship (Whalley 1996, methods became widely available. However, Edwards et al. 2003). The host plants become even the most sophisticated molecular more susceptible to their endophytic xylaria- identification technique will only produce ceous inhabitants after long periods of drought. inconclusive results in the lack of reliable When the host trees are subjected to water reference data. stress for a long time or in several consecutive Much information has become available

83 on apparent host specificity of certain Xylaria- inclu-ding Rosaceae and Salicaceae. From the ceae, which also relates to ecological aspects. view of a forest ecologist, they are “primary Such data are largely based on the apparent oc- colo-nisers”, and it was long assumed that the currence of the stromata. They need to be taken fungal spores only arrive shortly after the fire. with caution in view of the results on the In fact, the rapid production of stromata on the distribution of xylariaceous endophytes discus- freshly burnt wood rather indicates that the sed above. Nevertheless, floristic and chorolo- fungi were already present as endobionts. The gical studies on the prevailing host plants have damage to the host had apparently induced the already revealed interesting relationships production of reproductive stages. While such among the Xylariaceae and associated woody evidence is still circumstantial in many cases, angiosperms. Some examples for apparently this phenomenon was studied intensively by entirely different strategies are compared be- molecular methods, including methods of low. population genetics, by Johannesson (2000, Stromata of some apparently host-speci- 2001) and Guidot et al. (2003), for D. loculata fic Hypoxylon spp. (e.g., H. laschii Nitschke on in Sweden. In the latter study it was postulated Populus), are extremely rarely encountered. that pyrophilous insects associated with D. They have almost exclusively been found on loculata are essential vectors for the realization dead branches that were still attached to the of the sexual cycle of the fungus. By feeding host tree and apparently stopped growing as the on the conidia and flying between nearby trees branches became detached. inhabiting wood decay mycelia, these insects In Hypoxylon fuscum, there are several allow the opposite mating types to meet. predominant forms, which show apparent host- Indeed, the Hypoxyloideae are well-known for specificity for wood of different genera of their insect associations (see Hingley 1971, Betulaceae (Alnus, Betula, Carpinus, Corylus). Wheeler & Wheeler Jr. 1994 and other Certain morphological features such as asco- references compiled by Johannesson 2001). spore sizes have been correlated to these host Their stromata are inadvertently roamed by affinities (Petrini et al. 1987). This suggests the insects, and numerous insect species show host presence of a species complex that is about to preferences for certain Xylariaceae. Recently, evolve in co-evolution with the host plant Srůtka et al. (2007) and Pažoutová et al. (2010) family. reported xylariaceous anamorphs which The ubiquitous H. fragiforme (Fig. 1A) is showed high homologies in their 5.8S/ITS almost constantly associated with Fagus throu- nrDNA and β-tubulin DNA gene to Daldinia ghout the Northern hemisphere. Its correspon- decipiens and Entonaema cinnabarinum ding anamorph is invariably obtained from liv- (Cooke & Massee) Lloyd as apparently speci- ing, apparently undamaged beech trees (Petrini fic associates of woodwasps (genus Xiphydria). & Petrini 1985). According to our unpublished The putative anamorph of D. decipiens is results, H. fragiforme often will produce stro- constantly associated with a Xiphydria sp. that mata already in the first season, only some primarily colonises alder, whereas the other has weeks after the beech wood has been damaged a strong host preference for Salicaceae. The or felled. The fungus may then persist on the identity of the D. decipiens strains was confir- substrate for up to a decade as saprotroph and med by comparing its anamorphic morphology regularly produce stromata. and HPLC profile (S. Pažoutová & M.S., Many other species which preferentially unpublished results). Indeed, the stromata of D. produce their stromata on dead, decomposing decipiens and allies are constantly associated wood, appear to be less specialised and their with betulaceous hosts, including Alnus. The stromata do not show apparent host specificity. woodwasps appear highly suitable as vectors, Stromata of Daldinia fissa Lloyd and D. locu- as postulated by Guidot et al. (2003) for D. lata (Lév.) Sacc. grow preferentially on freshly loculata and other associated insects. burnt wood. Betula is said to be the preferred Woodwasps and other insects are known to host genus for the latter species, but we have have “ambrosia fungi” for a long time (Mueller also found the stromata of D. loculata on & Gerardo 2005). The role of Xylariaceae in several other genera of woody angiosperms, these associations remains to be further

84 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 clarified. Probably, entomologists will need to The Xylariaceae have traditionally raised become more strongly involved in order to great interest because they can be easily culti- obtain more information on the lifecycle and vated in the laboratory and studied for biologi- the ecology of the Xylariaceae. For instance, cally active and other secondary metabolites. the role of the invertebrates in the transmission Their massive, conspicuous stromata were also of endophytes should be studied further, found to constitute a good source for hitherto considering the possible role of insect vectors. unprecedented compounds. The first secondary Visser et al. (2009) revealed an interesting metabolites were already isolated from mem- phenomenon of co-speciation between termite- bers of the Xylariaceae prior to 1960. Mean- associated Xylaria spp. and their invertebrate while, several hundreds of metabolites have hosts. Even though these fungi are mor- been obtained, as summarised in Stadler & phologically highly variable, they appear to be Hellwig (2005). Since publication of this work, derived from a monophyletic lineage that might another 100 compounds have been isolated and have evolved parallel to the plant-inhabiting published from this fungal family. Xylaria species. These results point toward a There has been much speculation about close co-evolution between tropical insects and the natural functions of secondary metabolites their fungal symbionts, similar to the relation- in the past. But by now it is almost generally ship of the basidiomycete genus Termitomyces accepted that these compounds are not random- R. Heim, which has been studied more inten- ly produced, but have important functions for sively than the Xylaria spp. the biology of their producers. Finally, there are even mycophilic Xyla- Recent research has demonstrated that riaceae. Those may colonise the surface, or the biogenetic pathways for most of these com- grow inside the fruit-bodies of other fungi, in- pounds are encoded by gene clusters, such as cluding even the stromata of members of their polyketide synthases (PKS) and non-ribosomal own family. Some members of Xylariaceae, polypeptide synthetases (NRPS), and that their such as Chlorostroma subcubisporum A.N. production underlies a complex regulation Mill. et al. (Miller et al. 2007), apparently para- (Hoffmeister & Keller 2007). Recent studies on sitize stromata of other Xylariaceae. We have the genome of filamentous fungi revealed a repeatedly obtained “foreign” cultures of other surprising diversity of secondary metabolite xylariaceous genera from perithecial contents genes. In the genome of Emericella nidulans of herbarium specimens of the same family. (Eidam) Vuill. (often referred to as “Asper- These observations cannot be attributed to gillus” despite its teleomorph association is laboratory contaminations, as the isolation was known), each 27 NRPS and PKS gene clusters reproducible in several instances. Little is were already detected (von Döhren 2008). By known so far about the nature of these relation- far not all these gene clusters appear fully ships. It is also interesting to note that Hypoxy- functional in wild type strains, which express lon species often grow as consortia, and up to only a small percentage of the corresponding five species have been found to produce stro- secondary metabolite pathways. Numerous stu- mata simultaneously on the same woody sub- dies are ongoing, dedicated to their reactivation strate. and to the evaluation of possible functions of Undoubtedly, further interdisciplinary these genes in the few strains of filamentous studies making use of molecular ecology will fungi that have already been chosen for sequen- reveal further interesting relationships that cing of their genome. In the Xylariaceae, it is at highlight the co-evolution of the fungi and all present not possible to do similar studies, as organisms associated to them. However, as in not a single representative of the family has case of the endophytes, the availability of a been subjected to genomic sequencing, and not concise identification system that is indepen- even the order Xylariales has been tapped by dent from teleomorphic morphology will be an genomic approaches. Recently, the molecular absolute prerequisite to accomplish such tasks. structure of the tryptophan synthetase gene of nodulisporic acid was accomplished (Ireland et Secondary metabolites and practical al. 2008), which is to my knowledge the first importance study of this kind on a member of Xylariaceae.

85 However, the vast majority of the known Xyla- explained by the fact that most of the hitherto riaceae metabolites are polyketides. Some op- known compounds were obtained in the course tions for future exploitation of this matter will of screening projects or basic research on the be discussed below in relation to the results of mechanisms of fungal metabolism, including our “mycochemical” studies. their pigment chemistry. So far, not much ana- Natural functions of the secondary meta- lytical work has been dedicated to their chemi- bolites of the Xylariaceae become quite evident cal ecology. Accordingly, it has not been in case of the numerous antibiotics, which are proven conclusively at all whether the secon- produced by these fungi, or in other cases dary metabolites are produced by endophytic where the metabolites act against specifically Xylariaceae in planta. Even the most sophistic- associated organisms. For instance, the phyto- ated methods of analytical chemistry now avai- pathogenic Xylariaceae produce a large number lable have detection limits and may not work of phytotoxins that are probably involved in unless the respective metabolites accumulate parasitism and were discussed as important de- considerably. It could eventually become an terminants of virulence (see Stadler & Hellwig option to indirectly prove secondary metabolite 2005 for some examples). However, most production, using PCR-based detection me- phytotoxins of the Xylariaceae that were so far thods in planta for the fungal secondary meta- characterised exerted a rather broad-spectrum bolite genes. However, for Xylariaceae, even of bioactivities. They are not apparently speci- methods to detect the fungal producer orga- fic, but their action may still facilitate the pro- nisms in their hosts remain to be established at cess of parasitism by weakening the defence of this time. the host plants. In reality, the phytotoxins will With respect to their effect on human ci- likely act synergistically with lytic enzymes vilisation, the Xylariaceae could even be regar- and other features that were developed by the ded as beneficial. Nodulisporium spp. were parasites to colonise their host plants. occasionally detected as clinical isolates in pa- It even remains possible that the same tients suffering from cancer (Umabala et al. compound or compound class has different 2001). Otherwise these fungi can certainly not functions in different producer organisms, or be regarded as pathogenic to humans or mam- changes its function during the life cycle of its mals. Some antibiotics and phytotoxins of the producer organism. For instance, Rosellinia Xylariaceae including the cytochalasins, have necatrix produces cytochalasins which possess previously been identified from other fungi that phytotoxic as well as antibiotic effects (ten may colonise and poison food and are therefore Hoppen & Krauss 2006). The same class of regarded as “mycotoxins”. However, there is compounds are overproduced in laboratory cul- no xylariaceous fungus among the most impor- tures by various other fungi, including mem- tant food spoilage fungi. bers of the related genus Xylaria (Abate et al. But above all, the Xylariaceae have 1997, Espada et al. 1997). Those Xylaria spp. raised great interest in the pharmaceutical and are regarded as saprotrophic wood-destroyers agrochemical industry, since various interes- and frequently occur as endophytes in healthy ting metabolites are already known from their plants, but they have never been reported to cultures (see Stadler & Hellwig 2005 for com- cause disease symptoms. The cytochalasins prehensive overview). No metabolite of a xyla- would evidently serve well as means of de- riaceous fungus has so far made it to the fence for the saprobic stages of their xylaria- market. However, nodulisporic acid, PF-1022A ceous producers, to combat other micro-orga- and the sordarins (Fig. 3) reached developmen- nisms in the struggle for nutrient sources. How- tal candidate status as antibiotics or antipara- ever, it is most unlikely that they are overpro- sitic agents. Numerous other compounds, such duced in planta by the endophytic stages of the as hypoxyxylerone and the xyloketals (Fig. 3), Xylaria spp., because the plants might suffer, were taken as templates for chemists, to deve- too. lop mimetic or total syntheses based on the The lack of knowledge on the natural pharmacophores [the moiety of a given functions of secondary metabolites in the sa- bioactive molecule that is important (or even protrophic and endophytic Xylariaceae is easily essential) for a certain biological effect.] of the

86 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1

H H O O H O

OH O O O N H HO OH O H H O Nodulisporic Acid A O Xyloketal A

OH HO OH H O O O H HO O H O O O H H H H O O O OH OH O OH Hypoxysordarin Hypoxyxylerone O H O N H O O O H

O N H N O H H O O O H O N PF-1022A H O

Fig. 3 – Some metabolites with interesting biological activities from Xylariaceae.

Xylariaceae metabolites. Other compounds tively large amounts of material were then showed promising effects against biochemical required for structure elucidation. Only a few targets or in cellular in vitro assays, but were compounds, including the mitorubrins from apparently not further developed. As outlined Hypoxylon fragiforme (Steglich et al. 1974) by Larsen et al. (2005) and in agreement with and the pigments and other metabolites of a the proceedings outlined in Stadler & Hellwig Japanese Daldinia sp. (Hashimoto & Asakawa (2005), phenotypic taxonomy including 1998) had been known from the stromata of chemotaxonomic methodology, aided by Xylariaceae, prior to our studies (Fig. 4). Ear- HPLC-profiling can be extremely helpful in the lier attempts to characterise their stromatal discovery of unprecedented compounds. The pigment profiles by thin layer chromatography example of nodulisporic acid (Polishook et al. (TLC; Whalley & Whalley 1977) were hampe- 2001) also demonstrates that molecular red by the fact that no standard compounds and identification methods may be useful to find little information on the chemical structures of additional producers of a given compound and the detected pigments had been available at ultimately help to secure parent claims. that time. The availability of standards and Molecular identification techniques are now highly sensitive analytical HPLC-based me- increasingly being employed for characte- thods constituted a great advantage over the risation of fungal producers of novel com- TLC-based methodology. pounds. The majority of secondary metabolites PCR fingerprinting and molecular phyloge- known until 2000 from Xylariaceae had been netic studies derived from laboratory cultures, because rela- Not too many studies using molecular 87 O

O HO O O O O O

O O OH O O OH O O Mitorubrin Daldinin C

O OH O R1 R2 O O O O

HO OH

OH

Daldinal R1 = R2 = H: BNT R1 = R2 = CH3: Daldinol

Fig. 4 – Selected secondary metabolites from stromata of the Xylariaceae that were known from the literature at the beginning of our studies. methods based on PCR had been carried out on lariaceae were neglected for a rather long time. the Xylariaceae before our first papers dealing The first molecular phylogenetic studies using with such methodologies were published. For a significant number of morphologically well- instance, Yoon & Glawe (1993) used random characterised xylariaceous species were only amplified polymorphic DNA (RAPD) to discri- published several years later. Granmo et al. minate some isolates of Hypoxylon truncatum (1999) compared rDNA sequence data of a se- sensu Miller (1961). They recognised a taxon lection of Nemania, Entoleuca, and Hypoxylon that is now regarded as annu- species, but their sequences were unfortunately latum (Schweinitz) Y.M. Ju et al. (cf. Ju et al. never deposited in a public database. Johannes- 1996 as Hypoxylon; Hsieh et al. 2005). As son et al. (2000) and Sanchéz-Ballesteros sequencing costs rapidly declined and reference (2000) focused on the hypoxyloid Xylariaceae, data became increasingly available in public using morphologically verified specimens. databases, we also started to generate DNA se- Aside from some recent studies that relate to quences of Xylariaceae. Spatafora & Blackwell our own work in this field and will be treated (1993) generated ribosomal DNA sequences of below, the paper by Smith et al. (2003), in two representatives of Xylariaceae, Daldinia which the Xylariales were defined to be an concentrica and Xylaria hypoxylon, in their order comprising seven families, deserves men- early study on the molecular phylogeny of py- tioning. renomycetes based on nrDNA sequence data. The sequence data derived from a “X. hypoxy- Synthesis of our own contributions lon” strain, described first by Chacko & Rogers (1981) and deposited with ATCC were used in Morphological and chemotaxonomic studies numerous phylogenetic studies as representa- Several of the papers published in the tive of Xylariales and Xylariaceae. We have course of this work deal with biodiversity in- only recently clarified that this fungus does not ventories and taxonomic revisions of the gene- represent X. hypoxylon (see Peršoh et al. 2008). ra Daldinia and Hypoxylon. It was regarded Meanwhile, the availability of universal necessary to obtain sufficient data on herba- PCR primers for fungal rDNA (White et al. rium specimens for chemotaxonomic studies 1990) triggered numerous molecular phyloge- and to collect and culture a significant number netic studies. As compared to the Basidiomy- of representatives to evaluate the significance cota and other groups of ascomycetes, the Xy- of chemotaxonomic data. Even today, the revi-

88 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 sions by Ju et al. (1997) and Ju & Rogers  Verify current taxonomic concepts and (1996) have still not been broadly applied, and evolutionary hypotheses in the Xylaria- the respective chorological data were based on ceae by means of chemotaxonomic data. outdated taxonomic schemes. Our strategy to obtain a global survey of the biodiversity, with The genus Daldinia emphasis on the chemical variability, is The genus Daldinia was erected by illustrated below to show the extent of these Cesati & De Notaris (1863) to accommodate studies. Sphaeria concentrica Bolt. and S. vernicosa Aside from these herbarium specimens, Schwein. (= D. fissa in current nomenclature). considerable amounts of fresh material were It comprises stromatic Xylariaceae that may supplied by J. Fournier (mostly from France, form rather large, conspicuous stromata on and in collaboration with C. Lechat and R. dead wood of angiosperms, which are easily Courtecuisse, from the Caribbean). Numerous recognisable, based on their internal con- important specimens from different tropical centric zones. The generic status of Daldinia regions were provided by C. Decock. M. Pie- had been disputed by various taxonomists in penbring (Panama), C. Douanla-Meli (Came- the course of the past century, owing to the fact roon), T. Læssøe (Ecuador, Malaysia) and V. that the internal concentric zones are the only Kummer (South Africa) immediately after col- morphological feature to discriminate Daldinia lection. Many other colleagues also sent mate- from Hypoxylon. The morphology of the asci, rial that could be cultured in our laboratory. ascospores, and conidiophores is indeed highly My own field work encompassed forays in similar among these genera. Læssøe (1994) Germany, the Swiss and French Alps and the therefore also proposed to unite them. Ju et al. Spanish Pyrenees, Wales, Scandinavia, the (1997), however, chose to accept Daldinia as a Macaronesian Islands, mainland Spain and genus, arguing that the peculiar stromatal ana- Portugal, Hawaii and different regions of main- tomy relates to the biology of the genus. They land USA, and Japan. About 30% of the speci- pointed out that Daldinia has also affinities to mens hitherto studied belong to the genus Dal- other genera of Xylariaceae that Læssøe chose dinia, 40% to the genera Hypoxylon and Annu- to accept. Those genera [i.e., Entonaema lohypoxylon, and the remainder to other Xyla- Möller, Phylacia (Lév.) Sacc., Rhopalostroma riaceae. Over 300 cultures resulting from this D. Hawksw., and Thamnomyces Ehrenb.] will work were already characterised and deposited be treated in detail further below. Furthermore, in public collections (CBS, MUCL). Whalley & Edwards (1995) had just reported The objectives of these studies can be that Daldinia spp. differed from Hypoxylon by circumscribed as follows: the presence of naphthalenes in their cultures,  Establish a methodology based on analy- whereas various types of dihydroisocoumarins tical HPLC that works at high throughput prevailed in cultures of Hypoxylon and other to study a significant number of speci- Xylariaceae they studied concurrently. It there- mens of Xylariaceae for secondary meta- fore appeared practical to verify the generic bolites and allows for concise identifica- organisation of the Hypoxyloideae by comple- tion of these compounds using sophisti- mentary methodology. cated analytical methodology. In addition, various questions related to  Correlate chemotaxonomic data to mor- the taxonomy of Daldinia remained to be phological concepts and identify the pro- resolved at the infrageneric level. The mono- minent stromatal pigments of the Hypo- graph by Child (1932), who had confused the xyloideae that are deemed taxonomically type specimens of various important taxa (cf. significant. Ju et al. 1997) had been in use for over 50  Establish correlations between chemical years. The generic concept proposed by Child types of the secondary metabolites en- (1932) had changed drastically due to the re- countered, based on a comparison of their cent revision, and virtually all specimens iden- chemical structures and current know- tified using the latter monograph needed to be ledge on the biosynthesis of the corre- revised. sponding metabolite families. Concurrently, Frisvad and co-workers

89 had published exciting data on the chemotaxo- identified in the stromata by using standards, nomy of Penicillium (Samson & Frisvad 2005, which had been isolated de-novo and Frisvad et al. 2008, and references therein). characterised by NMR spectroscopy and mass Our work was strongly inspired by their con- spectrometry. This study provided the basis for clusive results, and we decided to use a similar various subsequently published papers, which methodology to study the Xylariaceae. We are discussed in a broader context further decided to start with Daldinia as it constitutes a below in the chemota-xonomic part. A relatively small but morphologically well-de- monograph employing the new concept in fined genus, which was treated controversially German language (Wollweber & Stadler 2001), in the literature nevertheless with respect to its included various field observations and taxonomic affinities. For our project, we fo- illustrations of several Daldinia spp., which cused on stromata as well as on cultures; there- were then recorded for Germany or other fore this study is the first one in fungal chemo- European countries for the first time. Material taxonomy where all developmental stages of a from America (Ju et al. 1997) and Papua New certain taxonomic group have been considered. Guinea (van der Gucht 1994, 1995) was also A pilot study, originally aimed at the re- studied for comparison. Another paper (Stadler vision of Daldinia in Germany and Europe et al. 2001e) introduced the HPLC profiling according to Ju et al. (1997), was initiated in technique, and also dealt with a preliminary collaboration with H. Wollweber (Wollweber characterisation of Daldinia spp. by PCR & Stadler 2001). However, while the type spe- fingerprinting methodology, using the ARDRA cies, D. concentrica, was reported in the recent technique based on restriction fragment analy- monograph to have yellow-brown stromatal sis of SSU rDNA. This technique provided spe- pigments, all European material studied by us cific results only for part of the species studied corresponding to the morphological description and revealed identical fragment patterns even by Ju et al. (1997) had purple pigments in among representatives of different genera, even KOH. We therefore established contact to Jack if fragment patterns obtained by using several D. Rogers, Thomas Læssøe, and Anthony J.S. different restriction polymerases were com- Whalley, to clarify the situation and also bined. These results were complemented by discussed our chemotaxonomic data based in minisatellite PCR fingerprinting, which proved HPLC profiling with them. Based on the much more specific for species discrimination rediscovery of a specimen that was originally (Stadler et al. 2001e). Concurrent morphologi- studied by Bolton (1789), and on material from cal studies resulted in two papers (Stadler et al. the herbarium of Anthony J.S. Whalley, Rogers 2001b,c) dealing with new species descriptions et al. (1999) revised the status of D. concen- (D. decipiens, D. albofibrosa M. Stadler et al., trica. They erected D. childiae J.D. Rogers & D. pyrenaica M. Stadler & Wollw., D. stegli- Y.M. Ju for the species that is most frequently chii M. Stadler et al.) and the characterisation encountered in America and selected an epi- of the anamorphic stage of D. loculata, which type for D. concentrica, based on a specimen had not been cultured before. Due to this work, from UK. For our own work, we used nume- the novelty of some species previously treated rous reference materials treated by Ju et al. tentatively by van der Gucht (1994) and Petrini (1997). The stromatal pigment colours in 10% & Müller (1986), respectively, under known KOH by comparing them with a botanical chart species sensu Child (1932), was established. (Rayner 1970) was correlated to HPLC profiles The morphological data were backed up by (Fig. 5), allowing for a better resolution. Hund- HPLC profiling. reds of additional specimens were revised Van der Gucht (1993) had compared according to the new taxonomic concepts, veri- ascospores of Daldinia spp. by scanning fying that HPLC profiling in mycology electron microscopy (SEM) and demonstrated constitutes an additional important tool to the utility of this technique for segregation of generate reliable and highly reproducible data morphologically similar taxa. This was for chemotaxonomic evaluation of Macro- confirmed by us, based on a large number of mycetes at rather high throughput. Some of the representatives for the first time (Stadler et al. prevailing stromatal pigments of Daldinia were 2002). Strong correlations were found between

90 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1

mAU Norm. BNT Hypoxylon macrocarpum 1400 700 5.827 1200 UV-VIS spectrum of BNT 600 1000 500 800 6.987 600 400 6.714 400 300 200 200 0 250 300 350 400 450 500 550 nm 100 0 12.138 12.540 0 2 4 6 8 10 12 14 min

BNT 10.091 Norm. Daldinia loculata 1750 1500 1250 1000 4.525 % Acetonitrile (0-100 %) 750 500 250 0 0 2 4 6 8 10 12 14 min

BNT Norm . Daldinia fissa 10.097 1200 1000 800 600 400 4.549 200 0 0 2 4 6 8 10 12 14 m in

Fig. 5 – Illustration of the HPLC profiling methodology for chemotaxonomic studies on the Xylariaceae. Comparison of three HPLC chromatograms from stromatal extracts of different Xylariaceae spp. containing binaphthalene tetrol (BNT; see arrow), chemical structure and DAD spectrum of the corresponding peak (cf. Stadler et al. 2001d). The compound was safely identified by matching its Identical DAD spectra and retention times with data from standard compounds stored in a spectral library. Mass spectrometric data as revealed by Electrospray HPLC-MS, were also recorded concurrently. This unpublished figure is taken from a lecture presented at the IMC8, Oslo, 2002). the ascospore ultrastructure as inferred from spores (Stadler et al. 2004d), dealt with Daldi- SEM and the secondary metabolite production nia grandis Child and related taxa, focusing on in the stromata of the investigated species. the Daldinia species of the Southern hemi- Furthermore, the results gave rise to study the sphere and other species featuring relatively teleomorphic and anamorphic morphology in large ascospores. Once again, it became evi- some specimens from the isle of Jersey, the dent that the concept established by Child Canary Islands, and Sicily. Five new cryptic (1932) had apparently caused much confusion, species (D. macarone-sica M. Stadler et al., D. which was not completely resolved by Ju et al. martinii M. Stadler et al., D. palmensis M. (1997) for lack of fresh material. Daldinia Stadler et al., D. raimundi M. Stadler et al., and grandis was restricted to a taxon that has up to D. vanderguchtiae M. Stadler et al.), all date not been found outside the Americas. Dal- appearing closely allied to D. concentrica s. dinia loculatoides Wollw. & M. Stadler and D. str., were described. They all deviate from the novae-zelandiae Wollw. & M. Stadler were type species in their anamorphic morphology recognised from new combinations of teleo- and/or in the morphology and ultrastructure of morphic and anamorphic characters. Another their asci and ascospores Stadler et al. 2004d). species with close relationships to D. concen- Another study, mainly based on compar- trica was recognised as D. dennisii Wollw. & ative morphology and ultrastructure of asco- M. Stadler, of which two varieties were

91 erected. In conclusion, the concept proposed by The species composition in the temperate Ju et al. (1997) was further elaborated due to Southern hemisphere appears to be entirely dif- these studies. Most of their species concepts ferent from that of the Northern hemisphere. were validated using a combination of chemo- Even though the predominant taxa of both he- taxonomy and PCR-based methods, and nume- mispheres are apparently related to one another rous additional taxa of Daldinia were already (e.g., both varieties of D. dennisii show high encountered and described. Fourteen new spe- morphological similarities to D. concentrica; cies were already erected, and there is evidence D. novae-zelandiae and D. bakeri have clear that numerous additional ones remain to be affinities to D. fissa) the deviations are signifi- described, especially from the tropics. With a cant enough to assume a long independent evo- single exception (D. bakeri Lloyd, see below lution of these species groups in the respective and Stadler et al. 2004b), the results reported geographic areas. For instance, D. dennisii is by Ju et al. (1997) on stromatal pigment co- the only taxon in the D. concentrica complex lours were correlated to genuine stromatal con- in which concentricols are apparently absent, stituents, which were unambiguously identified and D. fissa has much smaller ascospores and using HPLC profiling. It was also found that, at also significantly differs in its anamorphic mor- least in mature stromata of the same species, phology from D. novae-zelandiae. In general, the secondary metabolite profiles are surpri- the only Hypoxyloideae that were so far singly consistent with respect to the presence encountered in Europe as well as in Australia of major metabolites. No dependence from the and New Zealand were found on introduced substrate or the host plant was noted, aside host plants in the latter country. Out of the from those cases where deviations in the secon- tropical Daldinia species, D. caldariorum dary metabolites were also accompanied by Henn. and D. eschscholtzii have made it also to consistent morphological differences and appa- tropical Australia. The D. decipiens group, rent host affinities, as in case of D. petriniae comprising species with enteroblastic from different betulaceous hosts (Stadler et al. conidiogenesis, which are closely associated 2001c, d). This chemotaxonomic significance with betulaceous hosts, remains to be found in was further emphasised by the fact that the cha- the Southern hemisphere. On the other hand, racteristic marker compounds were still found the Southern hemisphere Daldinia spp. are intact in specimens that had been collected up associated with Nothofagus and other endemic to 200 years previously. This is important to plants of those geographic areas. note, because as shown for the type specimen The above examples only give some of D. bakeri (Stadler et al. 2004b) the pigment hints on what can be accomplished with our colours detected by using KOH in old herba- data. In the past years, numerous further speci- rium specimens may occasionally be falsified mens were examined, and a new world mono- by the presence of obvious artifacts, such as graph is now in preparation. However, this preservatives that were eventually added to the work was not yet finished, because it took quite specimens to prevent insect and mite contami- a long time to obtain some important type nation. materials, and the tropical species of Daldinia It was found that similar pigment colours appeared rather complicated. Recently, three may be due to the presence of entirely different additional species of the genus (D. barkalovii metabolites. In some cases (e.g., concentricols, Lar.N. Vassiljeva & M. Stadler, D. carpinicola cytochalasins), even compounds that are not Lar.N. Vassiljeva & M. Stadler, and D. govoro- pigments but only exhibit UV absorption, were vae Lar.N. Vassiljeva & M. Stadler) were des- revealed to be taxonomically significant, which cribed from Fareastern Russia (Vasilyeva & demonstrates the advantage of HPLC profiling Stadler 2008), a geographic region which had over conventional colour reactions. The speci- already shown earlier on to harbour highly in- ficity of secondary metabolite production in teresting Xylariaceae (cf. Ju et al. 1999). They cultures of Daldinia proposed by Whalley & all show an enteroblastic conidiogenesis and Edwards (1995), who had not given details on appear closely related to D. decipiens and D. the numbers of examined representatives and petriniae. species involved, were confirmed based on a

92 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 significant number of representative cultures. be widely distributed. However, many other Several types of metabolites were meanwhile species of Hypoxylon contained apparently found that do not occur in Hypoxylon cultures specific major metabolites. The interpretation while being omnipresent in Daldinia and of these HPLC profiling data (Mühlbauer et al. related genera (see also Bitzer et al. 2008). This 2002) was rather difficult. This changed as the also reinforced the generic status of Daldinia. chemical work described below was accom- Under standardised fermentation conditions, plished and the chemical structures of the me- the secondary metabolite profiles observed in tabolites detected in the crude extracts were cultures were highly specific for certain species elucidated. In addition to a HPLC profiling groups or even genera, but the prevailing stro- study, Mühlbauer et al. (2002) included the iso- matal metabolites were rarely detected in the lation and structure elucidation of the macro- mycelial cultures and vice versa. carpones. A panel of ca. 300 cultures of Daldinia The preliminary results appeared rather from around the world is now available to fur- promising, and by studying numerous additio- ther study the distribution and evolution of nal taxa, including over 100 type specimens, it morphological and chemotaxonomic traits in was meanwhile established that the specificity this group of ascomycetes. Along with ongoing of stromatal HPLC profiles is as high in Hypo- phylogenetic studies, the data available may ul- xylon as in Daldinia. Based on a revision of the timately also facilitate to draw further conclu- H. rubiginosum complex in Europe, a new spe- sions on their biogeography. cies (H. petriniae M. Stadler & J. Fournier) was described, and HPLC profiles of several Hypoxylon and Annulohypoxylon other Hypoxylon spp. were reported and com- Like Daldinia, the genus Hypoxylon had pared, revealing further species-consistent and just been thoroughly revised by Ju & Rogers specific features that relate to the production of (1996). Even after removal of the taxa with certain types of orange azaphilone pigments in Geniculosporium-like anamorphs (see Intro- the stromata (Stadler et al. 2004c). In addition, duction), it then still constituted a very large this paper included a newly elaborated key to conglomerate of taxa. Their common features Hypoxylon in Europe, taking the taxonomic include stromata with an essentially homoge- changes proposed by Ju & Rogers (1996) and neous context, the presence of stromatal pig- Granmo (1999, 2001), as well as our own ments and Nodulisporium-like anamorphs in all observations into account. Most of these data, species that had been cultured. Ju & Rogers including the above mentioned chemotaxono- (1996) divided the genus into sections Hypoxy- mic results as well as illustrations of the diag- lon and Annulata. The latter section was deli- nostic characteristics of Hypoxylon and other mited by the presence of ostioles that are encir- Xylariaceae and determination keys, are avai- cled with a truncate area, carbonized stromatal lable on http://pyrenomycetes.free.fr. The che- tissue that discretely encloses each individual motaxonomic evaluation of Hypoxylon conti- perithecium, and a thickening on the ascospore nued, based on a set of characteristic specimens perispore that is absent in most species of sect. kindly provided by Y.-M Ju and J.D. Rogers. Hypoxylon. European representatives of Hypo- Results were published along with the chemical xylon were already included for comparison in structure of hypomltin (Hellwig et al. 2005). our studies focusing on the chemotaxonomy of Meanwhile, Hsieh et al. (2005) had published a Daldinia (Stadler et al. 2001d,e). Several speci- molecular phylogenetic study of Xylariaceae fic metabolites including the binaphthalene te- that resulted in the segregation of the new trol BNT and the mitorubrins were isolated to genus Annulohypoxylon Y.M. Ju et al., being purity and their structures confirmed by mass identical with the former sect. Annulata of spectrometry and NMR spectroscopy. HPLC Hypoxylon sensu Ju & Rogers (1996). The profiling revealed a considerable metabolic di- erection of the new genus was based on the versity within the genus Hypoxylon. In some above mentioned morphological differences in species, such as H. fuscum (Pers.) Fr. similar conjunction with a comparison of α-actin and pigment profiles as in Daldinia childiae were β-tubulin DNA sequence data. Concurrently, encountered, and the mitorubrins were found to numerous taxa of Annulohypoxylon were stu-

93 died for stromatal HPLC profiles, and the are characterised by a hypoxyloid stromatal results (Quang et al. 2005c) backed up the new habit, with effused-pulvinate stromata and gra- taxonomic concept. Aside from the binaphtha- nules below the stromatal surface. However, lene BNT, which is present in numerous genera their asci are globose, lacking an amyloid api- of Xylariaceae, no common pigments were cal apparatus, and their ascospores are phaseo- encountered in the two sections. Azaphilones liform. Speer (1980) even excluded Pulveria were also detected in Annulohypoxylon, but from the Xylariaceae, lumped it with Phylacia those were shown to possess entirely different Lév. in an own family Phylaciaceae. Our stu- carbon skeletons from those of the mitorubrins dies of the type material of both genera and so- and other pigments of (sect.) Hypoxylon (see me recently collected specimens revealed the below for cohaerins and multiformins). following facts: Finally, in the paper by Stadler et al.  Pyrenomyxa and Pulveria (Malloch & (2008b), a part of the results on HPLC profi- Rogerson 1977) are indeed synonyms. ling of Hypoxylon spp. from around the world, Pulveria porrecta Malloch & C.T. focussing in the species of the subtropical and Rogerson is a later synonym of Hypoxylon temperate Northern hemisphere, were publi- piceum Ellis, for which the new shed. Results on HPLC profiling and morpho- combination Pyrenomyxa picea (Ellis) M. logical studies of a rather large number of an- Stadler et al. was made. cient specimens, including authentic and type  Pyrenomyxa invocans Morgan is indeed material from the herbaria of C. H. Persoon, F. highly similar to the above taxa but differs th Nitschke, and other mycologists of the 18 and in its HPLC profile and microscopic th 19 century were compared with fresh, conspe- characters. cific material. Once again, the results were  A third species, P. morganii M. Stadler et quite conclusive. Especially the specimens of al., was found from Eastern Russia. Its the H. rubiginosum complex appeared to be culture produced a Virgariella-like well suited for HPLC analyses. They still re- anamorph and 5-methlymellein, the vealed their characteristic metabolites even if characteristic marker metabolite of the H. they had been collected 200 years previously. rubiginosum complex. Aside from a survey of Hypoxylon in the Cana-  All three accepted Pyrenomyxa spp. ry Islands and the description of two new spe- produced orsellinic acid and azaphilones of cies (H. canariense J. Fournier et al., H. urrie- the mitorubrin type in their stromata. sii J. Fournier & M. Stadler), this paper Pyrenomyxa picea contained in addition (Stadler et al. 2008b) also included some preli- macrocarpone derivatives. minary data on some putatively new taxa. The genus Pyrenomyxa was therefore Those were, however, not yet officially des- emended (Stadler et al. 2005). A case could be cribed because they are so far only known from made to include it in Hypoxylon, as molecular old specimens. In other cases, certain species in data also point toward its close relationships to the concept by Ju & Rogers (1996) showed the H. rubiginosum complex. However, the heterogeneous HPLC profiles, along with type species and P. picea remain to be studied matching morphological deviations, suggesting further to assure that Pyrenomyxa is actually the involvement of species complexes. Data on monophyletic before further taxonomic some representatives of Hypoxylon that occur conclusions can be drawn. It still remains in tropical as well as subtropical regions were possible that Pyrenomyxa-like fungi have also included for comparison. However, the evolved more than once from Hypoxylon, bulk of HPLC profiling data we have obtained because the tendency to lose the amyloid apical from Hypoxylon spp. from tropical Africa, apparatus and other morphological features that America, and Asia still remains unpublished. point toward cleistocarpy are known from several, apparently unrelated taxa in Revision of Pyrenomyxa Hypoxylon. The genus Pyrenomyxa Morgan was dis- cussed to be a synonym of Pulveria Malloch & Revision of Entonaema C.T. Rogerson by Laessoe (1994). Both taxa The genus Entonaema A. Möller was

94 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 erected to accommodate two tropical Xyla- (Triebel et al. 2005). Other Xylaria spp. riaceae spp. with hollow, gelatinous, liquid- including X. telfairii in which xylaral was filled stromata (Möller 1901). Rogers (1981) concurrently detected did not appear reviewed the taxonomic history of this genus, particularly closely related to X. mesenterica as and subsequently (Rogers 1982) described a inferred from the molecular phylogeny. Xylaral typical Nodulisporium anamorph for E. was also detected in some taxa of Nemania and liquescens, the type species. Our paper (Stadler Stilbohypoxylon, and is therefore not of et al. 2004a) dealt with a comparison of HPLC taxonomic significance beyond species rank. profiles in stromata and cultures of various taxa The status of some other Entonaema spp. of Entonaema and other Xylariaceae that were sensu Rogers (1981), as well as affinities previously discussed to be related to this genus, between this genus and the presumably related such as Sarcoxylon (Jungh.) Cooke. Interes- genus Sarcoxylon, remain to be established. tingly, their stromatal pigments were of the Stromata of these taxa neither contain xylaral mitorubrin and rubiginosin types, which are nor any of the known metabolites of the widespread in the genus Hypoxylon, while the Hypoxyloideae (Stadler et al. 2004a, 2008a). A secondary metabolites of their cultures were culture in CBS, which did not produce reminiscent of Daldinia. The presence of conidiophores when studied by us, was naphthol and chromone derivatives instead of deposited by P. Martin as Penzigia compuncta mellein dihydroisocoumarins in E. liquescens (Jungh.) Sacc. & Paol., which is a synonym of and E. cinnabarinum also corresponded with Sarcoxylon compunctum. The 5.8S/ITS nrDNA molecular phylogenetic data, suggesting that sequence of this strain clustered in the the affinities of the genus are with Daldinia Xylarioideae (Fig. 7), but the corresponding rather than with Hypoxylon. specimen is not extant, the culture did not These data were complemented (Stadler produce an anamorph when studied in our et al. 2008a) by the recovery of authentic laboratory, and Martin apparently never material of Entonaema mesentericum A. published details on the material. Fresh Möller, the second species erected by Möller material of S. compunctum remains to be (1901). Even though this type specimen was found. Owing to the lack of “hypoxyloid” stored in ethanol for over 100 years, it was stromatal metabolites, and for the xylarioid shown to correspond with Entonaema pallidum morphology of its ascal apical apparatus, this G.W. Martin. The major stromatal constituent genus was included tentatively in the was identified as xylaral, a metabolite known Xylarioideae (Table 2). from (Pers.) Grev. (Gunawan et al. 1990). This compound was The genus Rostrohypoxylon detected by us in several species of Xylaria, During a recent foray in Northern Thailand, an and even in the concentrated spirit in which the interesting fungus was found, for which the type specimen of E. mesentericum had been erection of a new genus appeared justified. At preserved. Cultures (Fig. 6) of E. pallidum first glimpse, Rostrohypoxylon terebratum J. were obtained for the first time. They Fournier & M. Stadler (Fig. 8) appears resembled those of Xylaria, substantially morphologically rather similar to differing from other Entonaema spp., in their Biscogniauxia-like taxa because of its morphology, 5.8S/ITS nrDNA sequences, and erumpent carbonaceous stromata. However, HPLC profiles. Entonaema pallidum is thus stromata are unipartite, and show various regarded as a later synonym of E. mesen- characters that are unknown from other tericum, and the latter name was transferred to Xylariaceae. The most characteristic feature Xylaria [as Xylaria mesenterica (Möller) M. that does not occur in any other described Stadler et al.]. taxon of the family is the strongly uneven A phylogenetic tree (Fig. 7) illustrates the stromatal surface, owing to the presence of affinities of X. mesenterica to other stout ostiolar necks and cylindrical holes. A Xylariaceae inferred from 5.8S/ITS nrDNA detailed study showed that these “superficial” data, as compared to some representatives used features are accompanied by the lack of an for the phylogenetic tree published earlier on amyloid ascal apical apparatus, and the ability

95 to produce conidia directly from vegetative morphological structures that are present in the hyphae besides the presence of a typical latter genus, and by significant differences of hypoxyloid anamorph of the reduced its secondary metabolism to representatives of Nodulisporium type. The type species was Annulohypoxylon, which remain to be further included in a molecular study (Tang et al. explored by identification of the specific 2009, as “Xylariaceae sp.”), revealing metabolites. The fungus shows numerous phylogenetic affinities of the new taxon with features, suggesting its being derived from the three representatives of Annulohypoxylon. lineage comprising Xylariaceae with bipartite However, despite stromatal pigments were stromata, like Biscogniauxia. detected in the type specimen, the HPLC In the course of this work, two additional profile of R. terebratum did not reveal any species of the related genus Annulohypoxylon, known metabolites of the Hypoxyloideae. The A. bahnphadengense J. Fournier & M. Stadler ex-type culture was also studied by HPLC and A. maeteangense J. Fournier & M. Stadler, profiling, revealing 5-methylmellein aside from were also recognised and formally described. some unknown metabolites. This paper only constitutes the first of a series In conclusion. R. terebratum appears to which will deal which will deal with the represent a lineage of the Xylariaceae that has description of new taxa of tropical Xylariaceae further evolved from Annulohypoxylon, that we found in the past years. characterised by various reductions of

Table 2: Overview on the provenience of the specimens studied

Geographic Number of Herbaria (Important collections and publications) origin specimens studied

Europe ca. 3,500 B (Nitschke, Möller, Hennings); C (T. Læssøe); CWU (A. Akulov); L (Persoon); PAD (Saccardo); A.J.S. Whalley (personal herbarium; E, Whalley & Watling 1981); PRM (Pouzar); ST (G.J. Krieglsteiner); S, UPS (Nannfeldt); TROM (Granmo 1999, 2001); ZT (Petrini & Müller 1986); Herbarium of H. Wollweber (Wuppertal); GLM, KR, M, MA, TFC

North America ca. 1,500 BPI (C. G. Lloyd herbarium; material studied by Child 1932); GAM (Miller 1961); NY (Martin 1961; collections by G. Samuels; J.D. Rogers (personal herbarium; WSP (Ju & Rogers 1996, Ju et al. 1997); numerous specimens from F and FH.

South & ca. 1,000 C (T. Læssøe); LPS (Spegazzini herbarium); LIP (fresh material Central collected by C. Lechat and J. Fournier); S (Starbäck 1901, Möller America (1901)

Africa ca. 400 BR (Dennis 1961); CABI (Martin 1969) K (Deighton); Pers. herbarium of C. Douanla-Meli

Asia ca. 700 K (Dargan & Thind 1984, Petch 1924; Y.-M. Ju (personal herbarium; HAST); VLA (L. Vasilyeva)

Australia & ca. 200 PDD (Ju & Rogers 1996, Ju et el. 1997) New Zealand

Additional material from around the world, including numerous type specimens, was obtained from BPI, GAM, K, M, NY, S, and WSP.

96 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1

O

HO OH

O

O OH O

Xylaral

Fig. 6 – Type specimen of Entonaema mesentericum in herb. B, from spirit collection, and corresponding drawings by Möller (1901). Below: chemical structure of xylaral.

97 100% AM900591, „Entonaema pallidum“ (= Xylaria mesenterica ined.) xylarioid 55% AM900592, „Entonaema pallidum“ (= Xylaria mesenterica) agrKG24, Xylaria hypoxylon (Germany) 70% AM900590, Xylaria telfairii AM749930, sp. 100% 100% AM749929, Xylocoremium sp. AM900589, Xylaria enterogena 100% agrMS131, Penzigia compuncta (= Sarcoxylon compunctum, = X. compuncta) AF201714, Camillea obularia (B) 100% 100% AJ390415, (B) 100% AM749926, H. carneum (B) 100% 100% AM749935, H. perforatum (B) AM749936, H. rubiginosum 100% AM749920, Pyrenomyxa morganii (B) 100% 100% AM749923, Hypoxylon petriniae (B) AM749940, Nodulisporium hinnuleum (B) 100% 100% AY616693, H. fuscum (B) AY616690, H. fragiforme (C) 100% AM749928, H. howeanum (C) 100% AM749938, Annulohypoxylon annulatum (B) 100% AY616687, A. cohaerens (A/B) 100% AY616706, A. multiforme (A) AM749924, H. haematostroma (C1) hypoxyloid 100% 100% AM749922, H. nicaraguense (E) 100% 100% AM749941, H. polyporus (E) 100% AY616683, Daldinia concentrica (D) AM749925, D. grandis ss. str. (D) 100% AM749932, D. childiae (D) 59% AM749927, D. pyrenaica (D) daldinoid 67% AM749937, D. petriniae (D1) 100% AM749925, D. fissa (D) 100% AF176964, D. loculata (D) 100% AY616686, Entonaema liquescens (D) 100% 100% AM749939, „D. placentiformis“ (D, R. S-Africa, deviating morphochemotype; new species!) AM749933, „D. vernicosa“ ATCC 36660; Ulex, UK (= D. caldariorum; F1) 100% AM749934, D. caldariorum (Ulex, France, F1) 100% AM749919, Phylacia sagrana (F) 100% AY616684, D. eschscholzii (D) AM749921, D. placentiformis (D, Mexico, HPLC profile/morphology as the holotype) AM749931, D. clavata (D) AF192323, Diatrype stigma 0.10

Fig. 7 – Preliminary molecular phylogeny based on 5.8S/ITS nrDNA sequences of selected Xylariaceae representing different morphotypes and chemotypes, resulting from a Parsimony Ratchet analysis (20 replicates a 800 iterations). Majority consensus tree of the 15258 most parsimonious trees (395 steps) found. From a plenary lecture at the AMC 2007, Penang, Malaysia, summarising the results of Bitzer et al. (2008) and Stadler et al. (2008a, 2010a)

98 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 Phylacia, Rhopalostroma and Thamnomyces course of the phylogenetic evaluation of Type and authentic material of Phylacia, Entonaema (Stadler et al. 2008a) and the X. Rhopalostroma and Thamnomyces (Fig. 9) hypoxylon complex (Peršoh et al. 2008), were also studied concurrently with the respectively. These data served as prerequisite Entonaema spp. (Stadler et al. 2004a). These to evaluate the specificity of the compounds genera show characteristic features such as encountered in the hypoxyloid genera and reductions of their ascal apparatus and allowed for some additional conclusions on ascospore germ slit, suggesting their being chemotaxonomic differences between the large phylogenetically derived (cf. Table 3). groups of Xylariaceae. In contrast to the Representatives of all three genera hypoxyloid Xylariaceae, no large-scale contained BNT and other compounds that are fermentations resulting in the isolation of the prevalent in the stromata of Daldinia and characteristic major metabolites were carried Hypoxylon. This was in accordance with the out. Our ongoing studies were so far restricted Nodulisporium-like anamorphs reported earlier to a screening of these cultures by HPLC on by Hawksworth & Whalley (1985), profiling for the presence of known Rodrigues & Samuels (1989), Samuels & compounds. This helped to establish that the Müller (1980). Affinities between these genera Xylarioideae are entirely different from the had already been discussed by Hawksworth Hypoxyloideae with respect to their secondary (1977), Ju et al. (1997), and Medel et al. 2006) metabolome. Various apparently specific, Our chemotaxonomic study, in conjunction taxonomically significant compounds were with molecular data discussed further below, only detected in one of these subfamilies by provided strong evidence that these genera are HPLC profiling. Some compound classes, like very closely related to Daldinia. In case of the cytochalasins, occur throughout the family, Rhopalostroma, the data presented by Stadler but most of the characteristic metabolites et al. (2004a1) will need to be confirmed based detected in the Xylarioideae remain to be on a freshly isolated culture as the only culture isolated to purity and identified. available as now was derived from an old Stadler et al. (2008a) applied colour reactions herbarium specimen this has meanwhile been (NH3, KOH) of the ectostroma to a limited realised by Stadler et al. (2010b), and Whalley number of Xylaria spp. and other Xylarioideae, & Edwards (1995) reported griseofulvin and especially those species that contained derivatives from another species of that genus. xylaral derivatives showed such colour The cultures used by the latter authors, reactions. According to my knowledge, this however, are apparently not extant anymore in study provided first evidence that certain any public collection. species of the Xylarioideae may also contain pigments extractable in KOH. As xylaral was Preliminary chemotaxonomic studies of the also found in specimens of Nemania and Xylarioideae Stilbohypoxylon Henn. while being apparently Aside from Daldinia, Hypoxylon, and absent in Hypoxylon and allied genera, it may their allies, numerous representatives of other be a chemotaxonomic marker for certain taxa genera were also studied for characteristic of Xylarioideae. However, over 90% of the secondary metabolites. So far, about 200 xylarioid taxa so far studied were found devoid cultures of ca. 90 taxa of Xylariaceae genera of stromatal pigments. Therefore, HPLC with anamorphs other than Nodulisporium profiles of cultures appear more promising to (among those, the majority of cultures reveal useful chemotaxonomic traits to deposited with ATCC, CBS and MUCL) were segregate them. Whalley & Edwards (1995) studied for comparison. Some additional and their co-workers, as well as other research cultures were obtained by ourselves in the groups, had previously studied various cultures of xylarioid Xylariaceae and reported rather 1 A recent study in comparison with the culture of a interesting metabolites (see overview by Rhopalostroma sp. collected in fresh state revealed that the culture described by Stadler et al. (2004a) was Stadler & Hellwig 2005). Only few of these derived from Daldinia eschscholtzii, which had strains are deposited in public collections and apparently colonised the stromata of the Rhopalostroma could so far be re-examined (Bitzer et al. specimen, cf. Stadler et al. (2010b). 99

Fig. 8 – Stromatal habit of Rostrohypoxylon terebratum. (ex holotype, images by J. Fournier).

2008). Attempts to isolate and identify these significant number of isolates were studied. It and other metabolites are currently pending, to soon became evident that PCR fingerprinting extend our chemotaxonomic work to the would not remain the method of choice for Xylarioideae. The numerous metabolites molecular characterisation of fungi. A reported by Whalley & Edwards (1995) from collaboration with Dagmar Triebel (Botanische Xylaria and its allies would clearly be helpful Staatsammlung München) and Derek Peršoh as standards for these tasks. However, most of (University of Bayreuth) was initiated to them are probably not available anymore and explore the utility of rDNA sequences. The will need to be re-isolated from the respective objectives of these studies can be organisms. circumscribed as follows:

Application of PCR-based methods  Use DNA sequence data are to verify As already mentioned, our study relied morphological concepts in the Xylariaceae from the beginning also on PCR-based data for and obtain additional information on their characterisation of the Xylariaceae. Two of our phylogenetic relationships. early papers (Stadler et al. 2001d,e) dealt with  Evaluate the potential utility of rDNA the utility of PCR fingerprinting for sequence data to reflect the evolutionary discrimination of Xylariaceae, based on history of the Xylariaceae at generic and amplified ribosomal DNA restriction fragment species level. analysis (ARDRA) and minisatellite PCR,  Compare molecular phylogenies based on respectively. Especially the latter technique DNA sequence data to chemotaxonomic apparently provided specific results when a results.

100 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1

Fig. 9 – Stromata of Thamnomyces dendroidea and Phylacia sagrana. Image by J. Fournier.

 Provide the basis for safe molecular monophyletic clades for the sections Annulata identification techniques for endophytic and and Hypoxylon of Hypoxylon sensu Ju & insect-associated xylariaceous anamorphs. Rogers (1996), which gave rise to erect the Our first publication (Triebel et al. 2005) genus Annulohypoxylon Y.M. Ju et al. As appeared parallel to the study by Hsieh et al. confirmed from recent phylogenetic studies (2005). In both studies, a significant number of (Suwanassai et al. 2005, Pelaez et al. 2008, the hypoxyloid Xylariaceae were compared on Tang et al. 2009), it was so far not possible to similarities of their DNA sequence data. verify this taxonomy by rDNA sequence Whereas Hsieh et al. (2005) used α-actin and β- analyses, despite numerous additional data tubulin genes, our own work focused on the meanwhile became available. Representatives 5.8S/ITS region of the rDNA, taking the of Hypoxylon and Annulohypoxylon still previously published sequence data then became intermingled when their rDNA available from GenBank into account. sequence data were compared using different The phylogenetic tree based on 5.8S/ITS calculation methods. The same applies to other nrDNA data of Hypoxylon spp. was not suited genera of Xylariaceae, which can be segregated well to resolve the genus according to the conclusively by using morphological data [e.g., current morphological concepts. The matrix for Biscogniauxia Kuntze and Camillea Fr. this study was derived from the corresponding (Laessøe et al. 1989, Ju et al. 1998)]. DNA sequence data that had by then been The genus Daldinia was fairly resolved published in GenBank. We supposed that the from Hypoxylon by using nrDNA as well as α- poor resolution was due to the fact that too few actin and β-tubulin genes. All members of the representatives of this large genus had been genus studied by Hsieh et al. (2005) clustered sequenced at that time, while certain taxa were in a monophyletic clade, derived from within over-represented. In retrospective, the apparent Hypoxylon. The same was found based on incongruities of these phylogenetic studies 5.8S/ITS rDNA phylogeny (Triebel et al. were also due to the fact that about 30% of the 2005), however, members of Entonaema also sequences we had retrieved as background appeared in the clade comprising otherwise from GenBank were either obviously derived only Daldinia species. Representatives of other from misidentified isolates, or it is not possible genera that have similar metabolites in culture to verify their taxonomic status as no voucher as Daldinia, i.e., Phylacia and Thamnomyces, specimens are available. were included in the molecular phylogeny By contrast, Hsieh et al. (2005) found (Bitzer et al. 2008, Stadler et al. 2010a). The

101 results suggest that they are closely related to species was based on material from Europe, the latter genus, while the genera containing which was most probably collected in Sweden. mellein derivatives in their cultures are more Our comparison of molecular data based on closely related to Hypoxylon. The current status 5.8S/ITS nrDNA revealed that the data pub- of these attempts to reconstruct the phylo- lished in GenBank on X. hypoxylon obviously genetic affinities within the Xylariaceae is still correspond with many different species. Little rather preliminary because many of the known variations were observed among the 5.8S/ITS taxa have not been included, and different nrDNA sequences derived from the most com- portions of the DNA were used in independent mon European morphotype of this species studies, sometimes with contradictory results. complex, which were newly generated by us The data presently available appear insufficient based on material from France, Germany and to challenge the current generic organisation in Sweden. However, only a small percentage of the Xylariaceae. the published DNA sequence data was found Further clarifications could be obtained similar to the European genotype. Moreover, by the various cultures in public collections that had  inclusion of additional taxa, especially of been sequenced in the past were found to cor- those that show phenotypic traits that are respond with different species, or to be degene- interpreted as either basal or advanced; rate. This especially concerns the strains used  a concise multigene genealogy using a in the study on the molecular phylogeny of Xy- significant numbers of representatives of all laria by Lee et al. (2000). The genotype most known morphological lineages and chemo- frequently referred to in the literature corre- types; sponds to a culture which was deposited by  clarification of the relationship of genera Chacko & Rogers (1981) and only tentatively with Geniculosporium-like anamorphs to referred to as X. hypoxylon by the authors. the large, heterogeneous genus Xylaria, and DNA sequences derived from this material inclusion of other representatives of those were published by Spatafora & Blackwell genera currently included in the Xylariales (1993) and since then used as references (and showing intermediate morphological representative of Xylariaceae and Xylariales) in features between the Xylariaceae and other numerous molecular phylogenetic studies. In families. fact, this fungus most probably belongs to X. A thorough revision of the Xylariaceae longiana Rehm, which remains to be recorded using molecular phylogenetic data does not from Europe. The results of Peršoh et al. appear feasible at this time, because no (2008) gave impetus to attempt a concise re- representatives of important taxa, including ca. typification of X. hypoxylon, based on repre- 80% of the described species and about one sentative European material, which is currently third of the accepted genera remain to be pending. Our intention is to select a fresh sequenced. Not even the type species of representative specimen that can be cultured, important genera have been clearly defined by studied for anamorphic morphology and HPLC molecular data. This problem was addressed in profiles, sequenced, and used in modern another molecular study with emphasis on taxonomy and phylogeny. This might facilitate xylarioid taxa (Peršoh et al. 2008). It revealed the future revision of the genus Xylaria, once a that Xylaria hypoxylon, the type species of stable concept of the type species has become Xylaria, was ill-defined. This species has available. However, a morphological always been regarded as complicated and comparison of specimens from the collections complex, and cosmopolitan. Teleomorphic as of Linnaeus, E. Fries, and J. Dillenius, and a well as anamorphic features were reported critical review of the respective taxonomic independently by various researchers, but the literature needs to be carried out Such work is results are contradictory to some extent. A presently on going in preparation of a concise study of the holomorphic morphology subsequent publication. Three additional of various specimens from around the world European species of Xylaria, two of which are was never carried out before. As mentioned in similar to X. hypoxylon, have meanwhile been the Introduction, the first description of this described by Fournier et al. (2011).

102 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 Applications of analytical chemistry and systems which we have so far employed. Their biological assays natural function, and the reason why they From the results of the HPLC profiling specifically accumulate in stromata of D. study, it soon became apparent that it was not concentrica and related species remains possible to cope with the wealth of information obscure. provided by the analytical HPLC-DAD and Hypoxylon fuscum is one of the most HPLC-MS data, unless we would be able to get frequently encountered ascomycetes in Europe further information based on chemical struc- (cf. Petrini & Müller 1986, Granmo 1999). tures. Various apparently specific metabolites Fide Ju & Rogers (1996), it occurs throughout were detected in the stromata of these fungi, the Northern hemisphere. Stromata collected in which could not be identified for lack of stan- Germany from Corylus avellana were extrac- dards. Therefore, we started to collect larger ted and subjected to analytical studies (Quang amounts of the stromata on sites where we had et al. 2004b). They yielded BNT, daldinal A previously found them in abundance. Observa- and daldinin C, and two new azaphilones rela- tions throughout the vegetation periods to ana- ted to daldinin C, which we named daldinins E lyse the succession of metabolite production in and F (Fig. 11). Only the latter two compounds the stromata (see below) aided to determine the are apparently specific for H. fuscum and some optimal time for collection of material for pre- of its allies, whereas Daldinin C also occurs in parative studies. From collections of several H. rubiginosum, Daldinia childiae, and a series “common” species of Europe and Taiwan, of other daldinoid Xylariaceae. The daldinins sufficient quantities of stromatal extracts to represent the prevailing pigments in a broad allow for preparative studies were finally ob- range of other Hypoxylon spp. as will be out- tained. The results are summarised briefly for lined elsewhere. Collections of H. fuscum from each single species; extensive overviews on Alnus and Betula, as well as some specimens most of these results were published in the re- from Corylus in USA, however, contain smal- views by Stadler & Hellwig (2005) and Stadler ler amounts of the daldinins and have additio- & Fournier (2006). Only representative (and nal pigments which have so far not been identi- preferentially, novel) chemical structures are fied. These specimens with deviating HPLC shown here, out of a total of ca. 100 secondary profiles also differ in their ascospore size range metabolites that were isolated from the targeted from the “chemical race” on Corylus found in species. Europe. It was so far not possible to obtain a Daldinia concentrica was collected in conclusive segregation of the H. fuscum large amounts from a site in the Neanderthal complex, aside from the recognition of H. for preparative chromatographic work. In the porphyreum Granmo and other specimens, course of a diploma thesis, Baumgartner (2001) which are associated with Fagaceae, rather than isolated an unprecedented linear polyhydroxy- Betulaceae (Granmo 1999, Stadler et al. lated squalene, named concentricol (Stadler et 2008b). al. 2001a). This terpenoid (Fig. 14) is prevalent The characteristic stromatal pigments of in the stromata of D. concentrica and a group Hypoxylon rubiginosum, which has effused- of related species (i.e., the D. concentrica pulvinate stromata featuring orange stromatal complex). Subsequently, three further pigments and is widely distributed in the congeners of this type were identified from the Northern hemisphere, were isolated and stromata of D. concentrica, along with ten identified. The species was targeted because further metabolites, five of which were new to HPLC profiling revealed chromophores of the Science at the time of publication (Quang et al. major components, which were different from 2002a,b). One of the most interesting com- those of the known mitorubrins. The major pounds among the stromatal constituents of D. constituents were identified as novel natural concentrica is concentricol D, which bears a products, for which the trivial names, rather unique cyclisation in its ring system. The rubiginosin A–C (Fig. 11), were proposed concentricols are not produced in cultures of (Quang et al. 2004a). Another congeneric Daldinia spp. They did not exhibit any pigment of this type, entonaemin A, appears significant activities in the biological test chemically related to them. It was previously

103 obtained from an Entonaema sp. (Hashimoto & bond in the azaphilone ring system. The Asakawa 1998). The azaphilone backbone of chromophore is thus altered as compared to the the rubiginosins is identical to that of the “orange” rubiginosin and mitorubrin chromo- mitorubrins, while the substitution by orsellinic phore, which explains the different colours of acid via an ester bond occurs at a different this pigment (for DAD spectrum of hypomiltin, carbon atom. As shown in Fig. 10, DAD-based see Fig. 10). Hypoxylon trugodes and several HPLC profiling can easily discriminate the two other species of this genus, but even resulting types of mitorubrin vs. rubiginosin Pyrenomyxa picea, also contain hypomiltin, chromophores, despite both types of and a number of further derivatives with the azaphilones give orange colours in 10% KOH. same chromophore, but deviating molecular As such HPLC profiling data are species- masses were observed by HPLC profiling in consistent, the identification of the rubiginosins those species, which probably also correspond was an important prerequisite for the above with novel hypomiltins. described chemotaxonomic evaluation of the Hypoxylon macrocarpum Pouzar, only H. rubiginosum complex. Daldinin C (see segregated by Pouzar (1976) as a separate above) was also found in H. rubiginosum in taxon of the aggregate species “H. rubigi- small amounts. Rubiginosin A turned out to be nosum“ sensu Miller (1961), is actually quite widely distributed in the genera Hypoxylon and frequent in Europe and America but probably Entonaema and proved to be an important its stromata are still being frequently over- chemotaxonomic marker. looked. The species was found to contain BNT The relatively rare species Hypoxylon and three specific compounds, which were rutilum Tul. & C. Tul. is characterised by revealed to possess an unprecedented polyke- scarlet pigment granules beneath its stromatal tide carbon skeleton with an orsellinic acid-like surface. The scarlet pigments are the rutilins, moiety attached (Mühlbauer et al. 2002). The which were also detected in other Hypoxylon specificity of the macrocarpones chemical spp. which have similar features (Quang et al. structures see, e.g. macrocarpon (Fig. 11). H. 2005b). The rutilins (Fig. 11) are dimeric macrocarpum was meanwhile largely confirm- azaphilones composed of monomers, reminis- ed. They do apparently not occur in most other cent of mitorubrinol acetate and rubiginosin A, species of the H. rubiginosum complex. We which were also observed in rather large only found them in a specimen of Hypoxylon quantities as co-metabolites in the stromata. cf. anthochroum from tropical Africa (Stadler Interestingly the dimeric azaphilones were et al 2008b), and in Pyrenomyxa picea (Stadler found devoid of antimicrobial and cytotoxic et al. 2004a, as Pulveria porrecta). Interesting- effects, but Quang et al. (2006c) tested them ly, H. macrocarpum did not contain any positively as inhibitors of nitric oxide synthe- rubiginosin or mitorubrin type azaphilone sis. Hence, they apparently have pronounced pigments, as meanwhile established from antioxidant activities and may eventually HPLC profiling of more than 1000 specimens. become useful as templates for new cardio- Hypoxylon carneum Petch is another vascular drugs. species of the H. rubiginosum complex which Hypoxylon perforatum (Schwein.) Fr. is contains exceptionally small amounts of one of the species which was synonymised rubiginosins, which can only be detected by with H. rubiginosum by Miller (1961). It was HPLC-MS in the crude extracts. The species revealed by Ju & Rogers (1996) to have was studied by HPLC and found to contain greenish yellow stromatal pigments (rather BNT and a number of highly specific than the orange pigments of H. rubiginosum). compounds, which are not pigments, in large From this species, as well as from a Taiwanese amounts. Two of these compounds were collection of H. hypomiltum Mont., a major identified as carneic acids (Figs 11A, B), two pigment named hypomiltin (Hellwig et al. antibiotics chemically related to phomopsidine 2005) was obtained. This azaphilone (Fig. 11) (Quang et al. 2006a). At least ten further has a very similar molecular structure to putative novel carneic acid derivatives were mitorubrinol acetate. The only difference detected in the stromatal extract by HPLC-MS, between these molecules is a reduced double but we have yet to observe carneic acid in

104 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1

DAD1 C, Sig=210,8 Ref=550,100 (O:\MEASUR~1\HPLC\STMA\XYLAR\04110313.D) DAD1, 7.944 (277 mAU,Apx) of 04110313.D mAU mAU

Hypoxylon salicicola 250 7.932 7.932 Mitorubrinol acetate

200 200

150 150

100

100 50

50 0

8.1138.113 8.698

8.698 250 300 350 400 450 500 550 nm

7.7227.722

8.4068.406

9.0819.081

5.5265.526

7.2337.233

5.8465.846 6.9466.946

1.6711.671 6.5896.589

0.9570.957

5.3385.338

3.2583.258

10.15710.157

4.9304.930

4.4714.471 4.073

0 4.073

12.88412.884

0 2 4 6 8 10 12 14 min

DAD1 C, Sig=210,8 Ref=550,100 (O:\MEASUR~1\HPLC\STMA\XYLAR\10010507.D) DAD1, 7.704 (1041 mAU,Apx) of 10010507.D mAU mAU

Hypoxylon perforatum 1000

7.7017.701

800 800 Hypomiltin 600

600 400

400 200

0

200 250 300 350 400 450 500 550 nm

8.6868.686

11.21111.211

6.7026.702

8.1708.170

5.3215.321

8.4538.453

7.3207.320

6.4766.476 7.1467.146

5.8045.804 11.42411.424

5.6075.607

9.9449.944

1.6181.618

10.21710.217

10.48710.487

1.3041.304 1.4501.450

0.9620.962

11.93611.936

4.3614.361

4.7594.759 4.973 0 4.973

0 2 4 6 8 10 12 14 min

DAD1 C, Sig=210,8 Ref=550,100 (O:\MEASUR~1\HPLC\STMA\XYLAR\10010521.D) DAD1, 7.490 (891 mAU, - ) of 10010521.D mAU

mAU Hypoxylon rubiginosum 7.482 7.482 800 700 700 Rubiginosin A 600 600

500 500 400

400 300

200 300 100

200 0

250 300 350 400 450 500 550 nm 5.307

100 5.307

7.9947.994

8.9008.900

2.2312.231

8.5348.534

8.3728.372

10.69210.692

10.45610.456

10.89810.898

9.9449.944

6.3426.342

6.8106.810

1.3051.305

5.9365.936

5.7225.722

1.5101.510

2.9942.994

5.1015.101

4.2704.270 4.4214.421

4.6024.602 4.7684.768

12.27312.273 3.545 0 3.545

0 2 4 6 8 10 12 14 min

Fig. 10 – Comparison of the stromatal HPLC-UV chromatograms (210 nm) of three Hypoxylon species belonging to the H. rubiginosum complex, including characteristic UV-visible spectra of characteristic azaphilone pigments. The stromata of these species show a rather similar morphology, whereas their major extractable pigments are rather easy to discriminate by diode array analyses.

other species of Hypoxylon. from those of the pigments of Hypoxylon and Annulohypoxylon cohaerens (Pers.) Annulohypoxylon, and only the azaphilone Y.M. Ju et al. and A. multiforme (Fr.) Y.M. Ju chromophore is similar. Sassafrin D even et al. were also subjected to preparative studies, represents an unprecedented carbon skeleton. as the pigments of these species were found by Bitzer et al. (2007) have illustrated the HPLC profiling to differ very much from those methodology now available for effective of the above described species, except that dereplication of natural products by analytical BNT was also present in their stromata. A HPLC-DAD and HPLC-MS. “Dereplication” is series of azaphilones named multiformins or equivalent to the early stage identification of cohaerins (Fig. 12) after the respective secondary metabolites in crude extracts from producer species, were identified (Quang et al. natural sources, aided by spectral libraries and 2005c,e, 2006b). special software programs. As the methodology Creosphaeria sassafras (Schwein.) Y.M. was validated and further developed in part Ju et al. was included in the genus Hypoxylon, through our chemotaxonomic studies, the until Ju et al. (1993) noted its Libertella-like examples used for illustration of the derepli- anamorph. Indeed the hypoxyloid stromata cation process were taken from the Xylariaceae even contain pigment granula below the project. Aside from some metabolites of H. stromatal surface. These pigments were fragiforme, the evaluation of the tropical isolated and identified as sassafrins A-D species Annulohypoxylon urceolatum (Rehm) (Quang et al. 2006d). Interestingly, the carbon Y.M. Ju et al. was also described. This species skeleton of these compounds is quite different had shown a rather specific HPLC profile 105 O

HO CH3 O CH3 R R O O O CH O O 3 H3C OH O O AcO O OH Hypomiltin (R = OOCCH3) H3C O

Daldinin C (R = CH3)

Daldinin E (R = CH3CH=CH) HO H Daldinin F [R = CH3CH=C(CH3)] HO CH3 R

O O HO CH3

H3C H OH O O O R Rubiginosin B (R = OOCCH3) O OH O HO H C 3 O COOR Entonaemin A (R = OH( Rubiginosin A (R = OOCCH3)

O (R = H: Rubiginosic acid) R = O OH HO H C 3 O Rubiginosin C O OH

CH3 HO O OH OH H3C O O COOH OH O Macrocarpon A

Orsellinic acid

OH O O O H O HO O H O O OH O O Rutilin A OH H O OH Carneic acid A OH HO

Fig. 11 – Chemical structures of metabolites from stromata of Hypoxylon species.

(Hellwig et al. 2005). Aside from the urceolone are also present in Hypoxylon ubiquitous BNT and the naphthoquiinone monticulosum Mont. and H. submonticulosum derivative hypoxylone, a novel binaphthalene Y.M. Ju & J.D. Rogers, two species that were was obtained and named urceolone (Fig. 14), regarded basal in Hypoxylon by Ju & Rogers after the producer organism. These compounds (1996). Hypoxylone was reported by Bodo et are now available as standards for concise al. (1983) from stromata of Hypoxylon identification of hitherto unknown metabolites sclerophaeum Berk. & M.A. Curtis sensu in the crude extracts of tropical Xylariaceae in Miller (1961), which was resolved into several the course of our ongoing chemotaxonomic species by Ju & Rogers (1996). This compound work. Interestingly, compounds similar to constitutes a naphthoquinone, which is, like

106 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1

H3C H3C

O O O H C O OH 3 O O H3C O O CH3 H3C CH3 O

O CH3 cohaerin A O multiformin A

O CH3 O CH3

O OH H C 3 O OH O CH 3 HO CH3 H CH 3 CH3 O H MeOOC O OH OH O

sassafrin B sassafrin D

Fig. 12 – Chemical structures of stromatal pigments of Annulohypoxylon and Creosphaeria spp. urceolone, conceivably derived from the same Antimicrobial and other biological biogenetic pathway as BNT. The activities were already included in some of the chemotaxonomic significance of these above cited papers dealing with the isolation compounds is not yet fully understood, but and structure elucidation of the stromatal remarkably they both constitute naphtho- metabolites of Xylariaceae. An overview of a quinone derivatives. The biogenesis of such bioassay of several representatives against naphthoquinones from the 1,8-DHN (pentake- human pathogenic microbes (Quang et al. tide) pathway of melanin biosynthesis is 2005a) revealed significant biological activities conceivable. Their biosynthesis could be for most tested azaphilones. However, these accomplished via dimerisation of naphthol effects were not selective against fungi or precursors in a similar manner as that of BNT bacteria. and its oxidised derivatives and subsequent Two papers (Stadler et al. 2006, 2007) oxidations, as has been postulated for the deal with functional aspects of the secondary perylene quinones, daldinone A and trunca- metabolism in Xylariaceae, which are most tone. Interestingly, compounds with HPLC-MS remarkable in view of the possible ecological and HPLC-DAD spectra similar to those of role and the natural functions of these hypoxylone occur in the stromata of some compounds. These studies were initiated when Xylariaceae, which are devoid of daldinones HPLC profiling of stromata in different and truncatones but still show greenish developmental stages had revealed inconsisten- pigments with 10% KOH. Identification of this cies that could at first not be easily explained unknown putative naphthoquinones will be by morphological data. Only when the stage of important in the further chemotaxonomic development was correlated to the respective evaluation of those Xylariaceae taxa that HPLC profile, it became obvious that immature preferentially produce naphthalene-derived stromata and stromata bearing the anamorph compounds in their stromata. Those include, had different HPLC profiles than the mature for instance, Daldinia placentiformis (Berk. & stromata. We therefore decided on monitoring M.A. Curtis) Theissen, Hypoxylon nicaragu- the secondary metabolism of certain species in ense Ellis & Everh., and morphologically their natural environment throughout several similar species. consecutive vegetation periods and to pay more

107 OH

N H O O OH

Fragiformin B

Norm. mAU 7.739 ME 1750 1750

1500 ME Mycelial cultures 1500

1250 1250 5.988 1000 1000

ME

750 750

9.596

500 500

5.352

250 250

6.552

5.685

5.036 6.789 9.418 0 0 0 2 4 6 8 10 12 14 min 250 300 350 400 450 500 550 nm

DAD1 C, Sig=210,8 Ref=550,100 (XYLAR\14100501.D) DAD1, 8.704 (99.2 mAU, - ) of 14100501.D Norm. Mol DAD1, 8.437 (112 mAU, - ) of 14100501.D

DAD1, 8.170 (215 mAU, - ) of 14100501.D 6.798 Young stromata DAD1, 7.637 (215 mAU, - ) of 14100501.D 600

7.209 DAD1, 7.210 (560 mAU, - ) of 14100501.D Norm. 500 Cy

9.245 500 400 Cy Cy 400

300 9.657

300

10.790

7.631 8.163 200 11.022

OA 200

7.779

10.224

10.607

11.263

10.399

8.426

11.442

8.697 10.007

100 9.050

11.711

7.406 12.567

12.712 100

5.362

1.285

12.171

11.943

4.741

0.875

4.530

6.185

5.692

6.001

4.899 5.854

3.936 5.138

2.187

3.400

3.099 4.158 0 13.671 0

0 2 4 6 8 10 12 14 min 250 300 350 400 450 500 550 nm

DAD1 C, Sig=210,8 Ref=550,100 (J:\G_PHSDC\ANHPLC\PROTEAS\28030325.D)

mAU MAc 8.410 Mature stromata DAD1, 8.424 (1677 mAU, - ) of 28030325.D 1200 DAD1, 7.957 (839 mAU,Apx) of 28030325.D Mol DAD1, 6.904 (1452 mAU, - ) of 28030325.D 6.899 DAD1, 5.517 (94.5 mAU, - ) of 28030325.D Norm. 1000 M 1600 1400

800 7.942

1200

600 1000

800

400 10.798

10.997 600

200 OA 11.281

10.596 400

10.323

9.691

8.816

5.513

9.316

9.141

12.592

7.427

12.080

6.063

5.922

6.345

5.304

1.281

0.956

2.402

1.487 3.186 0 4.730 200

0 0 2 4 6 8 10 12 14 min 250 300 350 400 450 500 550 nm

Fig. 13 – HPLC-UV chromatograms of mycelial cultures, young, and mature stromata of Hypoxylon howeanum, illustrating the succession of secondary metabolite production in different developmental stages of the fungus. Major metabolites (as identified by HPLC-MS): Cytochalasins (Cy; as exemplified by the chemical structure of fragiformin B, depicted above the young stromata)), mellein dihydroisocoumarins (ME), mitorubrinol (Mol), mitorubrin (M), mitorubrinol acetate (MAc), and orsellinic acid (OA). Notably, numerous yet unidentified metabolites were detected in the young, maturing stromata of the fungus, which also exhibited the strongest antimicrobial activities. Image of mature stromata by J. Fournier. From a lecture presented at the IMC 9, Cairns, Australia (2006). attention to the stage of development when profiling, revealing changes in the composition interpreting stromatal HPLC profiles. during stromatal development (Stadler et al. Stromata of Hypoxylon fragiforme 2006). Three cytochalasins (among those the growing on different sites were studied for new natural products, fragiformins A–B, see three consecutive vegetation periods by HPLC chemical structure of a representative in Fig.

108 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 13) were identified as major constituents of the dary metabolites in its stromatal biomass. In young, maturing stromata, whereas mature, several species, significant antimicrobial ascogenous material yielded large amounts of effects were noted upon direct incubation of mitorubrins. The above compounds, further the fruiting bodies in an agar diffusion assay, cytochalasins from Xylariaceae and other i.e., without any need for extraction and fungi, and additional azaphilones of the concentration of active constituents. From mitorubrin type were compared on their these studies, we concluded that most of the nematicidal effects against Caenorhabditis characteristic constituents of subfamily Hypo- elegans and their antimicrobial activities xyloideae are involved in non-specific defence against Bacillus subtilis, and various fungi. The reactions that underwent specific permutations results confirmed data in the literature on in the course of evolutionary processes, broad-spectrum non-selective activities of resulting in a broad diversity of unique azaphilones and cytochalasins in biological polyketides and other secondary metabolites. systems. In contrast, the fruiting bodies of Interestingly, laboratory cultures of the representative species of Biscogniauxia and above Hypoxylon spp. mainly produced subfamily Xylarioideae generally contained no dihydroisocoumarin derivatives and were significant activities. Only cultures of most found devoid of mitorubrins and cytochalasins. Xylarioideae exhibited antimicrobial effects, A comparison of the HPLC chromatograms due to the presence of cytochalasins and other including illustrations of the different develop- toxins, while extracts from cultures of most mental stages, is provided in Fig. 13. The Hypoxyloideae were only weakly active. extracts from the young, growing stromata also Usually, the highest yields of secondary meta- yielded a series of further unknown compo- bolites and the strongest activities were nents that were neither detected in the cultures observed in the growing stromata of Hypoxy- nor in the mature stromata (see HPLC-UV loideae. chromatogram in the centre of Fig. 13). The strong antimicrobial activities detected in such samples provide some evidence on the natural Phylogenetic significance of secondary meta- function of this onset of secondary metabolism bolite production in cultures of Xylariaceae during stromatal ontogeny, which is also Some of our recent papers dealt with an associated with the rapid colonisation of the extensive polyphasic evaluation of specimens woody substrate. Certainly, these results were and cultures which had been studied over the also rather interesting for applied mycology, in past years. The paper by Bitzer et al. (2008) view of future options to fully explore the resulted from chemotaxonomic studies of over repertoire of fungi to produce unprecedented, 150 cultures selected from 600 strains of useful secondary metabolites and discover Xylariaceae. The fermentation of the fungi was novel lead compounds by targeting these optimised in several different culture media, organisms at their most productive stage of and samples were taken during the time course development. of fermentations to monitor the onset of The production of bioactive metabolites secondary metabolite production. Two new in young stromata is not restricted to the above chemotypes were observed in cultures of mentioned species pair, since similar results Phylacia sagrana Mont. (Fig. 16), and were obtained on a broad range of other species Hypoxylon nicaraguense (Fig. 15). and H. of the Hypoxyloideae. Over fifty representa- polyporus (Starbäck) Y.M. Ju & J.D. Rogers, tives of Xylariaceae and fifty of the above respectively. While the Phylacia culture mentioned secondary metabolites were tested appeared similar to Daldinia, the other two for antimicrobial and nematicidal activities. species strongly deviated from over 50 taxa The major constituents in the stromata studied concurrently of the genus Hypoxylon. were also quantified by HPLC-DAD, revealing This gave impetus to scale-up of fermentation, yields of up to 10% of a single component in isolate and identify the characteristic, the dry fruiting body biomass. Hypoxylon apparently specific metabolites. fuscum may even contain up to 20% of secon-

109 H H HO O O

HO HO HO OH O OH OH O OH OH O O

daldinone A truncatone daldinone B

OH O OH OH OH OH

OH O OH O hypoxylone urceolone

OH CH H C OH 3 3 CH3 HO OH

CH H C OH CH 3 3 3 concentricol (A) OH

OH O H H C HO 3 OCOCH3 CH CH3 H3C OH CH3 3

concentricol D

Fig. 14 – Chemical structures of characteristic stromatal pigments of the Xylariaceae derived from 1,8-dihydroxynaphathalene biosynthesis, and of the concentricol sesquiterpenes from D. concentrica.

The Phylacia culture was found to which an additional Phylacia sp. and three contain several major metabolites that also Thamnomyces spp. were cultured and studied prevail in Daldinia. In addition, it also in a similar manner, the genus Thamnomyces is produced eutypinol derivatives and was devoid also closely allied to Phylacia and Daldinia, of 8-methoxy-1-naphthol as major differences despite its aberrant stromatal morphology. The to Daldinia. A concurrent molecular study cultures of Phylacia were found to have a based on 5.8S/ITS nrDNA sequence data highly similar morphology and secondary demonstrated that these differences in the metabolism as those of Thamnomyces. The secondary metabolite profiles were reflected by relationships postulated by the previous similarity analyses of molecular data. comparison of stromatal HPLC profiles Interestingly, the Daldinia sp. that appeared (Stadler et al. 2004a) were thus confirmed. most closely related to P. sagrana (i.e., D. Interestingly, representatives of the genus caldariorum Henn.) as inferred from the Daldinia became split into two different clades molecular phylogeny also produced eutypinols in a phylogenetic tree included in Stadler et al. under the chosen, standardised fermentation (2010a). One of these clades comprised conditions. As meanwhile revealed from a representatives of Phylacia and Thamnomyces, subsequent study (Stadler et al. 2010a), in along with some tropical representatives,

110 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1

H OH OMe N O OH

HO

mAU 1600 O 1400 1200 N N 1000 H

800 mAU

600 1200

400 1000 200 C 800 0

250 300 350 400 450 500 550 nm 600 m A U 400 800 200 700 0

250 300 350 400 450 500 550 nm 600 N 500 400 300 C 200 100 0 14 m i n 0 2 4 6 8 10 m in Fig. 15 – HPLC-UV chromatogram of an ethyl acetate extract from a culture of Hypoxylon nicaraguense (HLX medium, 168h, revealing cochliodinol (C) and 8-methoxy-1-naphthol (N). Mellein derivatives were not detected. Modified from Bitzer et al. (2008).

OH O

6 O OH

HO O

O O OH mAU 5 800 600 5 mAU 400

500

200 400 1 O 0 300 250 300 350 400 450 500 550 nm HO O

200

100

OH OH 0 HO 250 300 350 400 450 500 550 nm

3 OH O mAU 6 2

mAU O 1600

1400 1200 4 5 200 1000

800 600 O 400

200 OH 0 150 250 300 350 400 450 500 550 nm 2

mAU 350 7 300

250

mAU 100 200 150 1 600 100 7 3 500 2 50 400

0 300 250 300 350 400 450 500 550 nm 4 50 200

100

0

250 300 350 400 450 500 550 nm

0

0 2 4 6 8 10 min Fig. 16 – HPLC-UV chromatogram (210 nm) of ethyl acetate extract prepared from a culture of Phylacia sagrana, including DAD spectra and corresponding chemical structures of some of the major components. Modified from Bitzer et al. (2008). whereas the other comprised clade comprised other phylogenetic studies based on 5.8/ITS the temperate Daldinia spp. and Entonaema nrDNA sequence data. They need to be liquescens. Furthermore, these two clades did confirmed through the inclusion of further taxa, not appear nested in the clade containing hence they are not discussed here at length. representatives of Hypoxylon and Annulo- The two above mentioned Hypoxylon hypoxylon. These results disagree with the species shared with Daldinia and Phylacia the Parsimony Ratchet analysis (Fig. 7) and with production of 8-methoxy-1-naphthol but

111 additionally produced aromatic alkaloids of the Hypoxylon, and their closest allies have been cochliodinol type. Similar compounds had identified and made available for HPLC pro- previously only been obtained from the filing studies. However, additional metabolites unrelated genus Cochliobolus (Pleosporaceae) remain to be identified, e.g., from cultures of and from a Nodulisporium sp. with unknown the Xylarioideae, and from stromata of certain teleomorph connections. In the preliminary species of Annulohypoxylon, Hypoxylon, and molecular phylogeny, their 5.8S/ITS nrDNA other genera. For instance, the blue-green sequences formed a cluster that appeared pigments of Chlorostroma subcubisporum and derived from the clades representing other Hypoxylon aeruginosum J.H. Miller and the members of Hypoxylon. pigments of Daldinia cuprea Starbäck are Two different specimens that matched rather specific according to unpublished HPLC the concept of Hypoxylon placentiforme sensu profiling results, and no close matches were Ju & Rogers (1996) and their corresponding found when their spectra and retention times cultures were studied for comparison, revealing were matched with the database of Xylariaceae several interesting facts: a) in accordance with metabolites. However, these species appear to their deviating stromatal metabolite profiles be extremely rare and/or have relatively small and ascospore size ranges, they clustered far stromata. The option to culture them in the apart from one another in the molecular laboratory and isolate their constituents from phylogeny; b) in agreement with the recent artificially produced stromata should be further revision by Hsieh et al. (2005), who transferred explored in future. H. placentiforme to Daldinia, resurrecting the Chemotaxonomic data on several thou- name D. placentiformis, they showed similar sands of specimens and several hundreds of HPLC profiles as all Daldinia spp. studied cultures of Hypoxyloideae have been correlated concurrently. The observed affinities of to the morphological concepts. This work has Daldinia, Phylacia, and Thamnomyces as also led to the recognition of further, revealed by chemotaxonomic and molecular undescribed taxa by resolution of species data gave rise to reject the concept of a family complexes. Some genera and species groups, “Phylaciaceae” (Speer 1980) for cleistocarpous however, still remain to be evaluated in-depth. genera with affinities to the Hypoxyloideae, as One taxonomic problem to be addressed in the the affinities of Pyrenomyxa to the H. near future is the redefinition of the boundaries rubiginosum complex (see above) were between Hypoxylon and Daldinia. The confirmed. Cleistocarpous stromata have inclusion of D. placentiformis in Daldinia as obviously evolved at least twice in separate inferred from molecular data and chemotaxo- lineages of the Hypoxyloideae. In Table 3, nomic results is problematic because this morphological and chemotaxonomic traits of species (which actually appears to constitute a the daldinoid and hypoxyloid Hypoxyloideae species complex as deduced from the high have been summarised. variability of morphological traits, HPLC profiles, and DNA sequence data) does not Outlook match the generic concept of Daldinia, which The above results have helped to settle was recognised and segregated from Hypoxylon various questions regarding the taxonomy of by Cesati & Notaris (1863) based on the the Xylariaceae, but there still remains a lot of presence of concentric zones. Therefore, a work to do. The steadily increasing matrix of revision and amendment of the generic molecular, morphological and chemotaxono- description of Daldinia will also be envisaged mic data will facilitate further monographic in the near future. The genus Rhopalostroma studies. The most important topics to be also remains to be studied further based on addressed in the near future have been fresh material (Stadler et al. 2010b), and the summarised below in relation to the objectives same holds true for certain tropical taxa of of our polyphasic studies. Hypoxylon, where the available morphological Many of the characteristic pigments and and chemotaxonomic data point toward the other chemotaxonomically significant secon- presence of additional species complexes. As dary metabolites of the genera Daldinia, demonstrated above by the revisions of

112 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 Entonaema and Pyrenomyxa, a taxonomic this context it is important to keep in mind that revision of such “problem taxa” makes more the biosynthesis of these secondary metabolites sense, once cultures and molecular data have is mediated by modular gene clusters, such as become available. However, this will afford the PKS and NRPS, the former being apparently availability of fresh material. Additional field more widely distributed in the species we have work, especially in tropical countries, will so far studied. These gene clusters contain therefore be indispensable. preserved moieties, which can be used as The concurrent molecular phylogenetic templates for primers to screen the genomic studies have also proved most useful to verify DNA of their producer organisms. For morphological concepts in the Xylariaceae. evaluation of the diversity of PKSs in a Xylaria Ribosomal DNA sequence data were shown to sp. (Amnuaykanjanasin et al. 2005), two reflect the evolutionary history of the degenerate primer sets were designed, to Xylariaceae at generic and species level in amplify reducing PKS and PKS-NRP synthe- many instances, and they were even found to tase hybrid genes, respectively. Seven genes be in accordance with chemotaxonomic results. predicted to encode proteins homologous to The results on the molecular phylogeny already highly reduced PKSs were identified, and published constitute only a small part of the additional four PKS gene fragments were molecular work that has already been accom- found using different, previously published plished, but remains to be disclosed. Ribosomal primers. Using these PKS fragments as probes DNA sequences of most of the strains to identify PKS genes from the genomic library belonging to the Hypoxyloideae studied for of the Xylaria sp. led to full-length sequences metabolite production in culture (Bitzer et al. for five PKS genes. The authors discussed the 2008) were already obtained. Representatives utility of their approach to further reveal the of some additional, small genera such as genetic potential of fungi with regard to the Lopadostoma Pouzar, Thuemenella Penz. & production of polyketides. Although they did Sacc., and Vivantia J.D. Rogers et al. were not finally correlate the function of any of the recently made available. Respective data for a PKS genes to the production of particular large number of representatives of xylarioid metabolites, the outcome of their study appears genera, such as Rosellinia and Nemania, that promising. The lack of detection of a certain had so far not been represented sufficiently, compound may relate to the absence of were also recorded from strains available in respective gene clusters, but it remains possible public collections as well as via our that the respective genes are present but down- collaboration network. The DNA sequence data regulated or the gene cluster has become non- will also serve to detect the respective taxa in functional. It remains to be proven whether the plant material and other environmental differences seen by HPLC profiling can be samples, using phylochips, microarrays and correlated to differences at the genetic level. related PCR-based methodologies to facilitate For instance, members of the genus Hypoxylon further studies aimed at clarification of the that produce mellein in culture could as well molecular ecology of these fungi. Another have genes for naphthol biosynthesis, as many option for future work could be to include data of them produce the binaphthyl BNT in their from additional genes, such as the large subunit stromata. of the ribosomal gene, the IGS region and non- Differences in these “phenochemotypes” ribosomal genes to create a multi-gene of various Xylariaceae become even more genealogy of Xylariaceae. evident when the carbon skeletons of the Various correlations could already be prevailing metabolites from their cultures (Fig. established between the observed chemotypes 17) and their stromatal pigments (Fig. 18) are and the current knowledge on the biosynthesis compared. The chemical structures of selected of the corresponding metabolite families that compounds have been reduced to the corres- are specifically produced by the Xylariaceae. ponding carbon skeletons in these figures a One task for the future could be to confirm similar manner as accomplished previously by these phenotype-based correlations by studying Turner (1970), who illustrated different types the genetic background of their biosynthesis. In of polyketides and interpreted the putative

113 biogenesis of many fungal polyketides, based They contribute substantially to the global on biosynthetic studies using radioactively biological diversity, but their cryptic lifestyle labelled precursors. As such studies yet remain may often render their detection rather to be done on the majority of Xylariaceae difficult. Mueller et al. (2004) have sum- metabolites, the discussion presented is highly marised the current state of the art on hypothetical. In Fig. 17 it is clarified that the identification methodology, and provided nu- melleins from cultures of Hypoxylon and other merous protocols and suggestions for con- genera are derived from entirely different duction of mycological biodiversity invento- folding types than those of the prevailing ries, from various kinds of habitats. This metabolites in cultures of Daldinia. In Fig. 17, manual demonstrates the great need of interdis- some possible origins of the polyketide-derived ciplinary teamwork for comprehensive moni- pigments from one or two PKS gene clusters toring of the mycobiota in a given habitat or have been postulated. Thus, BNT is assumed to geographic region. The use of standardised be derived from condensation of two mono- methodologies and the concise documentation meric naphthalene-like molecules, resulting in of specimens and data are strongly encouraged, the formation of new C-C bonds. For daldinin to facilitate the comparison of results from C and daldinal A, which co-occur in several different areas. Special emphasis is given on species of Daldinia and Hypoxylon, an octa- how to isolate fungal cultures from various ketide origin is assumed. The mitorubrins are environments and their preservation. probably hexaketides, esterified with the As fungi are associated with virtually all tetraketide, orsellinic acid, hence their bio- plants and animals, and even add substantially genesis would depend on two different PKS to the microbial communities in soil, gene clusters. It should be highly interesting to freshwater and marine habitats, “all taxa elucidate the genetic background of this mycodiversity inventories” are certainly not phenomenon, using methods of comparative easy to perform. Depending on the situation, functional genomics to elucidate the structures close collaborations of mycologists with other and functions of the PKS genes and study their biologists are indispensable, since host affini- regulation. Notably, the production of azaphi- ties and other aspects of ecology can otherwise lone pigments appears to be correlated with hardly be addressed. Where direct collabora- formation of stromatal primordia and precedes tions are not feasible, the Internet comes in the production of the teleomorph in those handy to summarise data from different cate- species that have been studied by HPLC gories, interlink them and thus make specialist profiling in the natural environment. knowledge available to non-specialists. The use of analytical chemistry in mycology also Discussion relates to specialist knowledge. Fungal pig- The work summarised in the present ments, volatiles and toxins are traditionally thesis relates to two major topics, which are important in taxonomy, and diagnostic closely related to one another: chemicals like Melzer’s reagent and KOH are  Employment of modern analytical che- widely used in fungal identification. Never- mistry to evaluate fungal chemotaxono- theless, modern analytical methods like HPLC my. are only available in a few mycological  Establishment of an integrative approach laboratories. No chapter on the utility of to characterise and assess the functional chemical profiling techniques is included in the biodiversity of fungi. above cited manual by Mueller et al. (2004). Even in the contemporary literature, the Based on the Xylariaceae example, these occurrence of specific secondary metabolites is topics will now be discussed in a broader still often being attributed to certain “formae context. specialis”, “host-specific varieties”, or “strain- As mentioned in the Introduction, there is specific” properties, and not regarded as accumulating evidence that fungi show a very taxonomically significant. Our own studies high diversity and have an immense confirmed that strain-specific metabolite importance for the functions of all ecosystems.

114 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1

O OH O OH

A O O OH 5-hydroxy-2-methyl- Ab-5046-A ? chromone

O OH OH O

8-methoxy-1- B OH naphthol isosclerone

OH O O

HO HO O eutypinol methyl Ab-5046-B C ether (6) D

O O OH O E OH O F mellein 5-methylmellein

Fig. 17 – Differences in the carbon skeletons among the putative polyketides, indicated by bold letters, that are prevalent in cultures of the genera Daldinia, Entonaema, Phylacia, and Thamnomyces (A-D) and Annulohypoxylon, Biscogniauxia, Camillea, Hypoxylon and Pyrenomyxa (E, F) respectively). One representative metabolite is depicted and connected by an arrow to the respective carbon backbones. For classification of these molecules and theoretical considerations, see Turner (1971). The fact that the occurrence of the carbon skeletons A-D is apparently restricted to those taxa that are devoid of the skeletons and folding types E-F is remarkable in view of the concurrently obtained molecular phylogenetic data. It is to be assumed that specific PKS gene clusters encode the biosynthesis of each carbon skeleton; whereas variations in the side chains may be mediated by different genes that are located outside these gene clusters.

production may occasionally occur, besides the riaceae. In fact, the reliability of chemotaxo- production of the chemotaxonomically signifi- nomic data will always be hampered by the fact cant marker compounds. For instance, if that they relate to a dynamic, physiological biosynthetic gene clusters are disrupted by process and is therefore highly dependent on mutation, this may lead to accumulation of standardised methodology. On the other hand, intermediate products of the original biosynthe- the same holds true for the classical morpho- sis. The degeneration of cultures upon frequent logy-based taxonomic concepts. Even molecu- subculturing may result in the loss of lar identification techniques will inadvertently production of the characteristic compounds and have to rely on the availability of data from was also occasionally observed in the Xyla- well-characterised “standard” organisms that

115 O

O O HO O O O O O O OH O OH O O O Daldinin C Mitorubrin

Hexaketide (Turner 1971) Methylated octaketide (?) (13 carbon atoms esterified 17 carbon atoms (esterified with orsellnic acid (tetraketide) with two different acids)

O OH O HO O HO OH

HO OH

OH BNT Daldinal A

Condensation product of Derived from decarboxylation two pentaketides (fide Turner 1971) of an octaketide precursor (?)

Fig. 18 – Chemical structures of four compounds that specifically occur in the stromata of certain Xylariaceae and their corresponding carbon skeletons, drawn after reduction of the chemical structures by omission of oxygen atoms and double bonds in a similar manner as proposed by Turner (1971). Accordingly, the mitorubrins are esters of the tetraketide, orsellinic acid, with a hexaketide-derived azaphilone carbon backbone. Daldinin C and Daldinal A could be octaketides. BNT and other binaphthyls are probably derived from condensation of two pentaketide units derived from biogenetic pathways related to that of the dhydroxynaphthalene melanin biosynthesis. These highly hypothetical assumptions remain to be proven by experimental data, including methods of functional genomics as well as classical labelling studies. need to be chosen first by using complemen- maceae (including Aspergillus and Penicil- tary data. lium), no other fungal family has so far been Frisvad et al. (2008) have highlighted the characterised by chemotaxonomic methodolo- importance of chemotaxonomy for the gy as intensively as the Xylariaceae. The model exploration of fungal biodiversity, but their character of this work may also be illustrated overview also shows that many groups of fungi by the following facts: remain to be studied. Except for the Trichoco- In contrast to other chemotaxonomic

116 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 studies of filamentous fungi, we have sity of medium polar low molecular secondary examined several genera concurrently, used metabolites (cf. discussion on “extrolites” by HPLC profiling techniques to study teleo- Samson & Frisvad 2005). However, they need morphic and anamorphic stages (i.e., stromata to be characterised by methods other than and cultures, in case of the Xylariaceae) in HPLC. concordance. Quantitative HPLC profiling, was The data presented here on the so far not used to correlate metabolite Xylariaceae should be viewed in a broader production during the ontogeny of sexual and context, which will be briefly explored at this asexual propagation stages. point. Not only for Xylariaceae, but for fungi in For the chemotaxonomic work on general, it now appears feasible to attain Aspergillus and Penicillium, numerous myco- unprecedented amounts of information that is toxins and antibiotics were readily available as useful for both basic and applied research in standards, due to their importance in agricul- mycology. An integrative assessment of biodi- tural and pharmaceutical research. In contrast, versity, considering both functional and non- our own work involved the de novo isolation functional aspects, now appears feasible for the and structure elucidation of the chemotaxo- first time for the entire fungal kingdom. Some nomically significant metabolites. Among important resources providing information on those were numerous novel natural products. fungi available on the Internet have become Our extensive studies of type material available in the past decade. They have already and other herbarium specimens by HPLC-MS been interlinked by certain Internet portals are also quite unprecedented in fungal (e.g., http://www.gbif.org for global informa- chemotaxonomy. It could not be expected that tion on biodiversity of all organisms; the characteristic marker metabolites may http://www.mycology.net/ for mycological remain stable in herbarium material for such a resources). The scheme in Fig. 19 illustrates long time. how synergistic work on different aspects of An attempt to correlate molecular fungal biology (including microbiology, bio- phylogenetic data with metabolomic studies to technology, physiology, ecology, genetics and identify phylogenetically significant “marker phytopathology) can ideally be accomplished molecules” is also quite unparalleled in by utilising these resources. mycology. Specific websites provide comprehensive Despite the innovative character of this information on certain groups of fungi (see for metabolomic approach, the chemical data the Xylariaceae e.g. http://mycology.sinica. would have little practical value, were it not edu.tw/Xylariaceae, or http://pyrenomycetes. possible to interpret them in a framework that free.fr). They contain online identification keys was elaborated by other mycologists through- and illustrations of macroscopic and micro- out the past centuries. The HPLC profiles were scopic morphological features which will often merely used as a vehicle, e.g., to link allow for concise identification of specimens. morphological evidence obtained in the past to Other websites provide information on biogeo- molecular data from the present, to verify and graphy and chorology. For instance, a good refine taxonomic concepts, and to allow for a overview on Xylariaceae from Ecuador, more concise revision of herbarium specimens. including illustrations and detailed morpholo- Along with concurrent studies on the biological gical descriptions of recently collected spe- activities of the respective secondary metabo- cimens, is provided at (http://www.mycokey. lites, studies on the distribution of the com/Ecuador.html). The websites dedicated to chemotaxonomic marker compounds allowed global biogeographic distribution patterns of for preliminary conclusions on their biological fungi might reflect the state of the art with functions. Other metabolites, which are specifi- respect to flowering plants and certain groups cally overexpressed, such as extracellular of basidiomycetes. However, they have enzymes or volatiles, might have a similar apparently not been updated, according to the importance for the biology of different groups current knowledge on taxonomy and chorology of fungi which do not show such a high diver- of Xylariaceae and many other groups of

117 Integrative (polyphasic) study • Morphological examination of herbarium material • Studies on occurrence and ecology in the field • HPLC and exoenzyme profiling etc. • Biological & biochemical assays • Molecular phylogenetic studies

Documentation & Collaboration Internet resources User groups • Morphology & chorology • Taxonomists & phylogenists • Taxonomy & nomenclature • Ecologists • Molecular data • Plant pathologists • Herbarium specimen catalogues • Physiologists & geneticists • Literature & patents • Biotechnologists & chemists

Fig. 19 – Importance of an integrative approach to fungal taxonomy and biodiversity, to be accomplished by interdisciplinary research: Mycological resources on the Internet and potential user groups for the resulting data.

Ascomycota. Other databases (http://www. catalogues of the international culture collec- indexfungorum.org; http://www.mycobank.org) tions. provide a comprehensive overview of data on Evidently, most of the user groups listed fungal taxonomy and nomenclature. Most in Fig. 19, and especially the taxonomists and mycological journals now request authors of phylogenists among them, are also potential new taxa to register them in these databases. contributors to the above resources. We have Mycobank even provides options to include contributed ourselves by adding specimens, morphological descriptions, pathogenicity, cultures, and data, which can now be utilised host-affinities, biogeographic, molecular, and by other mycologists. A generally accepted other relevant data. The global portal, Index taxonomic system, including well-defined herbariorum (http://sciweb.nybg.org/science2/ species concepts, will provide the prerequisite IndexHerbariorum.asp) contains links to the for communication between the different user websites of most herbaria, culture collections groups. Those who are interested in the and universities, which are relevant for physiological functions and products of the mycologists. Some of their inventories are respective fungal organisms, or who do not available for on-line in search; some have even dispose of molecular identification methods, progressed to add digital images and/or would evidently prefer species concepts, in morphological descriptions of their specimens. which practical aspects are strongly considered. Molecular data, ranging from the results of On the other hand, the availability of molecular genome sequencing projects to phylogene- data has certain advantages, and phylogenetic tically informative rDNA and other sequence aspects should ideally also be taken into data, are available on the Internet in GenBank account. and EMBL (http://www.embl-heidelberg.de). As outlined in the Introduction, the Other Internet websites, like Treebase current concepts in the taxonomy of the (http://www.treebase.org) contain molecular Xylariaceae are based on holomorphic morpho- phylogenetic trees. Even information relating logy. Chemotaxonomic features, including to beneficial and detrimental properties (e.g., stromatal pigment colours and HPLC profiles, production of secondary metabolites, extracel- also constitute characters of the phenotype. As lular enzymes; pathogenicity, etc.) is only alternative, Taylor et al. (2000) compared a occasionally included, e.g. in the online strain theoretical, phylogenetic approach to recognise 118 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 fungal species based on concordance of mul- cultures was regarded as indispensable for the tiple gene genealogies to the classical, practical eventual generation of more comprehensive concepts based on morphology and repro- molecular data (including multi-gene ductive behaviour. Indeed, molecular phylo- genealogies), as well as for studies on their genetic data are becoming increasingly impor- physiology and the metabolism of the tant at different levels of fungal taxonomy. respective species. By culturing as many Multi-gene genealogies, including information representatives and taxa as possible, we from genes encoding for ubiquitously available therefore were able to hit two birds with a proteins (including, e.g., α-actin and β-tubulin), single stone. The availability of concise have recently been favoured over the evalua- molecular data, along with corresponding tion of single genes (e. g, comparison of rDNA cultures, will facilitate future studies on sequence data). Notably, these alternative molecular ecology, e.g., to further elucidate the genes belong to the “housekeeping” category. life cycle and host affinities of endophytic They can from experience only be evaluated in Xylariaceae. Now it appears feasible to cultures or by destroying relatively large compare endophytic cultures of Xylariaceae amounts of specimens. Despite their analyses with their ascospore-derived counterparts that may in some instances provide more con- have similar rDNA sequences for possible clusive information than that of the rDNA, the differences in their physiology and morpho- broad application of such genes as informative logy. characters is out of the question for phylo- In Fig. 20, some data were compiled to genetic studies involving historical type give an overview on the current state of the art specimens, which do often not yield any DNA on biodiversity research of the Xylariaceae, suitable for PCR-based investigations at all. according to the above proposed workflow. For the same reason, molecular studies of Using the numbers of accepted species fide environmental samples, e.g., to detect endo- Kirk et al. (2008), the status has been phytic fungi in planta, should also best be illustrated for some important genera of the accomplished using rDNA primers, since the major subfamilies, as for the Hypoxyloideae rDNA is normally available in larger amounts (excluding the non-stromatic genus Anthosto- than the alternative genes. This poses less of a mella, in which a Nodulisporium type ana- problem if recent material that has been morph has only been identified for some cultured can be linked conclusively to the species, and molecular and chemical data are ancient type specimens; an epitype can then be widely amiss), vs. the non-hypoxyloid Xylaria- designated and the ex-type culture used for the ceae, and the entire family. It becomes evident more extensive molecular studies. immediately that the hypoxyloid genera have Our rationale was therefore to follow a been rather well evaluated, whereas the other rather pragmatic approach, realising that genera need further studies. practical and theoretical species concepts do Whereas the morphological diversity of not necessarily exclude each other. Grant Rosellinia is quite well-studied (Petrini 2003, (2003) commented on the historical struggle Petrini & Petrini 2005), only some of its between cladists and taxonomists, based on pathogenic species have been studied for examples from plant systematics. He concluded secondary metabolites and by molecular that molecular evidence should be combined methodology. For Xylaria, even molecular data with phenetic evidence, “in order to bring are now becoming increasingly available, more characters to bear on the question”. especially on Northern temperate representa- Evidently, this was realised in our study on tives. However, the genus is rather large and Xylariaceae. All species concepts hitherto extremely diverse, above all in the tropics. applied to the Xylariaceae are clearly practical, Many uncertain synonymies have been as they are based on phenotypic recognition. postulated for names in Xylaria that need to be However, in the course of our work, molecular verified by modern methodology, and even the data became increasingly available and were status of the type species remains to be settled. interpreted in conjunction with the phenotype- Likewise, most other genera of Xylarioideae based evidence. The availability of fresh remain to be studied in depth by molecular and

119 Total number of species (Kirk et al. 2008) Only morphological data from literature available Chemotaxonomy/morphology evaluated Cultures/molecular data available Host specificity/chorology evaluated (Europe)

1200

1000

800

600 Numbers oif species (ca.) species Numbersoif

400

200

0

Xylaria Daldinia Rosellinia Hypoxylon

Xylariaceae Total Annulohypoxylon Hypoxyloideae Total Xylarioideae & others Taxa

Fig. 20 – Overview of state of the art on selected genera of the Hypoxyloideae and Xylarioideae in comparison to the entire family Xylariaceae. Species numbers are given according to Kirk et al. (2008). Species numbers for Hypoxyloideae exclude the genus Anthostomella. chemotaxonomic methods. Only a few repre- In the (stromatic) Hypoxyloideae, how- sentatives of the Xylarioideae have already ever, the situation is much different. The been integrated into our HPLC profiling study, majority of their known species have already and even those largely remain to be evaluated been evaluated using the above proposed by molecular methods. In summary, the lack of approach. The taxa insufficiently studied from data on the Xylarioideae strongly accounts for this subfamily are mostly of tropical origin. the fact that an estimated two thirds of the Many of them belong to genera like Camillea currently accepted Xylariaceae species remain and Biscogniauxia that have no stromatal to be studied by our polythetic approach. pigments. As in the Xylarioideae, progress in

120 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 their further study will be highly dependent on in the panel of cultures available. In all the availability of fresh material and cultures. likelihood, their inclusion will in future gra- Due to the work by Y.-M. Ju and J.D. Rogers dually allow for the phenotype-based species and our own efforts, the number of well- concepts to become more congruent to the characterised Xylariaceae strains available in theoretical concept. HPLC profiles of newly public collections has at least doubled in the obtained material will continue to be valuable past decade. However, about 80% of these in establishing its identity with herbarium cultures belong to Annulohypoxylon, Hypoxy- specimens, and resolve the remaining species lon, and Daldinia. Morphological, chemotaxo- complexes. The revision of the herbarium nomic, molecular, and chorological data are inventories will also have to continue, available for many of the representatives of the especially if they have last been revised using above genera. A significant number of the outdated taxonomic concepts, such as that specimens cited in the last monographs (e.g., Ju by Miller (1961) for Hypoxylon species. For & Rogers 1996, Ju et al. 1997, Granmo 1999) resolution of the species complexes that were were already re-examined; several species were tentatively identified by HPLC profiling of cultured for the first time and studied by herbarium specimens, intensive field work and molecular methodology. Extensive chorolo- morphological studies will have to go on, gical data, backed up by morphological, above all in the tropics. molecular and chemical studies are so far The bulk of the chemotaxonomic work, available for over 40 taxa of Xylariaceae, most along with a thorough revision of morpho- of which are frequently encountered in their logical data, has been accomplished either in respective areas of distribution. For an our laboratory or in collaboration with other additional 30 taxa (including ubiquitous mycologists who are the co-authors of the cited species, plant parasites, or “model” species papers. The amount of molecular data contri- such as D. loculata, which were already buted by us in the course of this work presently studied in-depth by other working groups and accounts for less than 10% of the currently provided to us only for chemotaxonomic work published DNA sequences, but this will or as reference material), it can be safely substantially increase once the unpublished assumed from our corroborating data that their data have been released. chorology, morphology, chemotaxonomy and As pointed out by Wheeler (2004), who host specificity is well established by now. reviewed the situation in entomology, it is Most of those are distributed in Europe or the crucial not to neglect the classical morpho- Northern hemisphere. logical-based taxonomy, even in the era of Many of the other currently accepted molecular phylogeny. This certainly also holds species so far studied were not yet cultured at true for fungal biology. Well-trained fungal all, or only one or a few representatives are so morphologists, who are acquainted also with far available. Some of them are known to only microbiological, molecular, and ideally even rarely produce stromata; with some luck they chemical techniques, are desperately needed may soon be shown by molecular methods to even in the future. They are needed to fill the occur among the endophytes. Others are only above gaps, since morphological studies are known from remote areas of the tropics, especially important to generate data on the including regions that were once heavily xylarioid taxa, where chemical traits have so forested and were last collected in the 19th far not proved significant. Hence, it can only be century but are now used for housing or hoped that HPLC profiling of mycelial cultures agriculture. Their natural habitats may have will eventually provide additional valuable meanwhile been destroyed. Even in Central information. Chemotaxonomic data could Europe, there are species like Hypoxylon certainly also become important for characteri- commutatum Nitschke, which are only known sation of numerous other groups of Ascomy- from the type specimen despite they were cota and Basidiomycota which have proved to erected over 150 years ago. be creative secondary metabolite producers. Numerous morphological lineages of the The data matrix obtained and the Xylariaceae are still insufficiently represented methodology developed have meanwhile also

121 become most useful for applied mycology, in The complementarity of secondary meta- particular regarding the design of a library of bolite production in stromata vs. cultures and samples prepared from fermentation of fungal the high dependence of metabolite production cultures and fruiting body extracts for high on the developmental stages also gave strong throughput screening. Of course, it will never impetus for the future design of the natural be possible to do an in-depth characterisation product-derived screening libraries. The fact of all biological sources, prior to their inclusion that the onset of metabolite production is in a screening program aimed at the discovery associated with the morphogenesis of the of novel natural products, but some important anamorph was meanwhile also observed in improvements associated with the progress of other ascomycetes according to unpublished the pilot study on Xylariaceae that substantially results. Despite it has been known for a long helped to increase the probability of success time that mycelial cultures often show different became evident as the work proceeded. Three secondary metabolite profiles than the fruiting of those are mentioned further below. structures, this has never before been demon- As HPLC profiling results also agreed strated for an entire family. It could certainly with molecular phylogenies in other groups of not have been anticipated that no single filamentous fungi (e.g., Stadler et al. 2003) and Xylariaceae species contains the same major sequencing costs have substantially decreased metabolites in cultures and its stromata. in the past decade, it now appears worthwhile As mentioned in the introduction, this to characterise the strains used for screening by thesis was inspired by the work of Samson & molecular techniques prior to the screening Frisvad (2005), and continued the over their random selection. Generation of their chemotaxonomic evaluation by Whalley & 5.8S/ITS nrDNA sequences will give good Edwards (1995) of the Xylariaceae, which had correlations with known taxonomic groups. commenced at a time when no HPLC profiling This allow to assess the potential pathogenicity was possible at all and molecular techniques of unknown strains, and the early identification were still about to be developed. The of duplicates, which often occur among prerequisites to use a similar approach for populations of endophytic or insect pathogenic characterisation of fungi have certainly fungi, can also be recognised easily prior to the improved since then. Numerous other groups screening. Even desired redundancies can be of fungi, known to be rich in secondary revealed. It is possible to find additional metabolites, remain to be explored. I therefore producers of a certain metabolite (or even hope the work presented here will also be producers of additional, new compunds of a suitable to inspire others, and it will in future certain secondary metabolite family), by be possible to employ additional innovative comparing its DNA sequence data with those methodologies, including genomics and proteo- of strains available in public domain databases mics for broad characterisation of many fungal as well as in the in-house repositories. This organisms to further explore their biodiversity. may be helpful to gain further intellectual property. Conclusion The concentrations of secondary metabo- Since this thesis, which constitutes a lites in stromata of most Hypoxyloideae were review of our previous work on the found extremely high. In addition, over 90% of Xylariaceae, was presented for review to the the metabolites of the Hypoxyloideae isolated habilitation committee in 2009, I have been in the course of our work were found to encouraged by several colleagues to publish it constitute novel natural products. These obser- in a public domain journal. In view of the vations gave rise to study additional species recent discussions in the mycological with apparently inconspicuous stromata or community about the value of traditional vs. other types of fruiting bodies, which would molecular data in fungal taxonomy, and otherwise not have been regarded worthwhile especially of the changes in mycological to study because of their small size and nomenclature and taxonomy that have been therefore constitute hitherto untapped potential brought about at the International Botanical sources for novel lead compounds. Congress, Melbourne, 2011, it now appears all

122 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 the more practical to make this thesis available. I am extremely grateful to Prof. Dr. After all, it provides an example on how much Gerhard Rambold (Department Head of work still needs to be done in order to Mycology), for his kind co-operation and understand the taxonomic and phylogenetic manifold valuable advices throughout the past relationships in one of the most diverse and five years, and for allowing me to gain important groups of ascomycota, above all experience with teaching by letting me actively among the taxa from warmer climates and the participate in his mycology courses. Prof. southern hemisphere. Even after over 15 years Rambold and his colleagues in the mentorate, of intensive studies, which have meanwhile Prof. Dr. Konrad Dettner (Department Head of included several thousands of Xylariaceae Animal Ecology II/Chemical Ecology) and specimens, we are still far away from reaching Prof. Dr. Rainer Schobert (Department Head of conclusions on several important matters, Organic Chemistry), are also gratefully relating to the taxonomy and biology of these acknowledged for various helpful discussions fungi. and for having agreed to dedicate their valuable I hope that this paper will encourage time to supervise my activities at Bayreuth others to study other groups of tropical fungi, University. using a polythetic approach. Not only in the I became acquainted with fungi and their Xylariaceae, thousands of species may remain secondary metabolites during my Ph. D. studies to be found and described; hundreds of at Kaiserslautern University and the subsequent previously published names need to be epi- or post-doc project at the university of Lund, neotypified based on comparisons of type Sweden. Thus, my warmest thanks go to Profs. specimens with fresh material that can be used, Drs. (em.) Heidrun and Timm Anke, and Olov e.g., for molecular phylogenetic studies and to Sterner. They strongly influenced my early establish anamorph-teleomorph relationships. scientific career and supported my research in Even many of the known and frequently many ways. Prof. Dr. (em.) Wolfgang Steglich reported taxa do in fact constitute is also thanked for his continuous interest in heterogeneous species complexes, which our work, and for various fruitful discussions. remain to be resolved by a combination of Most of the work described was carried genotype- and phenotype-derived data. It out at the Bayer Research Centre Wuppertal appears all the more urgent to stop the current and (since February 2006) at InterMed trend to undertake premature name changes Discovery GmbH, Dortmund (IMD). I am and erect unnecessary genera based on rather grateful to both companies, especially to Dr. preliminary molecular data and in the absence Thomas Henkel, for giving me the opportunity of corroborating phenotype-derived evidence. to use the lab facilities. The same goes for my Hence, I am very grateful to Kevin D. colleagues at IMD, Bernard Becker and Drs. Hyde, Editor-in-Chief of CREAM for allowing Torsten Grothe and Matthias Gehling, for me to publish this work in an open access supporting me in many ways. Likewise I would mycological journal, for their help in like to thank my co-workers, Andrea transferring the contents of the thesis into an Rademacher, Beata Schmieschek, Petra adequate format for publication. Splittgerber, Claudia Wotsch, Michael Benfer, At the time when this thesis was Marcel Dressel, Dirk Müller, and Stefan Heke presented, the papers by Fournier et al. (2009) for expert technical assistance, and Manuela and Stadler et al. (2010a) had still not been Briegert, Marie Merrild and Manuela Kuhnert accepted, and the more recent studies on for their excellent performance during their Ruwenzoria and Rhopalostroma (Stadler et al. student projects, B.Sc. or M. Sc. theses on the 2010b,c) as well as the papers by Fournier et Xylariaceae in our laboratory. al. (2010, 2011) on Hypoxylon and Xylaria, and Prof. Drs. Yoshinori Asakawa, Veronika the paper by Læssøe et al. (2010) had not even Hellwig and Toshihiro Hashimoto and Drs. been submitted. Andrea Mühlbauer, Dang Ngoc Quang, and Jens Bitzer spent many hours to identify the Acknowledgments numerous novel natural products published in the cited papers by careful interpretation of

123 their spectral data. Dr. Bärbel Köpcke is Hartmund Wollweber and especially Jacques thanked for her efforts to build up the HPLC- Fournier, have been most instrumental in MS database of Xylariaceae metabolites, which developing the concepts presented here. I hope facilitated substantially the concise I will be able to rely on their aid for many identification of characteristic metabolites in years to come. trace amounts from the crude extracts. Thanks are also due to Profs. Jens C. Among the co-authors of the presented Frisvad, David L. Hawksworth, Dr. Gerald F. papers and my collaborators in other studies Bills, and countless other colleagues who have that are not yet published, are numerous heavily influenced my understanding of fungal mycologists to whom I am also deeply biology, phylogeny and taxonomy and thus the indebted. Profs. Drs. Jack D. Rogers (WSP) concepts brought forward in this thesis. Thomas Læssøe and Meike Piepenbring, Drs. Yu-Ming Ju and Alexander Akulov (CWU), References Juan M. Castro, Christian Lechat, Drs. Volker Kummer, Alfred Granmo, Larissa Vasilyeva Abate D, Abraham WR, Meyer H. 1997 − (VLA), Cony Decock (MUCL), Liliane E. Cytochalasins and phytotoxins from Petrini, Profs. Drs. Giuseppe Venturella (PAL) Xylaria obovata. Phytochemistry 44, and Anthony J. S. Whalley, Drs. Sylvie 1443−1448. Pažoutová, Clovis Douanla-Meli, Dieter Amnuaykanjanasin A, Punya J, Paungmoung Benkert and Norbert Amelang, Harry P, Rungrod A, Tachaleat A, Pongpat- Andersson, Prof. Dr. Hanns Kreisel, and Peter tanakitshote S, Cheevadhanarak S, Rönsch are thanked for sending material from Tanticharoen M. 2005 − Diversity of type their personal herbaria. The curators of public I polyketide synthase genes in the herbaria from all over the world - only to wood−decay fungus Xylaria sp. BCC mention here: Drs. Burghard Hein and Harrie 1067. FEMS Microbiology Letters 251, Sipman (B), Erin McCray (BPI), Drs. André 125−136. Fraiture (BR) and Robert Lücking (F), Arnold AE, Maynard Z, Gilbert GS. 2001 − Genevieve Lewis-Gentry (FH), Profs. Drs. Fungal endophytes in dicotyledonous Richard Hanlin (GAM) and Mieke Verbeken neotropical trees: patterns of abundance (GENT), Drs. Herbert Boyle (GLM), Deborah and diversity. Mycological Research 105, Lewis (ISC), Begoña Aguirre-Hudson and 1502−1507. Brian Spooner (K), Markus Scholler (KR), and Arnold AE, Mejía L, Kyllo D, Rojas E, Gerard Thijsse (L), Prof. Dr. Régis Maynard Z, Herre EA, 2003 − Fungal Courtecuisse (LIP), Drs. Vilma Rosato (LPS), endophytes limit pathogen damage in a Dagmar Triebel (M), Margarita Dueñas (MA), tropical tree. Proceedings of the National Ellen Bloch (NY), Drs. John Haines (NYS), Academy of Science USA 100, Rosella Marcuccci (PAD), James Lendemer 15649−15654. (PH), Joélle Dupont (PC), Eric McKenzie Barr ME, Rogers JD, Ju YM. 1993 − (PDD), and Jan Holeć (PRM), Anna-Lena Revisionary studies in the Calosphaeria- Anderberg (S), Drs. Esperanza Beltrán (TFC), les. Mycotaxon 48, 529−533. Geir Mathiassen (TROM), and Svengunnar Baumgartner M. 2001 − Morphologische, Ryman (UPS), Prof. Dr. Egon Horak (ZT), and chemotaxonomische und genetische many other colleagues and institutions have Untersuchungen bei Daldinia Ces & De kindly provided specimens. This list is by far Not. (Xylariaceae). Diplomarbeit (MSc not complete, and they all deserve my warmest Thesis) Friedrich Schiller−Universität thanks. I am also grateful to the curators of the Jena, Germany. culture collections CBS and MUCL for Bitzer J, Köpcke B, Stadler M, Hellwig V, Ju preserving well the cultures we obtained and YM, Seip S, Henkel T. 2007 − for provision of reference strains. Accelerated dereplication of natural pro- Out of many mycologists who are ducts, supported by reference libraries. involved in these studies, Drs. Jack D. Rogers, Chimia 51, 332−338. Yu-Ming Ju, Derek Peršoh, Thomas Læssøe,

124 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 Bitzer J, Læssøe T, Fournier J, Kummer V, Hypoxylon fragiforme: mechanisms in Decock C, Tichy HV, Piepenbring M, the early recognition and establishment Peršoh D, Stadler M. 2008 − Affinities of an endophytic symbiosis. Mycological of Phylacia and the daldinoid Xylaria- Research 97, 157−162. ceae, inferred from chemotypes of cul- Chesters CGC, Greenhalgh GN. 1964 − tures and ribosomal DNA sequences. Geniculosporium serpens gen. et sp. Mycological Research 112, 251−270. nov., the imperfect state of Hypoxylon Boddy L, Griffith GS. 1989 − Role of serpens. Transactions of the British endophytes and latent invasion in the Mycological Society 47, 393−401. development of decay communities in Child M. 1932 − The genus Daldinia. Annals sapwood of angiospermous trees. Sydo- of the Missouri Botanical Garden 19, wia 41, 41−73. 429−496. Boddy L, Rayner ADM. 1983 − Origins of Collado J, Platas G, Peláez F. 2001 − decay in living deciduous trees: the role Identification of an endophytic Nodu- of moisture content and a re-appraisal of lisporium sp. from Quercus ilex in central the expanded concept of tree decay. New Spain as the anamorph of Biscogniauxia Phytologist 94, 623−641. mediterranea by rDNA sequence analy- Boddy L, Gibbon OM, Grundy MA. 1985 − sis and effect of different ecological Ecology of Daldinia concentrica: Effect factors on distribution of the fungus. of abiotic variables on mycelial extension Mycologia 93, 875−886. and interspecific interactions. Transac- Dargan JS, Thind KS. 1984 − Xylariaceae of tions of the British Mycological Society India. VIII. Genus Daldinia Ces. & De 85, 201−211. Not. – a further segregation into two new Bodo B, Tih RG, Davoust D, Jacquemin H. subgenera. Kavaka 12, 113−110. 1983 − Hypoxylone, a naphthyl- Davis EC, Shaw AJ. 2008 − Biogeographic and naphthoquinone pigment from the fungus phylogenetic patterns in diversity of Hypoxylon sclerophaeum. Phytochemis- liverwort-associated endophytes Ameri- try 22, 2579−2581. can Journal of Botany 95, 914−924. Bolton J. 1789 − An History of Davis EC, Franklin JB, Shaw JA, Vilgalys R. Growing About Halifax III. Hud- 2003 − Endophytic Xylaria (Xylariaceae) dersfield. 93−138. among liverworts and angiosperms: Carroll G. 1988 − Fungal endophytes in stems phylogenetics, distribution, and symbio- and leaves: from latent pathogen to sis. American Journal of Botany 90, mutualistic symbiont. Ecology 69, 2−9. 1661−1667. Cesati V, De Notaris G. 1863 − Schema di Dennis RWG. 1959 − Further notes on tropical classificazione degli Sferiacei italici American Xylariaceae. Kew Bulletin 12, aschigeri più o meno appertenenti al 297–332. genere Sphaeria nell’ antico significato Dennis RWG. 1961 − Xylarioideae and attribuitogli da Persoon. Commentario Thamnomycetoideae of Congo. Bulletin della Societa Crittogamologica Italiana 1, du Jardin Botanique de l'État, Bruxelles 177−240. 31, 109–154. Chacko RJ, Rogers JD. 1981 − Cultural Dennis RWG. 1963 − Hypoxyloideae of characteristics of some species of Congo. Bulletin du Jardin Botanique de Xylaria. Mycologia 73, 415−428. l'État, Bruxelles 33, 317−343. Chapela IH, Petrini O, Petrini LE. 1990 − Dennis RWG. 1964 − Further records of Congo Unusual ascospore germination in Xylariaceae. Bulletin du Jardin Botani- Hypoxylon fragiforme: first steps in que de l'État, Bruxelles 34, 231−241. establishment of an endophytic symbio- Edwards RK, Jonglaekha N, Kshirsagar A, sis. Canadian Journal of Botany 68, Maitland DJ, Mekkamol S, Nugent LK, 2571−2575. Poshri C, Rodtong S, Ruchichachorn S, Chapela IH, Petrini O, Bielser G. 1993 − The Sangvichien E, Sharples GP, Sihanonth physiology of ascospore eclosion in P, Suwannasai N, Thienhirun S, Whalley

125 AJS, Whalley MA. 2003 − The Guidot A, Johannesson H, Dahlberg A, Stenlid Xylariaceae as phytopathogens. Recent J. 2003 − Parental tracking in the postfire Research Development in Plant Science wood decay ascomycete Daldinia 1, 1−19. loculata using highly variable nuclear Espada A, Rivera−Sagredo A, de la Fuente JM, gene loci. Molecular Ecology 12, Hueso−Rodríguez JA, Elson SW. 1997 − 1717−1730. New cytochalasins from the fungus Gunawan S, Steffan B, Steglich W. 1990 − Xylaria hypoxylon. Tetrahedron 53, Xylaral, a hydroxyphthalide derivative 6485−6492. from fruiting bodies of Xylaria polymor- Fournier J, Stadler M, Hyde KD, Duong LM. pha (Ascomycetes). Liebigs Annalen der 2009 − The new genus Rostrohypoxylon Chemie 8, 825−827. and two new Annulohypoxylon species Guo LD, Hyde KD, Liew ECY. 2000 − from Northern Thailand. Fungal Diver- Identification of endophytic fungi from sity 40, 23–36. Livistona chinensis (Palmae) using Fournier J, Köpcke B, Stadler M. 2010 − New morphological and molecular techniques. species of Hypoxylon from Western New Phytologist 147, 617−630. Europe and Ethiopia. Mycotaxon 113, Guo LD, Hyde KD, Liew ECY. 2001 − 209–235. Detection and identification of endo- Fournier J, Flessa F, Peršoh D, Stadler M. 2011 phytic fungi within frond tissues of −Three new Xylaria species from South- Livistona chinensis based on rDNA western Europe. Mycologcal Progress 10, sequence. Molecular Phylogeny and 33−52. Evolution 20, 1−13. Fries EM. 1823 − Systema Mycologicum. II. Hashimoto T, Asakawa Y. 1998 − Biologically Lundae. active substances of Japanese inedible Frisvad JC, Andersen B; Thrane U. 2009 − The mushrooms. Heterocycles 47, 1110– use of secondary metabolite profiling in 1121. chemotaxonomy of filamentous fungi. Hawksworth DL. 1977 − Rhopalostroma, a Mycological Research 112, 231−240. new genus in the Xylariaceae s.l. Kew Granmo A 1999 − Morphotaxonomy and Bulletin 31, 421–431. chorology of the genus Hypoxylon Hawksworth DL. 1991 − The fungal dimension (Xylariaceae) in Norway. Sommerfeltia of biodiversity, magnitude, significance, 26, 1−81. and conservation. Mycological Research Granmo A. 2001 − A new species of 95, 641−655. Hypoxylon (Xylariaceae). Sydowia 54, Hawksworth DL, Whalley AJS. 1985 − A new 44−52. species of Rhopalostroma with a Noduli- Granmo A, Læssøe T, Schumacher T. 1999 −. sporium anamorph from Thailand. Tran- The genus Nemania s.l. (Xylariaceae) in sactions of the British Mycological Norden. Sommerfeltia 27, 1−96. Society 84, 560–562. Grant V. 2003 − Incongruence between Hellwig V, Ju YM, Rogers JD, Fournier J, cladistic and taxonomic systems. Ameri- Stadler M. 2005 − Hypomiltin, a novel can Journa of Botany 90, 1263–1270. azaphilone from Hypoxylon hypomiltum, Greenhalgh GN, Chesters CGC. 1968 − and chemotypes in Hypoxylon sect. Conidiophore morphology in some Hypoxylon as inferred from analytical British members of the Xylariaceae. HPLC profiling. Mycological Progress 4, Transactions of the British Mycological 39–54. Society 51, 57−82. Hingley MP. 1971 − The ascomycete fungus, Griffith GS, Boddy L. 1990 − Fungal Daldinia concentrica as a habitat for decomposition of attached angiosperm animals. Journal of Animal Ecology 40, twigs I. Decay community development 17−32. in ash, beech and oak. New Phytologist Hoffmeister D, Keller NP. 2007 − Natural 116, 407−415. products of filamentous fungi: enzymes,

126 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 genes and their regulation. Natural Ju YM, Vasilyeva L, Rogers JD. 1999 − Product Reports 24, 393−416. Daldinia singularis, sp. nov. from eastern Hsieh HM, Ju YM, Rogers JD. 2005 − Russia, and notes on some other taxa. Molecular phylogeny of Hypoxylon and Mycotaxon 71, 405−412. closely related genera. Mycologia 97, Ju YM, Rogers JD, Hsieh HM. 2004 − New 914–923. Hypoxylon species and notes on some Hyde KD, Soytong K. 2008 − The endophyte names associated with or related to dilemma. Fungal Diversity 33, 163−173. Hypoxylon. Mycologia 96, 154−161. Ireland C, Peekhaus N, Lu P, Sangari R, Zhang Ju YM, Hsieh HM, Ho MC, Szu DH, Fang MJ. A, Masurekar P, An Z. 2008 − The 2007 − Theissenia rogersii sp. nov. and tryptophan synthetase gene TRP1 of phylogenetic position of Theissenia. Nodulisporium sp.: molecular charac- Mycologia 99, 612−621. terization and its relation to nodulisporic Kirk PF, Cannon PF, Minter DW, Stalpers JA. acid A production. Applied Microbiology 2008 − Dictionary of the Fungi, 10th edn. and Biotechnology 79, 451−459. CABI Publishing, Egham, UK. Johannesson H. 2001 − Ecology of Daldinia Læssøe T. 1994 − Index ascomycetum 1. spp. with special emphasis on Daldinia Xylariaceae. Systema Ascomycetum 13, loculata. Ph. D. thesis. Swedish Univer- 43–112. sity of Agricultural Sciences, Uppsala, Laessøe T, Spooner BM 1994 − Rosellinia & Sweden. Astrocystis (Xylariaceae): New species Johannesson H, Laessøe T, Stenlid J. 2000a − and generic concepts. Kew Bulletin 49, Molecular and morphological investiga- 1−70. tion of the genus Daldinia in Northern Læssøe T, Rogers JD, Whalley AJS. 1989 − Europe. Mycological Research 104, Camillea, Jongiella and light−spored 275−280. species of Hypoxylon. Mycological Johannesson H, Johannesson H, Stenlid J. Research 93, 121−155. 2000b − Development of a set of PCR Læssøe T, Srikitikulchai P, Fournier J, Köpcke primers to amplify conserved genes of B, Stadler M (2010) Lepraric acid Daldinia loculata for use in population derivatives as chemotaxonomic markers studies. Molecular Ecology 9, 375−378. in Hypoxylon aeruginosum, Chloro- Jong SC, Rogers JD. 1972 −. Illustrations and stroma subcubisporum and C. cyaninum, descriptions of conidial states of some sp. nov. Fungal Biology 114, 481−489. Hypoxylon species. Washington State Larsen TO, Smedsgaard J, Nielsen KF, Hansen Agricultural Experimantal Station Bulle- ME, Frisvad JC. 2005 − Phenotypic tin, Vol. 71. taxonomy and metabolite profiling in Ju,YM, Rogers JD. 1994 − Kretzschmariella microbial drug discovery. Natural Pro- culmorum (Cooke) comb. nov. and notes duct Reports 22, 672–695. on some other monocot-inhabiting xyla- Lee JSS, Kwan K, Jung HS. 2000 − riaceous fungi. Mycotaxon 51, 241−255. Phylogenetic analysis of Xylaria based Ju YM, Rogers JD 1996 − A Revision of the on nuclear ribosomal ITS–5.8S–ITS2 Genus Hypoxylon [Mycologia Memoir sequences. FEMS Microbiology Letters no. 20]. APS Press, St Paul. 187, 89−93. Ju YM, Rogers JD, San Martín F. 1997 − A Linnaeus C. 1753 − Species plantarum, revision of the genus Daldinia. Myco- exhibentes plantas rite cognitas, ad taxon 61, 243–293. genera relatas, cum differentiis specificis, Ju YM, Rogers JD, San Martín F, Granmo A. nominibus trivialibus, synonymis selec- 1998 − The genus Biscogniauxia. Myco- tis, locis natalibus, secundem systema taxon 66, 1−98. sexuale digestas. Salvius, Stockholm, Ju YM, San Martín F, Rogers JD. 1993 − Three 1182. xylariaceous fungi with scolecosporous Luchi N, Capretti P, Pinzani P, Orlando C, conidia. Mycotaxon 47, 219−228. Pazzagli M. 2005 − Real−time PCR detection of Biscogniauxia mediterranea

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128 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 Hypoxylon−Arten (Xylariaceae, ascomycete Daldinia concentrica. Jour- Sphaeriales) und verwandter Pilze. nal of Natural Products 65, 1869−1874. Mycologia Helvetica 1, 501−627. Quang DN, Hashimoto T, Tanaka M, Stadler Petrini LE, Petrini O. 2005 − Morphological M, Asakawa Y. 2004a − Constituents of studies in Rosellinia (Xylariaceae): the the inedible mushroom Hypoxylon first step towards a polyphasic taxonomy. rubiginosum. Journal of Natural Products Mycological Research 109, 569−580. 67, 1152−1155. Petrini LE, Petrini O, Sieber TN. 1987 − Host Quang DN, Hashimoto T, Tanaka M, Stadler specificity of Hypoxylon fuscum: A M, Asakawa Y. 2004b − Cyclic azaphi- statistical approach to the problem. lones daldinins E and F from the Sydowia 40, 227−234. ascomycete fungus Hypoxylon fuscum Petrini O, Petrini LE, Rodrigues K. 1995 − (Xylariaceae). Phytochemistry 65, Xylariaceous endophytes: an exercise in 469−473. biodiversity. Fitopatologica Brasiliensis Quang DN, Hashimoto T, Radulović N, Stadler 20, 531−539. M, Asakawa Y. 2005a − Antimicrobial Pirozynski KA. 1974 − Xenotypa Petrak and azaphilones from the xylariaceous inedi- Graphostroma gen. nov., segregates from ble mushrooms. International Journal of Diatrypaceae. Canadian Journal of Medicinal Mushrooms 7, 452−455. Botany 52, 2129−2135. Quang DN, Hashimoto T, Stadler M, Asakawa Pointing SB, Parungao MM, Hyde KD. 2003 − Y. 2005b − Dimeric azaphilones from the Production of wood−decay enzymes, xylariaceous ascomycete Hypoxylon mass loss and lignin solubilization in rutilum. Tetrahedron 61,8451−8455. wood by tropical Xylariaceae. Quang, DN, Hashimoto T, Nomura Y, Mycological Research 107, 231−235. Wollweber H, Hellwig V, Fournier J, Pointing SB, Pelling AL, Smith GJ, Hyde KD, Stadler M, Asakawa Y. 2005c – Cohae- Reddy CA. 2005 − Screening of rins A and B, azaphilones from the basidiomycetes and xylariaceous fungi fungus Hypoxylon cohaerens, and com- for lignin peroxidase and laccase parison of HPLC−based metabolite gene−specific sequences. Mycological profiles in Hypoxylon sect. Annulata. Research 109, 115−24. Phytochemistry 65, 797−809. Polishook JD, Ondeyka JG, Dobrowski, AW, Quang, DN, Hashimoto T, Radulovic N, Peláez F, Platas G, Teran AM. 2001 − Fournier J, Stadler M, Asakawa Y. 2005d Biogeography and relatedness of − Sassafrins A−D, new antimicrobial Nodulisporium strains producing azaphilones from the fungus Creo- nodulisporic acid. Mycologia 93, 1125– sphaeria sassafras. Tetrahedron 61, 1137. 1743−1748. Pouzar Z. 1985a − Reassessment of Hypoxylon Quang DN, Hashimoto T, Stadler M, serpens − complex I. Ceská Mykologia Radulovic N, Asakawa Y. 2005e – 39,15−25. Antimicrobial azaphilones from the fun- Pouzar Z. 1985b − Reassessment of the gus Hypoxylon multiforme. Planta Hypoxylon serpens − complex II. Ceská Medica 71, 1058−1072. Mykologia 39,129−134. Quang DN, Stadler M, Fournier J, Asakawa Y. Quang DN, Hashimoto T, Tanaka M, 2006a − Carneic acids A and B, two Baumgartner M, Stadler M, Asakawa Y. chemotaxonomically significant antimi- 2002a − Concentricols B, C and D, three crobial agents from the xylariaceous novel squalene−type triterpenoids from ascomycete, Hypoxylon carneum. Journal the ascomycete Daldinia concentrica. of Natural. Products 69, 1198−1202. Phytochemistry 61, 345−353. Quang DN, Stadler M, Fournier J, Tomita A, Quang DN, Hashimoto T, Tanaka M, Hashimoto T. 2006b − Cohaerins C−F, Baumgartner M, Stadler M, Asakawa Y. four azaphilones from the xylariaceous 2002b − Chemical constituents of the fungus Annulohypoxylon cohaerens. Tetrahedron 62, 6349−6354.

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130 Current Research in Environmental & Applied Mycology Doi 10.5943/cream/1/2/1 vista Iberoamericana de Micologia 23, holomorphic morphology, HPLC pro- 160−170. files, and ribosomal DNA sequences. Stadler M, Hellwig V. 2004 − PCR−based data Mycological Progress 7, 53−73. and secondary metabolites as chemo- Stadler M, Fournier J, Læssøe T, Chlebicki A, taxonomic markers in HTS for bioactive Lechat C, Flessa F, Peršoh D, Rambold compounds from fungi. In: Handbook of G. 2010a − Chemotaxonomic and phylo- Industrial Mycology (ed. Z An), Marcel genetic studies of Thamnomyces (Xyla- Dekker, New York, 269−307. riaceae). Mycoscience 51, 189−207. Stadler M, Hellwig V. 2005 − Chemotaxonomy Stadler M, Fournier J, Læssøe T, Chlebicki A, of the Xylariaceae and remarkable bio- Lechat C, Flessa F, Peršoh D, Rambold active compounds from Xylariales and G. 2010d − Chemotaxonomic and their associated asexual stages. Recent phylogenetic studies of Thamnomyces Resarch and Development in Phytoche- (Xylariaceae). Mycoscience 51, mistry 9, 41−93. 189−207. Stadler M, Baumgartner M, Grothe T, Mühl- Stadler M, Fournier J, Læssøe T, Decock C, bauer A, Seip S, Wollweber H. 2001a − Peršoh D, Rambold G. 2010c − Concentricol, a taxonomically significant Ruwenzoria, a new genus of the Xylaria- triterpenoid from Daldinia concentrica. ceae from Central Africa. Mycological Phytochemistry 56, 787−793. Progress 9,169–179. Stadler M, Baumgartner M, Wollweber H. Stadler M, Læssøe T, Simpson JA, Wollweber 2001b − Three new Daldinia species H. 2004b − A survey of Daldinia species with yellow stromatal pigments. Myco- with large ascospores. Mycological Re- taxon 80, 179−196. search 108, 1025−1041. Stadler M, Baumgartner M, Wollweber H, Stadler M, Læssøe T, Vasilyeva L. 2005 − The Rogers JD, Ju YM. 2001c − Daldinia genus Pyrenomyxa and its affinities to decipiens sp. nov. and notes on some other cleistocarpous Hypoxyloideae as other European Daldinia spp. inhabiting inferred from morphological and chemi- Betulaceae. Mycotaxon 80, 167−177. cal traits. Mycologia 97, 1129−1139. Stadler M, Baumgartner M, Ide K, Popp A, Stadler M, Henkel T, Müller H, Weber K, Wollweber H. 2002 − Importance of Schlecker H. 1998 − Identification of ascospore ornamentation in the taxonomy alkaloids and polyketides in an actinomy- of Daldinia. Mycological Progress 1, cete by a combination of analytical and 31−42. preparative HPLC and spectroscopic me- Stadler M, Fournier J, Beltrán−Tejera E, thods. Journal of Chromatography 818A, Granmo A. 2008b − The “red Hypoxy- 187−195. lons” of the Northern Hemisphere. In A Stadler M, Ju YM, Rogers JD. 2004a − Festschrift In Honor Of Professor Jack D. Chemotaxonomy of Entonaema, Rhopa- Rogers (eds DA Glawe, JF Ammirati). lostroma and other Xylariaceae. Mycolo- North American Fungi 3, 73−125. gical Research 108, 239−256. Stadler M, Fournier J, Gardt S, Peršoh D. Stadler M, Quang DN, Tomita A, Hashimoto 2010b − The phylogenetic position of T, Asakawa Y. 2006 − Production of Rhopalostroma as inferred from a poly- bioactive metabolites during stromatal thetic approach. Persoonia 25,11−21. ontogeny of Hypoxylon fragiforme. Stadler M, Fournier J, Quang DN, Akulov A. Mycological Research 110, 811−820. 2007 − Metabolomic studies on the Stadler M, Tichy HV, Katsiou E, Hellwig V. chemical ecology of the Xylariaceae 2003 − Chemotaxonomy of Pochonia (Ascomycota). Natural Products Commu- and other conidial fungi with Verticil- nications 2, 287−304. lium-like anamorphs. Mycological Pro- Stadler M, Fournier J, Læssøe T, Lechat C, gress 2, 95−122. Tichy HV, Piepenbring M. 2008a − Stadler M, Wollweber H, Fournier J. 2004c − Recognition of hypoxyloid and xylarioid A host-specific species of Hypoxylon Entonaema species from a comparison of from France, and notes on the chemo-

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