Resurrection and emendation of the Hypoxylaceae,recognised from a multigene phylogeny of the Xylariales

A multigene phylogeny was constructed, including a significant number of representative species of the main lineages in the Xylariaceae and four DNA loci the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, the second largest subunit of the RNA polymerase II (RPB2), and beta-tubulin (TUB2). Specimens were selected based on more than a decade of intensive morphological and chemotaxonomic work, and cautious taxon sampling was performed to cover the major lineages of the Xylariaceae; however, with emphasis on hypoxyloid species. The comprehensive phylogenetic analysis revealed a clear-cut segregation of the Xylariaceae into several major clades, which was well in accordance with previously established morphological and chemotaxonomic concepts. One of these clades contained Annulohypoxylon, Hypoxylon, Daldinia, and other related genera that have stromatal pigments and a nodulisporium-like anamorph. They are accommodated in the family Hypoxylaceae, which is resurrected and emended. Representatives of genera with a nodulisporium-like anamorph and bipartite stromata, lacking stromatal pigments (i.e. Biscogniauxia, Camillea, and Obolarina) appeared in a clade basal to the xylarioid taxa. As they clustered with Graphostroma platystomum, they are accommodated in the Graphostromataceae. The new genus Jackrogersella with J. multiformis as type species is segregated from Annulohypoxylon. The genus Pyrenopolyporus is resurrected for Hypoxylon polyporus and allied species. The genus Daldinia and its allies Entonaema, Rhopalostroma, Ruwenzoria, and Thamnomyces appeared in two separate subclades, which may warrant further splitting of Daldinia in the future, and even Hypoxylon was divided in several clades. However, more species of these genera need to be studied before a conclusive taxonomic rearrangement can be envisaged. Epitypes were designated for several important species in which living cultures and molecular data are available, in order to stabilise the taxonomy of the Xylariales.     

Recognition of hypoxyloid and xylarioid <Emphasis Type="Italic">Entonaema</Emphasis> species and allied <Emphasis Type="Italic">Xylaria</Emphasis> species from a comparison of holomorphic morphology,HPLC profiles,and ribosomal DNA sequences

The genus Entonaema comprises Xylariaceae with hollow, gelatinous stromata that accumulate liquid. Some of its species, including the type species, appear related to Daldinia from a polyphasic approach, comprising morphological studies, comparisons of ribosomal DNA sequences, and high performance liquid chromatography (HPLC) profiles with diode array and mass spectrometric detection (HPLC-DAD-MS). This methodology was used to study Entonaema pallidum. Its major stromatal constituent was identified as xylaral, a secondary metabolite known from Xylaria polymorpha. This compound was detected in several Xylaria spp., including the tropical X. telfairii and morphologically similar taxa, whose stromata may also become hollow and filled with liquid. Cultures of E. pallidum resembled those of Xylaria, substantially differing from other Entonaema spp., in their morphology, 5.8S/ITS nrDNA sequences, and HPLC profiles. The type specimen of E. mesentericum was located in the spirit collection of the herbarium B and found to agree morphologically with the nomenclatorily younger E. pallidum. Traces of xylaral were even detected by HPLC-DAD-MS in the spirit in which the fungus had been preserved. Entonaema pallidum is thus regarded as a later synonym of E. mesentericum. Therefore, the latter name is transferred to Xylaria. A key to entonaemoid Xylariaceae is provided. Colour reactions (NH₃, KOH) of the ectostroma were applied to a limited number of Xylaria spp., but metabolite profiles of cultures appear more promising as chemotaxonomic traits to segregate this genus. As xylaral was also found in Nemania and Stilbohypoxylon spp., while being apparently absent in Hypoxylon and allied genera, it may be a chemotaxonomic marker for Xylariaceae with Geniculosporium-like anamorphs.     

Macrocarpones,novel metabolites from stromata of <Emphasis Type="Italic">Hypoxylon macrocarpum</Emphasis>, and new evidence on the chemotaxonomy of <Emphasis Type="Italic">Hypoxylon</Emphasis> species

Several species of the genus Hypoxylon (Xylariaceae) were studied for morphological characters and HPLC-based secondary metabolite profiles. It was confirmed that Hypoxylon is divided into two groups of species, containing either Mitorubrin type azaphilones or binaphthyls, respectively, as main metabolites. In Hypoxylon species of the latter group, some metabolites that are known from the allied genus Daldinia (i.e., Daldinal A in H. fuscum and Daldinin C in H. fuscopurpureum), were encountered for the first time. Moreover, three novel aromatic polyketides, for which the trivial names Macrocarpones A, B and C are proposed, were isolated from ascostromata of Hypoxylon macrocarpum and identified by high resolution mass spectrometry and 2D-NMR spectroscopy. Orsellinic Acid was also identified from ascostromata of H. howeianum and detected by HPLC-MS and HPLC-UV/Vis fingerprinting methodology in H. fragiforme and H. rubiginosum, but not in other examined Hypoxylon species. Due to these studies, H. fuscopurpureum sensu Y.-M. Ju & J.D. Rogers 1996 was identified as a new record for Germany.     

Importance of ascospore ornamentation in the taxonomy of Daldinia

Representative specimens of fifteen Daldinia spp. were studied for ultrastructural characteristics of their ascospores by scanning electron microscopy (SEM). The ornamentation of their outermost spore layers was found to be species-consistent, confirming the results of concurrent studies on the morphology of their teleomorphs and anamorphs, secondary metabolite profiles and PCR-based genetic fingerprints. Daldinia spp. may either show smooth or transversally striated ascospores. The spores of the species within the latter group are always ellipsoid-equilateral to ellipsoid-inequilateral with narrowly rounded ends. Smooth, broadly ellipsoid to cylindrical ascospores were observed in all species (D. caldariorum, D. fissa and D. loculata) that are known to produce their stromata on substrates damaged by fire. The ascospores of D. concentrica differed from those of D. childiae (i.e., the cosmopolitan taxon previously regarded as D. concentrica ss. auct.) and other Daldinia spp. in showing a very faint ornamentation, which only became visible at 10000× magnification by SEM. A specimen collected on the isle of Jersey (Channel Islands, UK) showed morphological similarities to the pantropical D. eschscholzii, but its ascospores appeared smooth by SEM, and it may therefore represent a previously undescribed species. Dedicated to Professor Yoshinori Asakawa, Tokushima, Japan, on the occasion of his 60^th birthday PH-R Life Science Center Natural Products     

Affinities of Phylacia and the daldinoid Xylariaceae, inferred from chemotypes of cultures and ribosomal DNA sequences

A chemotaxonomic evaluation using hplc profiling was undertaken to resolve the infrageneric and intergeneric affinities of over 150 strains of Xylariaceae. Daldinia placentiformis, Hypoxylon nicaraguense, H. polyporus, and Phylacia sagrana were found to contain 8-methoxy-1-naphthol, which is apparently absent in Annulohypoxylon, Hypoxylon, and related genera with bipartite stromata. D. placentiformis and other species of Daldinia and Entonaema produced this naphthol, 5-hydroxy-2-methylchromone, isosclerone derivatives, and ‘AB-5046’ phytotoxins. Phylacia sagrana differed from most Daldinia spp., except for D. caldariorum, by producing eutypine derivatives in addition to the above compounds. Indolylquinones were observed in H. nicaraguense and H. polyporus. Isosclerones were also identified in the A. multiforme complex, but Hypoxylon and other Annulohypoxylon and most Hypoxylon spp. studied Annulohypoxylon spp. contained 5-methylmellein as the major metabolite of their cultures. Based on the occurrence of the above metabolites, further mellein-type dihydroisocoumarins, teleomorphic and anamorphic Xylariaceae with Nodulisporium-like anamorphs (‘Hypoxyloideae’) were divided into various chemotypes. A comparison of their 5.8S/ITS nuc-rDNA sequences agreed in some important aspects with the above results: H. nicaraguense and H. polyporus appeared basal to a clade comprising Daldinia, Entonaema, and Ph. sagrana. The latter species appeared allied to D. caldariorum, but was distantly related to Pyrenomyxa morganii and Hypoxylon s. str.     

Taxonomy of an anamorphic xylariaceous fungus from a termite nest found together with <Emphasis Type="Italic">Xylaria angulosa</Emphasis>

Two xylariaceous fungi were isolated from a nest of a termite, Odontotermes formosanus, that was incubated in a laboratory after collecting from Iriomote Is., Okinawa Pref., in Japan. One of the two fungi was identified as Xylaria angulosa on the basis of the morphology of branched stroma produced on medium, tiny asci, and ascospores having a germ slit. Another fungus is an anamorphic fungus that produces synnemata up to 50 mm long from which dendritic conidiophores branch out. Unicellular conidia are holoblastically produced on a sympodially proliferating conidiogenous cell. Such morphological characters resemble those of the genus Geniculosporium. However, its distinctive synnema formation and dendritic conidiophores do not assign the fungus to Geniculosporium or other known genera and warrant establishment of a new genus. The phylogenetic tree based on the ITS regions of rDNA shows that the fungus is nested in the cluster of the genus Nemania (Xylariaceae), whose species have mainly Geniculosporium-like anamorphs. We describe here the present anamorphic fungus as Geniculisynnema termiticola gen. et sp. nov., and discuss its phylogenetic and ecological relationships to xylariaceous fungi, especially termiticolous species.     

Bipolar heterothallism,a principal mating system of<Emphasis Type="Italic">Cordyceps militaris in vitro</Emphasis>

Interest inin vitro study of entomopathogenic fungi, includingCordyceps species, has been increasing due to their valuable bioactive compounds and biocontrol effects. AmongCordyceps species,in vitro stromata ofC. militaris has been successfully produced and cultivated for industrial purposes. However, genetic study onin vitro stromata formation ofC. militaris has not been carried out yet. Here, relationship between mating system and perithecial stromata formation ofC. militaris is reported. Mating system was determined by observing perithecial stromata formation from mono-ascospore cultures and their pair-wise combinations. Certain combinations of mono-ascospore strains produced perithecial club-shaped stromata, whereas other combinations produced either no stromata or only abnormal non-perithecial stromata. Similarly, monoascospore cultures without combination produced either no stromata or only abnormal nonperithecial stromata. Despite obvious heterothallism, self-fertility was occasionally observed in few strains ofC. militaris. These observations indicated thatC. militaris behaves as a bipolar heterothallic fungus and requires two mating compatible strains in order to produce regular clubshaped perithecial stromata, a fundamental requirement for its industrial cultivation.     

<Emphasis Type="Italic">Kretzschmaria varians</Emphasis> sp. nov., <Emphasis Type="Italic">Xylaria coremiifera</Emphasis> sp. nov. and <Emphasis Type="Italic">Xylaria umbonata</Emphasis> sp. nov. from Costa Rica

Kretzschmaria varians, a species apparently related to K. micropus, is described as new. It is distinguished primarily by having asci with 2 to 8 ascospores with inconstant germination slit length and remains of synnemata on stromata and surrounding substrate. Xylaria coremiifera, described here as new, bears small fragile coremia on pulvinate stromata and the surrounding substrate. Asci often have fewer than 8 ascospores, most frequently 4. Xylaria umbonata, described here as new, produces perithecia around a central umbo that appears to be the remains of a synnema. Ascospores have long spiralling germination slits.     

A New Species of <Emphasis Type="Italic">Rhytisma</Emphasis> Causes Tar Spot on <Emphasis Type="Italic">Comarostaphylis arbutoides</Emphasis> (Ericaceae) in Panama

A fungus causing tar spots on leaves of Comarostaphylis arbutoides (Ericaceae) in Panama is described as a new species, Rhytisma panamense. The fungus forms gregarious black stromata on pale yellow spots on the adaxial side of leaves. Its ascomata develop from unilocular or multilocular stromata. An analysis of a combined dataset of DNA sequences from LSU to ITS rDNA supports the placement of the species in the genus Rhytisma.     

Known and two new species of <Emphasis Type="Italic">Rhytisma</Emphasis> (Rhytismatales,Ascomycota) from China

Three species of Rhytisma are described based on recently collected specimens from the Anhui province, China. Rhytisma anhuiense is a new species causing a serious tar spot disease on Rhododendron simsii. Rhytisma yuexiense is a new species, which develops its stromata only on fallen leaves of Rhododendron ovatum. Rhytisma himalense on Ilex fargesii is a known species and probably widely distributed in China. Including the two new species, 11 species of Rhytisma are known from China which are presented in a key.     

Study of endophytic Xylariaceae in Thailand: diversity and taxonomy inferred from rDNA sequence analyses with saprobes forming fruit bodies in the field

A study of the diversity, taxonomy, and ecology of endophytic Xylariaceae (Ascomycota) was carried out. In this study, we obtained isolates of Xylariaceae from healthy, attached leaves and teleomorphic stromata on decayed plant materials in a permanent plot at Khao Yai National Park (Thailand). In addition, strains deposited beforehand were selected in which both endophytic strains isolated from living plant tissues and saprobic strains from fruit bodies were included. Consequently, 405 strains of Xylariaceae (273 endophytic and 132 saprobic strains, including identified strains) were studied to reveal the diversity and taxonomy of endophytes and the relationships between those endophytes and saprobic Xylariaceae in Thailand that have been recorded according to fruit-body formation on decayed plant materials. Analysis of 28S rDNA D1/D2 sequences revealed 21 xylariaceous species inhabiting tropical foliage at the site, and several species that are already known as saprobes appear to be among those isolated from living leaves. Furthermore, several clades that consisted of only endophytic strains were found, and some of these have no known matches in public DNA sequence banks.     

Association of slugs with the fungal pathogen Epichloë typhina (Ascomycotina: Clavicipitaceae): potential role in stroma fertilisation and disease spread

Epichloë spp. are endophytes of grasses, and form epiphytic external stromata on flowering tillers. E. typhina was first noticed infecting Dactylis glomerata (= orchardgrass, cocksfoot) stands in the Willamette Valley in 1996, and soon became the primary factor limiting the longevity of seed production fields. Several species of slugs are present in these fields, and we investigated their role in E. typhina biology. Pre‐dawn surveys of D. glomerata fields in 2009 and 2010 found Prophysaon andersoni and Arion subfuscus slugs feeding on the fungal stromata. When unfertilised and fertilised immature stromata predominated, approximately 80% of the individuals of these two species that were seen on plants were found on the stromata. As the majority of stromata reached maturity the presence of these species on stromata declined to between 20–40%. The common agricultural slug pest, Deroceras reticulatum, was on stromata only 20% of the time early in the season, and declined to <5% at stromata maturity. Observations of frass from slugs determined that the most common constituent was the food sources upon which the slug species was usually found during these surveys. Typically 100% of the frass from P. andersoni and A. subfuscus contained stroma material, compared to 25% for D. reticulatum. Spermatia, and ascospores later in the season, were commonly seen in the frass of slugs that consumed stromata. Some slugs that had no stroma material in their frass appeared to have consumed spermatia and ascospores from the leaf surface. A multiple‐choice laboratory test confirmed the different proportional preferences of P. andersoni and D. reticulatum for stroma (0.72 vs 0.20) and leaf (0.07 vs 0.38), respectively. Two laboratory multiple‐choice tests, and a field survey, found that P. andersoni preferred unfertilised and immature stroma over mature stroma. D. reticulatum is the most common and abundant slug in Willamette Valley grass seed fields, yet it is the least likely to move spermatia between unfertilised stromata, or ascospores to uninfected plants. P. andersoni and A. subfuscus are mycophagous, frequently transport viable spermatia and ascospores in their frass; yet they are generally confined to field edges. Data and observations suggest the role of slugs in the epidemiology of E. typhina is small compared to other factors.     

Biology of the Epichloë–Botanophila interaction: An intriguing association between fungi and insects

Epichloë fungi (Ascomycota: Clavicipitaceae) are endophytes of grasses that can produce epiphytic stromata on the culms of their hosts. The fungal stromata are visited by Botanophila flies for feeding and egg laying. We review research that has documented the heterothallic mating system of Epichloë, the mutualistic service of spermatization flies provide for the fungus, and host selection by flies. Flies display an active, stereotypical behavior immediately following oviposition by which spermatia are transferred endozoochorously to stromata. After eggs hatch larvae feed on developing perithecia. Several studies have focused on the cost (consumption of ascospores) to the fungus of engaging in the mutualism with its insect visitors. Generally, researchers have found benefits outweigh costs suggesting the mutualism is stable, however exceptions have been reported for Epichloë infecting some commercially cultivated grasses. Because Epichloë fungi are obligate outcrossers and flies are the major (perhaps only) vector of spermatia, the possibility exists that flies could promote reproductive isolation among Epichloë species through specific behavior. This idea has been tested using different approaches including observations of flies within screened cages containing Epichloë species and an analysis of fly gut content, which revealed no or very moderate selectivity by flies. Volatile fungal compounds are responsible for fly attraction and differ among Epichloë species. However, in a field bioassay using species-specific blends of the two predominant volatiles – a sesquiterpene alcohol (chokol K) and a methyl ester – flies showed no preference for specific blends. That is, flies do not appear able to distinguish between different fungal species based on their odor profiles. Thus, it appears that the flies' role in maintaining reproductive isolation among Epichloë species may be minor and that mechanisms of post-zygotic reproductive isolation are more important in keeping compatible species distinct. However, ethological mechanisms such as ‘stroma constancy’ favoring intraspecific mating may operate at a local scale and need to be investigated further.     

Phylogenetic relationships of Nemania plumbea sp. nov. and related taxa based on ribosomal ITS and RPB2 sequences

During a survey fungal diversity of xylariaceous fungi in Thailand, a new Nemania species, N. plumbea, was identified. Nemania plumbea is characterized by soft-textured grey stromata on a persistent mat of white hyphae, pale brown ascospores with a short germ-slit on the more convex side. It also produces a Geniculosporium-like anamorph in culture. In order to evaluate its phylogenetic relationships among related species and genera, ITS-5.8S rDNA and RPB2 were analysed separately and simultaneously. Results from the phylogenetic analyses indicate that there is close phylogenetic association between N. plumbea and N. aenea. A preliminary account into the natural grouping of Xylariaceae based on ITS-5.8S rDNA and RPB2 sequences is also discussed.     

Two new hyphomycetes parasitic on leaves of Maianthemum species in Panama

Two species of dematiaceous hyphomycetes parasitic on leaves of Maianthemum gigas or M. paniculatum (Liliidae) in tropical mountain rainforests in Western Panama are described as new. One species is morphologically intermediate between Periconiella, Pseudocercosporidium, and Stenella. It is tentatively accommodated in Periconiella because of strongly branched conidiophores and verruculose external hyphae. It differs from other Periconiella species by stromata embedded in the substomatal chamber of the leaves. The other species belongs to Pseudocercospora and is described as new since no similar species of Pseudocercospora is known on species of Maianthemum or related genera. Both species are illustrated in detail with drawings based on light microscopy and images based on scanning electron microscopy. Taxonomic novelties: Periconiella maianthemi R. Kirschner, Pseudocercospora maianthemi R. Kirschner     

Lepraric acid derivatives as chemotaxonomic markers in Hypoxylon aeruginosum, Chlorostroma subcubisporum and C. cyaninum, sp. nov

Hypoxylon aeruginosum (Xylariaceae), an infrequently encountered predominantly tropical pyrenomycete, of which two varieties are known to science, is characterised by having a cyan blue stromatal surface or subsurface. In the course of our ongoing chemotaxonomic evaluation of the Xylariaceae, specific profiles of H. aeruginosum were observed by high performance liquid chromatography, coupled with diode array detection and mass spectrometry (hplc-DAD/MS). By comparison with an authentic standard, lepraric acid and several yet unidentified metabolites with similar hplc-DAD/MS characteristics were detected in the stromata of the type material and other specimens of this species. Interestingly, lepraric acid was hitherto only known from lichenised ascomycetes. Hypoxylon aeruginosum, which is here reported first from Africa and Asia, contained none of the metabolites that were previously detected in other Xylariaceae, except for stromata growing hyperparasitically on other Hypoxylon species. A different lepraric acid derivative was also detected in the type specimen of Chlorostroma subcubisporum, which differs from H. aeruginosum by having a green stromatal surface, cuboid ascospores, and in lacking an amyloid ascal apical apparatus. A second species of Chlorostroma, which showed essentially the same metabolite profile as H. aeruginosum, is described from Thailand. We conclude that Chlorostroma and H. aeruginosum are closely related. However, no taxonomic conclusions are drawn from these findings because no cultures have so far become available to study their anamorphic morphology, their secondary metabolites in culture, and their molecular phylogeny. Taxonomic novelty: Chlorostroma cyaninum L?ss?e, Srikitikulchai & J. Fournier, sp. nov.     

The Botanophila–Epichloë association in cultivated Festuca in Oregon: evidence of simple fungivory

We investigated the Botanophila (Diptera: Anthomyiidae)–Epichloë (Ascomycetes: Clavicipitaceae) interaction in cultivated Festuca spp. (fine fescue) in Oregon in western USA. Epichloë spp. are endophytic fungi of grasses in the subfamily Pooideae. They develop a felt‐like stroma on the surface of grass culms and a dense mycelium within the culms that typically prevents seed head emergence. As a result, seed yields are suppressed, and hence the disease is known as choke. Studies of Epichloë spp. on wild grasses indicate that the fly–fungus interaction is an obligatory mutualism. During oviposition, Botanophila transfers Epichloë spermatia between stromata of opposite mating types, and the perithecia that develop after fungal fertilization serve as food for Botanophila larvae. In the current study, we surveyed 19 cultivated fields of Festuca spp. in Oregon, and observed choke caused by Epichloë festucae Leuchtmann, Schardl and Siegl in 10 of these. However, perithecia were observed in only four fields, and on only 1.0–2.6% of stromata. Perithecial development was also low, and rarely covered 50% of the stroma surface. Despite the absence or low frequency of fertilized stromata, Botanophila lobata Collin larvae were present in all choke‐infested fields. Infestation levels ranged from 2.5 to 70.7%, based on an examination of 175–200 stromata from each field. Only eight (= 2%) of the 450 stromata with B. lobata had perithecia, and the greater majority of B. lobata larvae completed development and exited from unfertilized stromata. This is the first report of the B. lobata–E. festucae association in the USA, and of B. lobata larvae developing successfully on unfertilized Epichloë stromata. The average pupal weight (0.0032 g) did not differ significantly from pupae (0.0030 g) originating from larvae that had developed on fertilized stromata of E. typhina on Dactylis glomerata in a neighboring field. This result indicates that in cultivated fine fescue fields in Oregon, B. lobata forages on E. festucae, but fly development is not dependent on the fertilized stromata of Epichloë.     

Diversity and distribution of Neotyphodium-infected grasses in Argentina

In the present work, we studied the presence of “epichlo?/neotyphodium endophytes” in native grasses from Argentina. An extensive area of this country, representative of several different environments, was studied. Nine new host species of the genera Briza, Bromus and Poa were observed to be infected with asexual seed-borne endophytes. Epichlo? stromata were not observed on any grass species. The incidence of infection in natural populations was highly variable among host species and among populations of the same host species. Morphological characterization revealed differences among the endophytes of different host species and among endophytes of different populations of the same host species. We also summarize the previous knowledge and present unpublished data on host diversity and distribution of these fungi in Argentina. Our results are discussed and compared with previous studies on endophytes in Argentina. This work supports the hypothesis that sexual species (Epichlo?) are not present in the southern hemisphere, and suggests the existence of a high diversity of asexual endophytes in South America.     

<Emphasis Type="Italic">Desmosorus</Emphasis>, a new rust genus (Uredinales)

Uredo behnickiana (syn. Hemileia americana and H. oncidii) is known on orchids of the genera Cattleya, Epidendrum, Laelia, Lophiaris and Oncidium. Previously, only the uredinial stages of these fungi have been described. The telial stage of the rust species was found for the first time on Oncidium marshallianum. Because of a unique combination of morphological characters the fungus could not be assigned to Hemileia or any other known rust genus. Desmosorus is proposed, therefore, as a new genus with Desmosorus oncidii as the single known species. Desmosorus is characterised by (1) suprastomatal sori on which the spores are formed at the ends of branched hyphae, by (2) subglobose to ovoid, echinulate urediniospores with a smooth patch around the hilum and by (3) thin-walled, oblong elliptical to cylindrical teliospores with a rounded apex.