Phialide arrangement and character evolution in the helotialean anamorph genera Cadophora and Phialocephala.

The dematiaceous hyphomycete genera Cadophora and Phialocephala are anamorphs associated with mollisioid inoperculate discomycetes (Helotiales) and are delineated based on the complexity of the phialide arrangement with members of Cadophora producing solitary phialides and species of Phialocephala producing complex heads of multiple phialides. A third phylogenetically related taxon, Leptodontidium orchidicola, produces mostly indehiscent conidia that may represent non-functional phialides. Morphological characteristics of both sexual and asexual states of these and other fungi in a focal group of helotialean taxa were re-examined, in light of relationships shown by molecular phylogenetic analyses of rDNA ITS sequences, to determine the evolutionary significance of phialide arrangement. The focal species of Phialocephala formed a monophyletic clade, while five of six species of Cadophora including the type were in a separate clade along with L. orchidicola. C. finlandica was placed in a third clade with species of Meliniomyces and Rhizoscyphus. We hypothesized that the ancestral state for species in Cadophora and Phialocephala is the production of sclerotium-like heads of multiple phialides, which has been retained in most species assignable to Phialocephala. A reduction to solitary phialides occurred in the lineage leading to the clade containing most of the Cadophora species. Two possible reductions to non-functional phialides were identified: one in the Meliniomyces-C. finlandica-Chloridium paucisporum clade and another in the L. orchidicola and Mollisia "rhizophila": clade. A reversion to increased phialide complexity might have occurred in the clade containing C. finlandica and Ch. paucisporum. Our data and analyses also show a previously unrecognized relationship between teleomorph and anamorph morphology in that Mollisia species with smaller asci would be expected to have Phialocephala states while those with larger asci would be expected to have Cadophora states. Based on morphology and phylogenetic placement, L. orchidicola and C. hiberna are transferred respectively to Cadophora and Phialocephala.

Endophoma, a new didymellaceous endoconidial genus from bat-cave soil.

A new endoconidial taxon, Endophoma elongata gen. et sp. nov., isolated from bat-cave soil, is reported from Alberta, Canada. It is morphologically unique in producing two forms of unilocular, endoconidial conidiomata (i.e. a superficially Phoma-like spherical, often ostiolate form and a cylindrical, non-ostiolate, often setose cleistopycnidial form). Locules of both forms are pseudoparenchymatous, filled with hyaline, thin-walled, endoconidial conidiogenous cells. Endoconidia are hyaline and unicellular. One- or two-celled chlamydospores are abundant in culture. Phylogenetic analysis of the LSU, ITS and ß-tubulin regions indicates Endophoma is a member of the Didymellaceae and remote from all other endoconidial genera. Endoconidiogenesis has not been reported previously within the Didymellaceae, and Endophoma represents the first report of a coelomycetous, endoconidial genus in the Pleosporales.

In vitro degradation of the moss Hylocomium splendens by three pleosporalean fungi.

Three darkly pigmented species of conidial fungi of the family Pleosporaceae isolated from plants colonizing the Saskatchewan Glacier forefield were examined for potential roles in the degradation of moss gametophytes. Curvularia inaequalis and Ulocladium atrum isolated from bryophytes Ditrichum flexicaule and Tortella tortuosa , respectively, and Chalastospora gossypii from Saxifraga oppositifolia were inoculated onto autoclaved gametophytes of the moss Hylocomium splendens. All three species of fungi caused mass losses of the moss gametophytes. In vitro enzymatic tests revealed that all three fungi degraded cellulose, while none degraded insoluble polyphenols. When this material was examined by scanning electron microscopy, it was evident that the fungi had eroded the outer wall layer of the moss leaf cells to some extent but not the inner layer containing more lignin-like compounds. Once the outer wall layer was removed, the cells easily disarticulated. It is proposed that accumulations of these phenolics-rich leaf fragments subsequently ameliorate the rooting environment for vascular plants and have the potential to support the growth of basidiomycetes and other fungi, potentially mycorrhizal with pioneer vascular plants.

Endomembrane system of aspen root cells plays a key role in defense against a common fungal root endophyte, Cryptosporiopsis radicicola.

The host-endophyte interaction between roots of aspen (Populus tremuloides) and Cryptosporiopsis radicicola was examined primarily by transmission electron microscopy. Hyphae growing on the exterior of the inoculated roots had a thick, electron-dense, adhesive sheath. At hyphal contact and penetration, host epidermal cells exhibited a series of defense responses (viz. formation of papillae and partition walls, general wall thickening and walling-off of internal hyphae). In papilla formation, loop-shaped, rough endoplasmic reticula (rER) gave rise to globose secretory vesicles that accumulated around and then fused to the developing papilla. Unlike papillae, general wall thickening was associated with the Golgi apparatus (GA) that produced cell wall materials; 1-3 layers of Golgi cisternae were in contact with or in the immediate proximity (mostly within 0-0.5 microm) of and lying parallel to the host cell wall, where they budded out numerous subglobose vesicles that fused directly to the host cell wall and made it thicker. Partition wall formation and walling-off of internal hyphae also were common; the former was associated with an extended single cisterna, which was indistinguishable from rER or individual cisternae of GA, and in the latter phenomenon internal hyphae were encased by electron-dense material containing numerous ribosomes and membranous elements that were derived apparently from proliferated rER. These pronounced defense responses protected the stele and contributed to making C. radicicola endophytic rather than pathogenic.

Phialocephala urceolata, sp. nov., from a commercial, water-soluble heparin solution.

Phialocephala urceolata sp. nov. was isolated from a black film that had developed on a water-soluble proprietary heparin solution (pH 2.5). Morphological and enzymatic characters, along with phylogenetic analyses of rDNA sequence data, indicated that the conidial fungus is closely related to species of Phialocephala known primarily as endophytes in the roots of vascular plants (e.g., Acephala applanata, P. fortinii and P. sphaeroides) or as associates of persistent plant organs such as the stems and needles of woody plants (e.g., P. compacta, P. dimorphospora and P. scopiformis). Phialocephala urceolata is distinctive in having urn-shaped phialides that are sparsely distributed along the conidiophore axis, a slow growth rate in culture and in exhibiting a unique combination of reactions on enzymatic test media (i.e., it acidifies casamino acids medium and is gelatinase negative). Partial sequence data from the small subunit (SSU) rDNA indicated that P. urceolata is among the Helotiales and close to the type species of Phialocephala. Sequence data from the internal transcribed spacer (ITS) region places P. urceolata closest to P. sphaeroides. The source of this contaminant is unknown but its taxonomic relationship with other root endophytic species and its ability to produce polyphenol oxidases suggest that the natural habitat of this species is possibly woody plant tissues or soil enriched with lignocellulose.

Morphology and development of Nigrosabulum globosum, a cleistothecial coprophile in the Bionectriaceae (Hypocreales).

Recent DNA sequence analyses indicated that Nigrosabulum globosum is a cleistothecial representative of the Bionectriaceae in the Hypocreales, but morphological characters supporting this relationship are unknown. Using light and electron microscopy we followed the development of the ascomata of this species, from the formation of gametangia through to the development of mature ascospores, and observed a series of characters that confirmed its hypocrealean affinities. These included the formation of a gel-filled centrum during early stages of ascoma development, the subsequent appearance of hyaline peridial tissue enclosed within a layer we interpret as representing a melanized uniloculate stroma, apically derived paraphyses, and an ascogenous system that gives rise to asci that were both cylindrical to clavate and globose. Ascospores, previously reported to be smooth, were ornamented with a honeycomb-like reticulum and were able to germinate within the ascoma. The carbonaceous outer (stromatic) walls of the mature, grit-like cleistothecia indicate possible resistance to UV radiation and desiccation. Furthermore, the complement of germinated ascospores would enable mature ascomata to function as propagules that could quickly initiate new growth when transferred to fresh substrate. Our reexamination of N. globosum also provides data that support the hypothesized close relationship with other bionectriaceous, cleistothecial coprophiles, i.e., species of Hapsidospora, and Bulbithecium in particular.

Pathogenesis of bryophyte hosts by the ascomycete Atradidymella muscivora.

Atradidymella muscivora (Pleosporales) is a bryophyte pathogen that infects the mosses Aulacomnium palustre, Hylocomium splendens, and Polytrichum juniperinum. Light and scanning electron microscopy and extracellular enzyme production were used to characterize the interactions between this fungus and its native hosts and the model host Funaria hygrometrica. Penetration was direct via hyphae or appressoria, and hosts responded by forming layered, darkly pigmented deposits at penetration sites, similar to the papillae formed by vascular plants in response to fungal infection. Infected hosts gradually became chlorotic as hyphae grew intracellularly, presumably killing host cells. Pycnidia of the Phoma anamorph (P. muscivora) and uniloculate pseudothecia were initiated as tightly packed masses of stromatic dematiaceous hyphae within a single host cell. Mature pycnidia and pseudothecia were erumpent. A new microniche among bryophilous fungi is described, whereby A. muscivora supplants the gemmae of Aul. palustre and exploits the normal nutrient-flow of the moss gametophyte. Atradidymella muscivora produced both cellulases and soluble polyphenolic oxidases, allowing it to also function as a saprobe and degrade the cell walls of bryophytes. The saprophytic and pathogenic abilities of A. muscivora suggest it may play a role in nutrient cycling, population dynamics, and small-scale disturbances in boreal ecosystems.

Atradidymella muscivora gen. et sp. nov. (Pleosporales) and its anamorph Phoma muscivora sp. nov.: A new pleomorphic pathogen of boreal bryophytes.

During a survey of bryophilous fungi from boreal and montane habitats, 12 isolates of a hitherto unknown plant pathogenic member of the Pleosporales were recovered from Aulacomnium palustre, Hylocomium splendens, and Polytrichum juniperinum, and described as Atradidymella muscivora gen. et sp. nov. Atradidymella is characterized by minute, unilocular, setose pseudothecia having 2-3 wall layers; brown, fusiform, 1-septate ascospores; and a Phoma anamorph. The genus is distinguished from all other pleosporalean genera with brown, fusiform ascospores on the basis of ascospore and pseudothecium morphology and a highly reduced stroma that is localized within a single host cell. Atradidymella muscivora is distinguished by its minute pseudothecia (<115 µm) and ascospores that are slightly allantoid and constricted at the septum with the upper cell often wider than the lower. Its anamorph, Phoma muscivora sp. nov., is morphologically distinguishable from P. herbarum in having smaller conidia. Parsimony analysis of the ITS rDNA region indicates A. muscivora has affinities to the Phoma-Ascochyta-Didymella clade that is sister to the Phaeosphaeriaceae within the Pleosporales.

Evidence of apothecial ancestry in the cleistothecial ascomata of Pleuroascus nicholsonii.

Ascomata of Pleuroascus nicholsonii, a rarely reported cleistothecial ascomycete, show little overt evidence of a putative affiliation with the Leotiomycetes. However, close examination of the plectomycetous centrum reveals a distorted hymenium arising from a system of branched ascogenous hyphae, and twisted or coiled uniseriate ascospores enclosed within what appears to be the remains of the spore investing membrane of a clavate ascus precursor. Abundant sterile elements arising from the inner wall layer of the peridium and interspersed throughout the centrum are interpreted as representing vestiges of apically branched paraphyses. Whole ascomata show limited signs of polarity, although the characteristic, tightly coiled appendages generally arise along or below the equatorial region and there is a marked thinning of subicular hyphae over the crown of the cleistothecium. The mature peridium, which consists of a thin, melanized outer layer of squamulose cells, splits irregularly along intercellular grooves when disturbed. The adaptive significance of these characteristics is unknown, but the persistent paraphyses, the easily fractured and darkly pigmented membranous peridium, an ascospore mass that is dry at maturity, and the tendency for ascomata to cling together in clumps can be rationalized in the context of a coprophilous life-style. Collection data for P. nicholsonii provide further support for this supposition because five of the six reported specimens are from rodent dung.

Evidence that the gemmae of Papulaspora sepedonioides are neotenous perithecia in the Melanosporales.

Papulaspora sepedonioides produces large multicellular gemmae with several, thick-walled central cells enclosed within a sheath of smaller thin-walled cells. Phylogenetic analysis of the large subunit rDNA indicates P. sepedonioides has affinities to the Melanosporales (Hypocreomycetidae). The development of gemmae in P. sepedonioides was characterized by light and scanning and transmission electron microscopy and was similar to previous ontogenetic studies of ascoma development in the Melanosporales. However instead of giving rise to ascogenous tissues the central cells of the incipient gemma became darkly pigmented, thick walled and filled with lipid globules while the contents of the sheath cells autolysed, leaving them empty and deflated at maturity. Both central cells and pre-autolytic sheath cells produced both germ tubes and new gemmae primordia, suggesting microcyclic conidiogenesis occurs in this species. Mature gemmae were non-deciduous or seceded by schizolytic secession and appear to have both perennating and disseminative potential. The evolution of these neotenous perithecial propagules may be driven by life-history and ecological factors selecting for functional versatility.

Margaretbarromyces dictyosporus gen. sp. nov.: a permineralized corticolous ascomycete from the Eocene of Vancouver Island, British Columbia.

A single, permineralized ascoma resembling a pseudothecium assignable to the Pleosporales is described from the Eocene Appian Way fossil locality on Vancouver Island, British Columbia. The ascoma is globose, ostiolate, and erumpent on a fragment of the bark from an unidentified seed plant. Basally arranged asci contain large, multicelled, obovate ascospores within a single cavity or locule enclosed by a two-layered pseudoparenchymatous tissue that ostensibly represents ascostroma. Given this interpretation of the specimen's morphological features, Margaretbarromyces dictyosporus gen. sp. nov. represents the first report of a corticolous pleosporalean ascoma in the fossil record.

Development and dehiscence of the cephalothecoid peridium in Aporothielavia leptoderma shows it belongs in Chaetomidium.

The development of the cephalothecoid peridium of Aporothielavia leptoderma was examined using light and electron microscopy. Early stages in ascoma initiation were consistent with previous reports for other species in the Chaetomiaceae. However, as young cleistothecia increased in size, clusters of peridial cells in the outer textura angularis elongated in a radial pattern around a central cell or cell cluster to form rosettes of relatively thick-walled segments that marked the central areas of incipient cephalothecoid plates. The external flank along median portions of the radial cells became thin walled and swelled outwards so that each plate became concave and was separated from adjacent plates by a more or less circular to polygonal ridge of knuckle-shaped swellings. When dry, mature peridia split apart along some of the ridges demarcating individual plates but an internal mechanism for liberating ascospores from the confines of the ascoma was not observed. Physical disturbance of mature cleistothecia by beetles, when enclosed together in a Petri dish, shattered the peridia, liberating the ascospores. Smaller insects were unable to cause disarticulation of the cephalothecoid plates. Because of the presence of an apical germ pore in the ascospores and morphological similarity to Chaetomidium arxii, the new combination Chaetomidium leptoderma (syn. Thielavia leptoderma) comb. nov. is proposed.

A new species of Cladophialophora (hyphomycetes) from boreal and montane bryophytes.

During a survey of bryophilous fungi from boreal and montane habitats in central Alberta, a hitherto undescribed species of Cladophialophora was recovered from Polytrichum juniperinum, Aulacomnium palustre, and Sphagnum fuscum. On potato dextrose agar (PDA) colonies grew slowly, attaining a diameter of 25 mm after 30 d, were dark grey, velvety, radially sulcate, and convolute and cracked at the centre. Micronematous conidiophores gave rise to branched chains of small (1-2 x 8-22 microm), cylindrical to fusiform conidia with truncate, swollen scars at each end. Phylogenies built on the ITS and ribosomal SSU regions indicate the isolates form a monophyletic clade within the family Herpotrichiellaceae (Chaetothyriales) that is composed of two geographically based groups, each with 99% within-group sequence similarity and 97-98% between-group sequence similarity. A teleomorph has not been found but would likely be similar to species of Capronia. In vitro inoculation of the isolates onto axenically grown P. juniperinum produced no discernible host symptoms, and host penetration could not be detected using light microscopy. The production of polyphenol oxidases by the fungus and the role of other Cladophialophora species as latent endophytes and saprobes suggest that a potential role for the fungus is the degradation of the polyphenol-rich cell walls of mosses. A dichotomous key to species of the genus Cladophialophora is provided.

Ascoma development and phylogeny of an apothecioid dothideomycete, Catinella olivacea.

Catinella olivacea is a discomycetous fungus often found fruiting within cavities in rotting logs. Because this habitat would lack the air currents upon which discomycete species normally rely for the dispersal of their forcibly ejected ascospores, we suspected an alternative disseminative strategy might be employed by this species. An examination of the development of the discomycetous ascomata in pure culture, on wood blocks, and on agar showed that the epithecium was gelatinous at maturity and entrapped released ascospores in a slimy mass. We interpreted this as an adaptation for ascospore disperal by arthropods. Developmental data also showed that C. olivacea was unusual among other discomycetes in the Helotiales (Leotiomycetes). For example, the ascoma developed from a stromatic mass of meristematically dividing cells and involved the formation of a uniloculate cavity within a structure better considered an ascostroma than an incipient apothecium. Furthermore, the ascus had a prominent ocular chamber and released its ascospores through a broad, bivalvate slit. These features, along with phylogenetic analyses of large subunit and small subunit rDNA, indicated that this unusual apothecial fungus is, surprisingly, more closely affiliated with the Dothideomycetes than the Leotiomycetes.

Two new species of Pseudogymnoascus with Geomyces anamorphs and their phylogenetic relationship with Gymnostellatospora.

Two new psychrophilic Pseudogymnoascus species with Geomyces anamorphs are described from a Sphagnum bog in Alberta, Canada. Pseudogymnoascus appendiculatus has long, branched, orange appendages and smooth, fusoid to ellipsoidal ascospores with a faint longitudinal rim. Pseudogymnoascus verrucosus has short, subhyaline appendages and warty peridial hyphae and ascospores, and both smooth to asperulate and irregularly warty conidia. Both species produce asci in chains, a feature that supports the distinction between this group and Myxotrichum, which produces asci singly. The discovery of species intermediate between Pseudogymnoascus and Gymnostellatospora, in having both ornamented ascospores and Geomyces anamorphs, prompted a re-evaluation of the genera. Sequence analysis of the internal transcribed spacer regions (ITS) of the nuclear ribosomal DNA indicates that the two genera remain distinct and comprise a monophyletic group. Pseudogymnoascus species have smooth to warty or lobate-reticulate ascospores while species of Gymnostellatospora have walnut-shaped spores with distinct longitudinal crests and striations. Anamorphs assignable to the form genus Geomyces are allied with both genera. A key is provided to the four species and varieties of Pseudogymnoascus.

In vitro decomposition of Sphagnum by some microfungi resembles white rot of wood.

The abilities of some ascomycetes (Myxotrichaceae) from a Sphagnum bog in Alberta to degrade cellulose, phenolics, and Sphagnum tissue were compared with those of two basidiomycetes. Most Myxotrichaceae degraded cellulose and tannic acid, and removed cell-wall components simultaneously from Sphagnum tissues, whereas the basidiomycetes degraded cellulose and insoluble phenolics, and preferentially removed the polyphenolic matrix from Sphagnum cell walls. Mass losses from Sphagnum varied from up to 50% for some ascomycetes to a maximum of 35% for the basidiomycetes. The decomposition of Sphagnum by the Myxotrichaceae was analogous to the white rot of wood and indicates that these fungi have the potential to cause significant mineralization of carbon in bogs.

Leptographium piriforme sp. nov., from a taxonomically diverse collection of arthropods collected in an aspen-dominated forest in western Canada.

During a survey of fungi associated with arthropods collected in a southern boreal mixed-wood forest in Alberta we obtained 29 isolates of a unique species of Leptographium. This species displayed a distinct combination of characteristics, including curved conidia on short-stipitate conidiophores, a secondary micronematous conidial state, stalked pear-shaped cells and an optimal growth rate at 35 C, and is described as Leptographium piriforme sp. nov. The isolates were most similar morphologically to L. crassivaginatum, but ITS sequence comparisons indicate that our isolates cannot be assigned to this or any other sequenced species in the genus. Initial observations on the pear-shaped cells in feeding experiments with Sancassania berlesei show that these structures may act as a nutritional incentive for visiting arthropods. Most arthropods carrying this new species were caught in traps baited with dung which, in light of its optimum growth temperature, suggests a coprophilous phase in the life cycle of this species. Additional isolates from woody species typical of the survey area might clarify whether Leptographium piriforme in its forest habitat occurs as a plant pathogen or saprobe.

Patterns of genetic variation in Phialocephala fortinii across a broad latitudinal transect in Canada.

Dark septate root endophytes (DSE) are an artificial assemblage of fungi that have darkly pigmented, septate hyphae and that are frequent or distinctive intracellular associates of roots of apparently healthy plants. Based on isolates obtained from the roots of Salix spp., the distribution of a common DSE fungus, Phialocephala fortinii, was examined along a latitudinal transect in Canada running from the high arctic to the 49 degrees N parallel. Non-sporulating isolates were provisionally identified as P. fortinii through analysis of DNA sequence data of the ITS2 region of rDNA. P. fortinii was isolated frequently from boreal and arctic habitats, but rarely from grassland habitats. Patterns of genetic variation were examined through analysis of amplified fragment length polymorphisms (AFLP). All AFLP profiles were unique with the majority of genetic variation occurring among individuals within the collecting sites at each latitude. Neighbour-joining analysis of genetic distances yielded eight well-supported clusters, three of which included individuals from more than one latitude. Some linkage disequilibrium, possibly due to partial clonality, was detected.

Cretaceous and Eocene poroid hymenophores from Vancouver Island, British Columbia.

Two fossil poroid hymenophore fragments, one from the Cretaceous Period and the other from the Eocene Epoch, are described. The permineralized specimens were obtained from marine calcareous concretions on Vancouver Island, British Columbia, Canada, and were studied using the cellulose acetate peel technique. Size and distribution of pores in the hymenophores, as well as the hyphal anatomy of the dissepiments and some hymenial elements, were examined. In the Cretaceous specimen, Quatsinoporites cranhamii sp. nov., pores are round to elliptical, three per mm, and 130-540 µm diam. Dissepiments consist of narrow, simple septate, hyphae. Neither basidia nor basidiospores are present, but acuminate hymenial cystidia, up to 54 µm in length, are common. The Eocene specimen, Appianoporites vancouverensis sp. nov., has a pore density of six per mm and pores are 130-163 µm in diam. Dissepiments consist of narrow, simple septate, thin-walled hyphae. Neither basidia nor basidiospores are present, but acuminate, thick-walled hymenial cystidia, up to 32 µm in length, are common. The poroid hymenophore is a characteristic of a number of extant basidiomycete taxa, including the Boletales, Polyporales and Hymenochaetales. It is unlikely that the fleshy, ephemeral, terrestrial basidiomata of the Boletales would be preserved in a marine environment, and thus the specimens are interpreted as belonging to basidiomycete lineages, with persistent, leathery or corky basidiomata. The simple septate hyphae, the minute pores and presence of cystidia most closely resemble taxa of the Hymenochaetales. These fossils unequivocally push back the minimum age of homobasidiomycetes and extend their paleogeographical range.

Microcosm tests of the effects of temperature and microbial species number on the decomposition of Carex aquatilis and Sphagnum fuscum litter from southern boreal peatlands.

Increased decomposition rates in boreal peatlands with global warming might increase the release of atmospheric greenhouse gases, thereby producing a positive feedback to global warming. How temperature influences microbial decomposers is unclear. We measured in vitro rates of decomposition of senesced sedge leaves and rhizomes (Carex aquatilis), from a fen, and peat moss (Sphagnum fuscum), from a bog, at 14 and 20 degrees C by the three most frequently isolated fungi and bacteria from these materials. Decomposition rates of the bog litter decreased (5- to 17-fold) with elevated temperatures, and decomposition of the sedge litters was either enhanced (2- to 30-fold) or remained unaffected by elevated temperatures. The increased temperature regime always favoured fungal over bacterial decomposition rates (2- to 3-fold). Different physiological characteristics of these microbes suggest that fungi using polyphenolic polymers as a carbon source cause greater mass losses of these litters. Litter quality exerted a stronger influence on decomposition at elevated temperatures, as litter rich in nutrients decomposed more quickly than litter poorer in nutrients at higher temperatures (8.0%-25.7% for the sedge litters vs. 0.2% for the bryophyte litter). We conclude that not all peatlands may provide a positive feedback to global warming. Cautious extrapolation of our data to the ecosystem level suggests that decomposition rates in fens may increase and those in bogs may decrease under a global warming scenario.