Neale L. Bougher

Multiple origins of sequestrate fungi related to Cortinarius (Cortinariaceae).

The aim of the present study was to investigate the phylogeny and evolution of sequestrate fungi (with gastroid or partially exposed basidiomes) in relation to their gilled relatives from the Cortinariaceae (Basidiomycetes). Phylogenetic analyses of 151 ITS sequences from 77 gilled species and 37 sequestrate taxa were performed using maximum parsimony and maximum likelihood methods. Results show that sequestrate basidiome forms occur in all three major ectomycorrhizal lineages of Cortinariaceae: the clades Cortinarius, Hebeloma/Hymenogaster/Naucoria, and Descolea. However, these forms do not appear within the saprobic outgroup Gymnopilus, indicating multiple origins of sequestrate forms from ectomycorrhizal ancestors. Additionally, within the Cortinarius clade sequestrate forms have multiple origins: emergent Cortinarius spp., Thaxterogaster, Quadrispora, Protoglossum, and two Hymenogaster spp. (H. remyi, H. sublilacinus) share common ancestors with Cortinarius spp., but these sequestrate genera are not closely related to each other (with exception of Thaxterogaster and Quadrispora). Hymenogaster sensu stricto, Setchelliogaster, and Descomyces were placed in the two other major clades. Thus, sequestrate taxa evolved independently many times within brown-spored Agaricales. Furthermore, emergent, secotioid, and gastroid forms have evolved independently from each other, and so are not necessarily intermediate forms. After their establishment, these apparently morphologically stable taxa show a tendency to radiate.

Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests

Abstract. A molecular survey of basidiomycete ectomycorrhizal fungi colonising root tips at a site in Eucalyptus marginata (jarrah) forest revealed the presence of many fungal species which could not be identified from a database of ITS-PCR-RFLP profiles from morphologically identified species. Three of these unidentified taxa were among the six most frequently encountered profiles. Phylogenetic analyses of ITS and nuclear LSU sequences revealed a close relationship among the three fungi and that they belong to the family Sebacinaceae (sensu Weiß and Oberwinkler 2001). The possibility that DNA of non-ectomycorrhizal rhizosphere or endophytic fungi had been amplified selectively by the basidiomycete-specific primers was tested by amplification with fungal-specific primers. A single PCR fragment was amplified in all but two of the 24 samples tested and digestion with two restriction enzymes produced RFLP profiles which matched those from the Sebacinoid sequence. We conclude, therefore, that at least three species of Sebacinaceae are common ectomycorrhizal associates of E. marginata.

Sequestrate (truffle-like) fungi of Australia and New Zealand

Sequestrate fungi are a polyphyletic, diverse group of macrofungi with truffle-like, underground (hypogeous) or emergent fruit bodies, which are well represented in Australia and New Zealand. The first species in the region were described in 1844, but sequestrate fungi have been poorly documented until recent times. Regional diversity of sequestrate fungi is high in comparison to other parts of the world: for ascomycetes and basidiomycetes 83 genera and 294 species are currently known in Australia and 32 genera and 58 species in New Zealand. Only an estimated 12–23% of species are known for Australia and 25–30% for New Zealand. On that basis, between 1278–2450 species may occur in Australia and 193–232 in New Zealand. Centres of diversity for some groups of sequestrate fungi occur in the region, e.g. Russulaceae (five known genera, 68 species) and Cortinariaceae (eight genera, 33 species). Some other groups are less diverse than in the northern hemisphere, e.g. sequestrate Boletaceae (seven genera, 25 species). More than 35% of Australian sequestrate genera and 95% of species are endemic; for New Zealand about 45% of sequestrate genera and 80% of species are endemic. Australia and New Zealand share similarities in sequestrate fungi at generic level (11% of total) but do not share many of the same species (4% of total). Knowledge of biogeographical distributions is limited by incomplete taxonomic knowledge and insufficient collections. Some Gondwanan, Australasian and widespread/cosmopolitan patterns are evident. Some exotic sequestrate fungi have been recently introduced and some fungi indigenous to the region occur world-wide as exotics with eucalypt plantings. Within Australia and New Zealand, there is evidence that characteristic suites of fungi co-occur in different climatic and vegetation types. Mycorrhizas of Australian and New Zealand taxa have a range of morphological and physiological attributes relating to their effect on plants and broader roles in ecosystem nutrient cycling and health. Spores of sequestrate fungi are dispersed by a range of fauna. There are tripartite inter-dependent interactions between mycorrhizal plants, sequestrate fungi and native mammals and birds that use the fungi as food. Major environmental influences affecting the distribution, diversity and abundance of sequestrate fungi include climate, topography, soil, vegetation and animals. Imposed upon such influences are a range of natural and human-induced disturbance factors which alter habitat heterogeneity, e.g. fire, fragmentation and replacement of native vegetation and exotic organisms. Rare and endangered sequestrate fungi are likely to occur in Australia and New Zealand, but for most taxa there is insufficient data to determine rarity or commonality. In the face of poor knowledge, assemblage-based and habitat-based approaches are the most appropriate for conservation and management of sequestrate fungi. Habitat heterogeneity may be important for the fungi at scales ranging from different climatic and vegetation types to local topographic-related variations.

Interspecific and intraspecific variation of ectomycorrhizal fungi associated with Eucalyptus ecosystems as revealed by ribosomal DNA PCR–RFLP

Gondwanan vegetation, and the Australian region in particular, is species rich for ectomycorrhizal fungi in epigeous and hypogeous forms with over 100 species recorded in small (1 ha) patches of forests. Distinguishing co-occurring ectomycorrhizal fungi as root associations in native (natural or wildlands) vegetation or plantations and discriminating them from other larger basidiomycetes, e.g. wood and leaf litter decomposer fungi, places large demands on molecular identification, especially if interspecific similarities and intraspecific variation occur in target sequences. One hundred and nine species of larger basidiomycetes from a single forest location were characterised by PCR-RFLP profiles of two genomic regions (nuclear rDNA ITS and mtLSU). Over one-third of the species for which multiple isolates were tested showed intraspecific variation in either one or both genomic regions. This remarkably high variation questions previous assumptions about intraspecific ITS sequence variation and highlights the value of integrated molecular and morphological databases including voucher specimens. It also emphasises the value of molecular investigations that use more than one genomic region. Interspecific similarities were common among the Cortinariaceae, especially in the ITS region. Discrimination of most Cortinariaceae species was achieved using variation in the mtLSU region in conjunction with the ITS. This new information raises the possibilities that the ITS sequence is more conserved and the mtLSU more variable than among species of the other 23 families. In the other families, interspecific ITS variation was greater and the mtLSU profiles grouped species within families. The high variation in the two genomic regions indicated possible differences in the fungal population structure between two adjacent, differently managed blocks of Eucalyptus marginata forest. The significance of this variation to ecology, biodiversity assessment and ecosystem management are discussed.

Consideration of the taxonomy and biodiversity of Australian ectomycorrhizal fungi

Mycorrhiza management in forestry must be predicated on an understanding of fungal biology and ecology. A fundamental building block of the biology and ecology of any organism is accurate identification and an understanding of its relationship to other organisms.The taxonomy of the larger fungi has been largely based on morphological classification of sexual structures but now Taxonomy routinely incorporates mating studies, and biochemical and molecular data. Taxonomy may not revolutionize theories on mycorrhiza but can clarify some of the inconsistencies due to misrepresentation or over-generalizations and inappropriate conclusions drawn from studies with inaccurately identified fungi. To illustrate this, we discuss and example where incorrect fungal names were repeatedly erroneously applied in morphological and physiological research reports on this fungus, e.g. Laccaria laccata. In this case subsequent taxonomic study revealed the reason for the conflicting research results reported for this fungus.We discuss the status of identifying the ectomycorrhizal fungi in various forest communities in Australia and the relationships of this process to assessing their use in forestry. Recent intensive efforts to collect, isolate and identify Australian ectomycorrhizal fungi have revealed an enormous and unique species diversity, e.g., for truffle-like fungi, over 2000 collections from the last five years alone have yielded 2 new families, 24 new genera, and about 184 new species. Nearly 95% of the described and undescribed fungi from Australia are novel, with some 22 genera and 3 families endemic. In most cases the current systematic knowledge of mycorrhizal fungi is inadequate to support clear framework for Australian taxa. This reflects the traditional Northern Hemisphere view of the world, the uniqueness of the Australian fungal flora, and how poorly it is known. For example, the genus Hymenogaster had been widely acknowledged as the most reduced member of the Cortinariaceae. However recent work on Hymenogaster species from the Southern Hemisphere has offered a number of alternative affinities to various species.We also discuss the role proper identification of the organisms involved plays in understanding the ecosystem. Emphasis should be placed on how species diversity equates with physiological and genetic diversity and how a sound taxonomic understanding of species and their systematic position is essential to properly manage them. Accurate taxonomic information will continue to be required as the basis for assessing the role of ectomycorrhizal fungi in sustained ecological development. Of particular significance is the role of ectomycorrhizal fungi in maintenance of plant diversity in natural ecosystems and those disturbed by management. In conclusion, we present some key research areas involving the use of taxonomy that need priority attention.

Specificity, sensitivity and discrimination of primers for PCR-RFLP of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and plantations

Techniques to rapidly identify the basidiomycete fungal partner of ectomycorrhizal associations would be a major advantage for ecological, fungal population dynamics and life history studies of epigeous and hypogeous forms in plantations, forests, wild lands and other native or natural vegetation. PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) identification of DNA regions is an available technique; however, primers which have a high probability of amplifying only the basidiomycete DNA are needed. Here we have assessed the specificity, sensitivity and discrimination of six different primer pairs, three targeting nuclear and three mitochondrial regions, for use in identification of Australian basidiomycete fungi from Eucalyptus forests by matching PCR-RFLP patterns to morphologically defined species. Two sets of primers, one newly designed and targeting the nuclear ribosomal DNA internal transcribed spacers (ITS) and the other amplifying a fragment of mitochondrial large subunit ribosomal DNA met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of larger basidiomycetes, with no amplification of plant, bacterial or ascomycete DNA. The specificity of the ITS primer pair was compared with that of ITS1-F/ITS4-B. PCR-RFLP of the two regions discriminated fungi to species level for 91 fungal species from 28 families. Hence these two DNA regions and the specific primers are a potential practical PCR-RFLP tool for identifying basidiomycetes associated with plants from field samples.

Ganoderma and Amauroderma species associated with root-rot disease of Acacia mangium plantation trees in Indonesia and Malaysia

Fungal sporocarps and cultures associated with signs and symptoms of root-rot disease were collected from Acacia mangium and other tropical hardwood species. The collections were identified by either morphological characters and/or by phylogenetic analysis based on DNA sequences as Ganoderma philippii, G. mastoporum, G. aff. steyaertanum, G. australe and Amauroderma rugosum. Phylogenetic analysis unequivocally placed in the G. philippii clade four sequences amplified from A. mangium root and butt tissue showing clear signs of red root-rot disease (roots are covered by a red rhizomorphic skin). Whereas G. philippii was the most frequently encountered fungal species in A. mangium with red root-rot disease, this study indicates that other fungal species related to G. mastoporum may cause root-rot disease with very similar symptoms. An isolate (FRIM 138) that had caused red root-rot disease in artificial inoculations carried out before this study and was presumed to be G. philippii, is here determined to be closely related to G. mastoporum, G. cupreum and G. sinense. A Ganoderma species associated with a yellow-brown root-rot disease killing trees in an A. mangium plantation in Central Java, previously identified as G. lucidum, is shown by phylogenetic analysis to be closely related to G. steyaertanum, though some morphological characters vary from the original description of that species.

Laccaria fraterna, a common ectomycorrhizal fungus with mono- and bi-sporic basidia and multinucleate spores: comparison with the quadristerigmate, binucleate spored L. laccata and the hypogeous relative Hydnangium carneum

Examination of Laccaria collections from Western Australia revealed that the most common species L. fraterna had basidia which were monosporic, bisporic or very occasionally trisporic, in contrast to L. laccata which was quadrisporic. For L. fraterna , all post-meiotic nuclei migrated into the spores leaving basidia anucleate. Spores of monosterigmate basidia had four nuclei, and bisterigmate basidia usually had two. Post-meiotic mitosis occurred after spores were delimited and prior to morphological maturation. Mature spores had up to eight nuclei. That most spores had compatible mating-type nuclei was indicated by most single germinated spores having clamped mycelium with dikaryotic cells. In Hydnangium carneum , a hypogeous relative of Laccaria , post-meiotic mitosis occurred in the basidium and two nuclei migrated into each of the four spores. Migration patterns indicated that some spores had compatible mating-type nuclei, as in L. fraterna . Allocation of compatible mating types to spores partly explains why L. fraterna ‘weed-like’ behaviour and is a pioneer in plantations of eucalypts on former farmland or rehabilitated mine sites. Mycorrhizas of these fungi have been synthesized aseptically with several species of Eucalyptus .

Delimitation of Hymenogaster sensu stricto and four new segregate genera

Since Vittadini first described Hymenogaster in 1831, a heterogeneous assemblage of truffle-like Basidiomycetes has been assigned to the genus. As a consequence, the boundaries of Hymenogaster became inflated even beyond Vittadinis original broad concept, and the genus came to represent more than one phylogenetic line. This paper clarifies the generic limits of Hymenogaster and challenges a prevailing notion that Hymenogaster represents the hypogeous member of a phylogenetic line linked through Thaxterogaster to Cortinarius. On the basis of both macromorphological and micromorphological characters of basidiomes, Hymenogaster sensu stricto is redefined. Selected species are allocated to four new genera: Cortinomyces, Descomyces, Quadrispora, and Timgrovea. A key to these genera is provided. Four of the eight original Vittadini species are excluded from Hymenogaster: H. rufus (type lost), H. citrinus (to Gautieria), H. luteus (to Hysterogaster), and H. niveus (to Cortinomyces). The remaining four species have large, thick-walled, broad ellipsoid to fusiform spores bearing a large, cupped hilar appendix and are designated as the core of Hymenogaster since they include the type species H. bulliardi. Also included with the type are H. olivaceus, H. lycoperdineus and H. griseus. The relationships of these Hymenogaster species to other fungi are not known, but the spore type does not indicate a close relationship with Cortinarius and Thaxterogaster. The remaining Vittadini species H. niveus is placed in the new genus Cortinomyces by virtue of its smaller, warty spores. Cortinomyces is largely distinguished from Hymenogaster by having cortinarioid spores. Numerous other characters, such as peridial pigments and structure, suggest that Cortinomyces fits into a phylogenetic series with Thaxterogaster and Cortinarius. Hymenogaster cribbiae, H. effodiendus, H. luteus (non Vittadini), H. niveus, H. purpureus, H. violaceus, and H. viscidus are recombined to Cortinomyces. Descomyces has distinctive spore morphology (e.g., a smooth rostrum and ornamentation embedded in the perisporium) and peridium structure (e.g., two layered and with swollen cells). This peculiar combination of peridial and spore characteristics also occurs in Setchelliogaster and in Descolea. It is proposed that Descomyces (with hypogeous angiocarpic basidiomes and a loculate hymenium) represents the truffle-like form in a phylogenetic series that also includes Setchelliogaster (subhypogeous, pseudoangiocarpic basidiomes) and Descolea (epigeous bivelangiocarpic basidiomes and a lamellate hymenium). Further supporting evidence of this relationship is obtained from examination of mycorrhizae and axenic cultures of these fungi. Species transferred to Descomyces include H. albellus, H. albus, and H. javanicus. H. albellus and H. albus are maintained as separate species, the former is considered to include collections having a polycystoderm (epithelium). Quadrispora includes species with assymetrical spores that adhere in tetrads after release from the basidium. H. oblongisporus is recombined into the new genus, and Q. musispora is described as new. The relationships of Quadrispora to other fungi are not known. Finally, Timgrovea is proposed to accommodate species with reticulate spores, T. reticulatus, T. macrosporus, T. subtropicus, and T. ferrugineus from Australia, and T. kwangiensis from China. The relationships of Timgrovea probably occur outside the Cortinariaceae. A possible relationship of Timgrovea to the Boletaceae is discussed.

Nuclear behaviour in the basidiomes and ectomycorrhizas of Hebeloma westraliense sp. nov.

Hebeloma westraliense is described, and its identity as a new species established by comparison with other species of the genus. It formed ectomycorrhizas in aseptic and pot cultures with species of Eucalyptus and Allocasuarina , and basidiomes were produced in pot cultures with Eucalyptus camaldulensis, E. teretecornis and E. diversicolor . Septa in the stipe and pileus including those delimiting the basidium had clamp connexions, and cells in both juvenile and mature tissues were dikaryotic. Basidia were consistently quadrisporic and spores had two nuclei. In developing basidia a post-meiotic nucleus migrated into each spore, and a post-meiotic mitosis occurred in spores after septum delimitation but prior to morphological maturation. Mature spores should therefore be homokaryons and this view was supported by observations of spore germination. mYcelium produced by single spores was monokaryotic and without clamp connexions.