Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus‐avis (L.) L.C.M. Rich. and neighbouring tree ectomycorrhizae

Several achlorophyllous orchids associate with ectomycorrhizal hymenomycetes deriving carbon from surrounding trees for the plant. However, this has not been shown for achlorophyllous orchids associating with species of Rhizoctonia, a complex of basal lineages of hymenomycetes that are the most common orchid partners. We analysed Neottia nidus‐avis, an achlorophyllous orchid symbiotic with a Rhizoctonia, to identify its symbionts by internal transcribed spacer (ITS) sequencing. Analysis of 61 root systems from 23 French populations showed that N. nidus‐avis associates highly specifically with a group of species of Sebacinaceae. Their diversity emphasizes the need for further investigations in the Sebacinaceae systematics. Sebacinoid ITS sequences were often identical within orchid populations and a trend to regional variation in symbionts was observed. Using ITS and intergenic spacer (IGS) polymorphism, we showed that each root system harboured a single species, but that several genets colonized it. However, no polymorphism of these markers was found among portions of each root: this is consistent with the putative mode of entry of the fungus, i.e. from the rhizome into roots but not repeatedly from the soil. In addition, ectomycorrhizae were always found within the N. nidus‐avis root systems: 120 of the 144 ectomycorrhizae typed by ITS sequencing were colonized by a sebacinoid fungus identical in ITS sequence to the respective orchid symbiont (even for the IGS polymorphism in some cases). Because sebacinoids were demonstrated recently to be ectomycorrhizal, the orchid is likely to derive its resources from surrounding trees, a mycorrhizal cheating strategy similar to other myco‐heterotrophic plants studied to date.

Related papers:

title: Phylogeny and classification of the orchid family
abstract: 1.  Structure and other evidence of relationship 3. Orchid phylogeny 4. Orchids with two or three fertile anthers 5. Primitive orchids with a single fertile anther 6. Lady's tresses and relatives, the subfamily Spiranthoideae 7. Orchis and its allies, the subfamily Orchidoideae 8. The advanced Epidendroideae, general discussion and phylogeny 9. The cormous or cymbidioid phylad 10. The reed-stem or epidendroid phylad 11. Misfits, parallelisms, and miscellaneous problems 12. Classification and phylogenetic analysis Glossary Appendices Literature cited Index.

title: Terrestrial Orchids: From Seed to Mycotrophic Plant
abstract: Acknowledgements Introduction 1. Properties of 'dust' seeds 2. Seed development 3. Seed survival 4. Requirements for germination 5. Fungi 6. Germination processes 7. Underground organs 8. Orchid mycorrhiza 9. Abiotic factors in growth and development 10. Life history and phenology 11. Propagation 12. Effects of orchid mycorrhiza 13. Descriptions of genera Appendix A: nutrient substrates mentioned in the text Appendix B: names and synonyms References Index.

title: The biology of myco-heterotrophic ('saprophytic') plants.
abstract: More than 400 species of vascular plants, in 87 genera, are acholophyllous and heterotrophic, but not directly parasitic upon autotrophs. They are usually, but incorrectly, described as 'saprophytes'since they are in fact nourished by means of specialized mycorrhizal associations. Although distributed world-wide, they are most abundant and show the greatest species-richness in the Neotropics and Palaeotropical regions. Their aerial parts range in size from a few centimetres to extensive liane types up to 40 m long. With few exceptions, their habitats are dense moist forests in which there is a surface accumulation of leaf litter, often in situations which are too shaded for autotrophic growth. Although the achlorophyllous mycorrhizal mode of life has evolved independently many times and in widely disparate taxonomic groups, such plants show strong convergent evolution in particular adaptations to their peculiar mode of life. Most prominant amongst these are reductions in the size of seed and embryo, and the lack of differentiation of the embryo at maturity. The number of seeds produced by each flower is typically very large and the shape, structure and surface features of seeds involving adaptation for wind dispersal show remarkable parallels in many species. Specific adaptations for zoochory are rare but well developed in a small number of genera, some of which produce scents like fungal fruit bodies or floral parts which mimic fungal sporocarps. Vegetative parts are often even more conspicuously reduced. Most myco-heterotrophs are entirely subterranean for most of their lives and these stages exhibit adaptations consistent with a change in function from organs of absorption to organs of storage, shown by the almost universal loss of root hairs, decrease in surface area as exhibited in short cylindric'vermiform'and tuberous roots or, in extreme cases, the complete suppression of roots and the formation of a swollen tuber or rhizome. Increased width of the root cortex often accommodates mycorrhizal infection and stores of carbohydrates and other materials obtained from the fungal symbiont. Mycorrhizal infection is confined to the below-ground parts of the plants but may be found there in modified stems as well as in roots. In many genera, stems are exceptionally slender and thread-like and their vascular tissues are either reduced to a single narrow cylinder of bicollateral bundles or, minimally, to four or six narrow bundles in the cortex. Secondary thickening is poorly developed in all but a tiny minority of species, lignification being confined to annular or, rarely, a few scalariform xylem vessels. Phloem is present in very small amounts and then mainly as parenchyma with sieve tubes frequently recorded as narrow and possibly with abherent sieve plates. Leaves are typically reduced to widely spaced achlorophyllous scales on the inflorescence axis. Occasionally, they are present only on underground rhizomes or tubers. The vascular supply to the leaf-scales, normally reduced to a single trace, may be absent. Vestigial stomata are sometimes found on leaves and, in a few species which retain traces of chlorophyll, on shoots but, in most fully heterotrophic species, stomata are absent from aerial parts. Since their seeds are very small and contain minimal reserve carbohydrates, the germination of myco-heterotrophs in nature would appear to depend upon infection by an appropriate symbiotic fungus at an early stage. The nature of the carbohydrates transferred from the fungus to the plants has not been determined. Once acquired from The fungal partner, most plants store carbon in a variety of forms, the most common of which is starch, although other compounds including glucomannan, fructan and calcium oxalate art important in some specks. Asexual reproduction is frequently important with root tubers, tubercles and rhizomes providing the means of vegetative spread. Nonetheless, all the angiospermous species recorded to date also reproduce sexually. Floral structures show varying degrees of reduction concomitant with myco-heterotrophy. Inflorescences are typically small, often with a single terminal flower, and the floral parts often show extreme simplification, with the production of unilocular or, more rarely, bilocular and trilocular ovaries. In some of the most highly adapted species, there is reduction of integumentary layers surrounding each ovule from the normal bitegmic condition to unitegmic or, occasionally, ategmic. With the principal exception of the Monotropaceae, placentation is typically trimerous and parietal. Flowers normally appear to be cross-pollinated and are brightly coloured. nectiferous, occasionally scented, and can demonstrate extreme morphological adaptations which attract insects as in the production of lone caudate tepals or fungus-mimicking structures. Much is still to be learned about the adaptive features and especially about the physiology of these plants and of their early developmental stages during which the essential associations with fungi are established. Similarly, studies of the taxonomy and physiology of most of their fungal partners are still in their infancy. Contents Summary 171 I. Introduction 172 II. Taxonomic and phylogenetic relationships of myco-heterotrophic plants 174 III. Distribution patterns 180 IV. Habitats 183 V. Embryology 185 VI. Characteristics of seeds 186 VII. Mycorrhizal infection 192 VIII. Morphologies of roots 196 IX. Characteristics of shoots 199 X. Carbon assimilation and storage by mycoheterotrophic plants 202 XI. Reproduction 208 XII. Mutualism or parasitistn? 210 XIII. Future directions for research in mycoheterotrophic plants 210 XIV.  Acknowledgements 211 References 211.

title: High root concentration and uneven ectomycorrhizal diversity near Sarcodes sanguinea (Ericaceae): a cheater that stimulates its victims?
abstract: Sarcodes sanguinea is a nonphotosynthetic mycoheterotrophic plant that obtains all of its fixed carbon from neighboring trees through a shared ectomycorrhizal fungus. We studied the spatial structuring of this tripartite symbiosis in a forest where Sarcodes is abundant, and its only fungal and photosynthetic plant associates are Rhizopogon ellenae and Abies magnifica, respectively. We found disproportionately high concentrations of Abies roots adjacent to Sarcodes roots compared to the surrounding soil. Rhizopogon ellenae colonizes the vast majority of those Abies roots (86-98%), and its abundance tends to decrease with increasing distance from Sarcodes plants. At 500 cm from Sarcodes plants we did not detect R. ellenae, and the ectomycorrhizal community instead was dominated by members of the Russulaceae and Thelephoraceae, which are commonly dominant in other California pinaceous forests. The highly clumped distribution of Abies-R. ellenae ectomycorrhizas indicates that Sarcodes plants either establish within pre-existing clumps, or they stimulate clump formation. Several lines of evidence favor the latter interpretation, suggesting an unexpected mutualistic aspect to the symbiosis. However, the mechanism involved remains unknown.

title: In vitro germination of nonphotosynthetic, myco-heterotrophic plants stimulated by fungi isolated from the adult plants.
abstract: Sarcodes sanguinea and Pterospora andromedea (Ericaceae, Monotropoideae) are nonphotosynthetic mycoheterotrophic plants. Recent studies have shown that the roots of the adult plants are always associated with closely related but exclusive sets of Rhizopogon species (Basidiomycota, Boletales) from section Amylopogon. We have isolated Rhizopogon species that were associated with the adult plants and used them to germinate seeds under gnotobiotic conditions. All Rhizopogon species isolated from either plant species were capable of stimulating seed germination in both Sarcodes and Pterospora. Under the primary conditions used, germination varied from 9 to 73% in the case of Sarcodes and 0 to 13% in that of Pterospora. The single Rhizopogon strain that failed to elicit germination in Pterospora under these conditions did stimulate germination under slightly dierent conditions. By contrast, seeds failed to germinate on all media which lacked these Rhizopogon species, or in the presence of six other genera of basidiomycetes. Seed germination could be stimulated either through cellophane or at the edge of fungal colonies without direct fungus‐seed contact. These results suggest that a diusible or volatile compound that is unique to Rhizopogon stimulates germination of these plant seeds. Seed lots of Sarcodes from two successive years had similar germination levels. Sarcodes seeds that had overwintered under natural conditions were also stimulated to germinate. These results demonstrate the potential for long-term dormancy. We suggest that a combination of dormancy and the use of specific germination cues might increase the opportunities of these plants for recruitment. In addition, the specific germination response explains at least a part of the specialized associations observed in the adult plants. Nevertheless, the seeds respond to a slightly broader range of Rhizopogon species than has been observed to be associated with the adult plants; thus other factors must also be involved with specificity under natural conditions.

title: Extreme specificity in epiparasitic Monotropoideae (Ericaceae): widespread phylogenetic and geographical structure
abstract: The Monotropoideae (Ericaceae) are nonphotosynthetic plants that obtain fixed carbon from their fungal mycorrhizal associates. To infer the evolutionary history of this symbiosis we identified both the plant and fungal lineages involved using a molecular phylogenetic approach to screen 331 plants, representing 10 of the 12 described species. For five species no prior molecular data were available; for three species we confirmed prior studies which used limited samples; for five species all previous reports are in conflict with our results, which are supported by sequence analysis of multiple samples and are consistent with the phylogenetic patterns of host plants. The phylogenetic patterns observed indicate that: (i) each of the 13 plant phylogenetic lineages identified is specialized to a different genus or species group within five families of ectomycorrhizal Basidiomycetes; (ii) mycorrhizal specificity is correlated with phylogeny; (iii) in sympatry, there is no overlap in mature plant fungal symbionts even if the fungi and the plants are closely related; and (iv) there are geographical patterns to specificity.

title: Phylogeny and Ecology of Mycotrophic Achlorophyllous Angiosperms
abstract: Achlorophyllous genera of angiosperms, such as Galeola (Orchidaceae), Leiphaimos (Gentianaceae), and Monotropa (Pyrolaceae), have evolved highly specialized, mycotrophic root systems, along with chlorosis and complex floral morphologies. Productivity rates of most such plants cannot be satisfactorily explained by the saprophytic niche which is often assumed as theirs but is inadequately nutritious. Rather, they parasitize their mycorrhizal fungi. When a mycorrhizal fungus is shared by roots of an achlorophyllous angiosperm an those of a photosynthesizing plant, the former can indirectly parasitize the latter via the connecting mycelium (epiparasitism). The functional mycelial connection between the mycorrhizae of epiparasites and those of associated host trees has been demonstrated by radiotracer techniques. Some epiparasites are very locally distributed. The ecological requirements of their mycorrhizal fungi, such a suitable host trees, may explain much of this localization. A taxonomic survey of the Orchidaceae and Pyrolaceae reveals coherent, continous lines of evolution from green, leafy taxa with fibrous root systems to progressively more chlorotic taxa with reduced root systems. The widely distributed genera Hypopitys and Montropa of north temperate forests represent highly successful occupants of a widespread, mycotrophic-epiparasitic niche for angiosperms.

title: Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids.
abstract: We have investigated the mycorrhizal associations of two nonphotosynthetic orchids from distant tribes within the Orchidaceae. The two orchids were found to associate exclusively with two distinct clades of ectomycorrhizal basidiomycetous fungi over wide geographic ranges. Yet both orchids retained the internal mycorrhizal structure typical of photosynthetic orchids that do not associate with ectomycorrhizal fungi. Restriction fragment length polymorphism and sequence analysis of two ribosomal regions along with fungal isolation provided congruent, independent evidence for the identities of the fungal symbionts. All 14 fungal entities that were associated with the orchid Cephalanthera austinae belonged to a clade within the Thelephoraceae, and all 18 fungal entities that were associated with the orchid Corallorhiza maculata fell within the Russulaceae. Restriction fragment length polymorphism and single-strand conformational polymorphism analysis of ectomycorrhizal tree roots collected adjacent to Cephalanthera showed that (i) the fungi associated internally with Cephalanthera also form typical external ectomycorrhizae and that (ii) ectomycorrhizae formed by other Basidiomycetes were abundant where the orchid grows but these fungi did not associate with the orchid. This is the first proof of ectomycorrhizal epiparasitism in nature by an orchid. We argue that these orchids are cheaters because they do not provide fixed carbon to associated fungi. This view suggests that mycorrhizae, like other ancient mutualisms, are susceptible to cheating. The extreme specificity in these orchids relative to other ectomycorrhizal plants agrees with trends seen in more conventional parasites.

title: Symbiotic germination and development of myco-heterotrophic plants in nature: ontogeny of Corallorhiza trifida and characterization of its mycorrhizal fungi.
abstract: The processes of symbiotic germination and seedling development were analysed in the myco-heterotrophic orchid Corallorhiza trifida, seeds of which were buried in 'packets' either adjacent to or at varying distances from adult plants in defined communities of ectomycorrhizal tree species. Germination occurred within eight months of burial under Betula-Alnus and within seven months under Salix repens. It was always associated with penetration of the suspensor by a clamp-forming mycorrhizal fungus. Four distinct developmental stages were defined and the rates of transition through these stages were plotted. There was no evidence of a relationship between extent of germination or rate of development and the presence of naturally distributed plants of C. trifida at the spatial scale of 1 m. The best germination and the most rapid rate of development of C. trifida seedlings occurred in a Salix repens community located at a considerable distance from any extant C. trifida population. Determination of internal transcribed spacer (ITS) RFLPs and of gene sequences of the fungi involved in symbiotic germination and growth of C. trifida, revealed them to belong exclusively to the Thelephora-Tomentella complex of the Thelephoraceae. These fungi are known also to be ectomycorrhizal associates of trees. It is hypothesized that the rate of growth of the C. trifida seedlings is determined by the ability of the fungal symbionts to transfer carbon from their ectomycorrhizal co-associates.

title: War in the world of lichens: parasitism and symbiosis as exemplified by lichens and lichenicolous fungi
abstract: Lichens are typically formed by the association of two partners. The fungus (mycobiont) usually comprises most of the lichen, while the photosynthetic algal or cyanobacterial partner is referred to as the photobiont. Many lichens contain more than one photobiont and other associates, the lichenicolous fungi, may also be involved. The spectrum of interactions is fascinating and ranges from mutualism, through commensalism, to situations where one, or sometimes more, of the associates is doomed by the antagonistic activities of an invading fungus. Furthermore, these interactions are dynamic over time. Many mycologists assume that once a lichen ascospore has contacted a suitable photobiont, or once a dual propagule such as a soredium, isidium or lichen fragment has landed on an appropriate surface, a lichen thallus will develop. Accepted dogma is that the thallus will grow to form a mature lichen surviving for tens, if not hundreds or even thousands of years. These are over-simplifications; in fact there is war in the world of lichens. Following a brief consideration of parasitism and other forms of symbiosis, this war will be illustrated under the headings of algal slaves, exploiters of two kingdoms, alien invaders, cosy niche seekers and take-over specialists. It is hoped that this review will encourage mycologists to study these interactions which, as yet, remain largely unexplored.

title: EVIDENCE FOR A FUNGAL LIAISON BETWEEN CORALLORHIZA TRIFIDA (ORCHIDACEAE) AND PINUS CONTORTA (PINACEAE)
abstract: Corallorhiza trifida Châtelain, or pale coral root orchid, is a heterotrophic, leafless, rootless, terrestrial orchid with a circumboreal distribution. Because of its relative inability to photosynthesize, the orchid obtains energy through the digestion of fungal hyphae that grow within the cells of its contorted, yellowish, coralloid rhizomes. Recently, we isolated and cultured strains of a slow-growing basidiomycete with bright yellow, clamped hyphae that are typical of the fungal cells present in C. trifida endomycorrhizas from different treed habitats at widely distributed locations in the northern hemisphere. By inoculating the roots of Pinus contorta Douglas ex Loudon seedlings with this fungus we were able to demonstrate its ability to form distinctive ectomycorrhizas with an ectotrophic, woody plant. The formation of endomycorrhizas with C. trifida and ectomycorrhizas with P. contorta indicates that in nature a triple symbiosis, with a circumboreal distribution, exists among certain trees, the cor...

title: The Rhizoctonia endophytes of Rhizanthella (Orchidaceae)
abstract: The endophytes of the endemic Australian underground orchids, Rhizanthella gardneri from Western Australia and R. slateri from New South Wales and southern Queensland, are rhizoctonias that form endomycorrhizas with the orchids and ectomycorrhizas with many other plants. Isolates from the two orchids differ in cultural characters but whether they are different species cannot be decided on anamorphs alone. The isolates from R. gardneri formed a teleomorph that is described as Thanatephorus gardneri sp. nov. In Western Australia Rhizanthella gardneri has only been found closely associated with the shrub Melaleuca uncinata as ectomycorrhizal companion plant. Seeds of the orchid, however, germinated and formed protocorms in association with Thanatephorus gardneri on 4 species of Eucalyptus . Whether R. gardneri can grow to maturity in association with eucalypts remains unresolved.

title: Mycorrhizal associations of isolates of Sebacina vermifera
abstract: Isolates of Sebacina vermifera Oberwinkler (Tremellaceae), identified by their teleomorphs, from orchid and non-orchid hosts have been examined for their mycorrhizal associations. All isolates stimulated germination of seed of species of Microtis (Orchidaceae). Three isolates from ectomycorrhizal hosts formed ectomycorrhizas on Melaleuca uneinata R. Br. ex Alton I. and other ectomycorrhizal plants. None of the orchid isolates nor an isolate which was endophytic in Phyllanthus formed ectomycorrhizas but in addition to species of Micron's they stimulated germination of one or more species of Caladenia, Cyrtostylis or Glossodia but not other terrestrial orchids. 
 
Protocorms of Microtis developed at depth in soil in the presence but not the absence of ectomycorrhizal S. vermifera on M. uneinata. Young albino plants of Microtis rara R. Br. grew for 4 months in the presence of ectomycorrhizal S. vermifera with M. uneinata but mycorrhizal albino plants on their own all died, suggesting a possible tripartite mycorrhizal association.

title: Population, habitat and genetic correlates of mycorrhizal specialization in the ‘cheating’ orchids Corallorhiza maculata and C. mertensiana
abstract: Unlike photosynthetic plants, several distantly related nonphotosynthetic plants are highly specialized toward their mycorrhizal fungi. It is unknown whether this specialization varies geographically or is influenced by the environment. We have investigated these questions in the nonphotosynthetic orchids Corallorhiza maculata and C. mertensiana by amplifying fungal internal transcribed spacer (ITS) fragments from widespread mycorrhiza samples and then discriminating putative fungal species using ITS restriction fragment length polymorphisms (RFLPs). Three fungal species were found across 27 plants representing seven populations of C. mertensiana; 20 species were found across 104 plants and 21 populations of C. maculata. All fungi belonged to the Russulaceae, an ectomycorrhizal family. Partitioning of Simpson’s diversity showed that 48% of the variance in occurrences of fungal species coincided with population boundaries in C. mertensiana, vs. 68% in C. maculata. This differentiation coincided with geography but not habitat in C. mertensiana. In contrast, likelihood ratio tests showed strong associations between fungal occurrence and both habitat and phenotype in C. maculata. For example, C. maculata populations growing under oaks had no fungi in common with nearby populations growing under conifers, and those above 2000 m had no fungi in common with those below 2000 m. However, plant genetic differentiation may underlie some of these patterns. C. mertensiana and C. maculata never shared fungal species, even when growing intermixed at the same site, demonstrating genetic control that was independent of habitat. Similarly, intermixed normal and pale‐coloured variants of C. maculata had no fungal species in common. These results demonstrate fine‐scale genetic influences and geographical mosaicism in a mycorrhizal interaction.

title: SOME OBSERVATIONS WITH LIGHT AND ELECTRON MICROSCOPE ON THE ENDOTROPHICMYCORRHIZA OF ORCHIDS
abstract: The endotrophic mycorrhiza of four heterotrophic and four autotrophic orchid species have been investigated by light and electron microscopy. Contrary to what has been found in some other plants, the cell wall of the cortex cells of the orchids lacks lignin which might preclude the invasion of fungi. Cellulose and pectin are attacked by the mycorrhizal fungi; these were all found to produce cellulase and pectinase. Within the cells of the plant, the hyphae of the fungus were found always to be enveloped within the invaginated plasmalemma which continues forming pectin and cellulose and, in the case of exodermal cells, may also form suberin. These pectin and cellulose layers are rather thin or even seem to be lacking in cells with young and active mycorrhizal fungi; on the other hand they are clearly visible in cells with decaying hyphae. It may be inferred that the active fungus digests pectin and cellulose about as fast as they are formed by the plant cell plasmalemma. 
 
 
 
Decaying hyphae and the pectin and cellulose layer around them are usually found in the central part of the fungus-digesting cells and form a clot, which is also enveloped by a pectin-cellulose layer. During the process of digestion the plasmalemma shows pinocytosis.

title: A phylogenetic analysis of the Orchidaceae: evidence from rbcL nucleotide.
abstract: Cladistic parsimony analyses of rbcL nucleotide sequence data from 171 taxa representing nearly all tribes and subtribes of Orchidaceae are presented here. These analyses divide the family into five primary monophyletic clades: apostasioid, cypripedioid, vanilloid, orchidoid, and epidendroid orchids, arranged in that order. These clades, with the exception of the vanilloids, essentially correspond to currently recognized subfamilies. A distinct subfamily, based upon tribe Vanilleae, is supported for Vanilla and its allies. The general tree topology is, for the most part, congruent with previously published hypotheses of intrafamilial relationships; however, there is no evidence supporting the previously recognized subfamilies Spiranthoideae, Neottioideae, or Vandoideae. Subfamily Spiranthoideae is embedded within a single clade containing members of Orchidoideae and sister to tribe Diurideae. Genera representing tribe Tropideae are placed within the epidendroid clade. Most traditional subtribal units are supported within each clade, but few tribes, as currently circumscribed, are monophyletic. Although powerful in assessing monophyly of clades within the family, in this case rbcL fails to provide strong support for the interrelationships of the subfamilies (i.e., along the spine of the tree). The cladograms presented here should serve as a standard to which future morphological and molecular studies can be compared.

title: ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts
abstract: We have designed two taxon‐selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1‐F and ITS4‐B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4‐B, when paired with either a ‘universal’ primer ITS1 or the fungal‐specific primer ITS1‐F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1‐F/ITS4‐B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the ‘universal’ ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1‐F/ITS4‐B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust‐infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.

title: Phylogenetic relationships in Auriculariales and related groups: hypotheses derived from nuclear ribosomal DNA sequences
abstract: In order to estimate phylogenetic relationships in the Auriculariales sensu Bandoni (1984) and allied groups we have analysed a representative sample of species by comparison of nuclear coded ribosomal DNA sequences, applying models of neighbour joining, maximum parsimony, conditional clustering, and maximum likelihood. Analyses of the 5’ terminal domain of the gene coding for the 28 S ribosomal large subunit supported the monophyly of the Dacrymycetales and Tremellales, while the monophyly of the Auriculariales was not supported. The Sebacinaceae, including the genera Sebacina, Efibulobasidium, Tremelloscypha, and Craterocolla, was confirmed as a monophyletic group, which appeared distant from other taxa ascribed to the Auriculariales. Within the latter the following subgroups were significantly supported: (1) a group of closely related species containing members of the genera Auricularia, Exidia, Exidiopsis, Heterochaete, and Eichleriella; (2) a group comprising species of Bourdotia and Ductifera; (3) a group of globose-spored species of the genus Basidiodendron; (4) a group that includes the members of the genus Myxarium and Hyaloria pilacre; (5) a group consisting of species of the genera Protomerulius, Tremellodendropsis, Heterochaetella, and Protodontia. Additional analyses of the internal transcribed spacer (ITS) region of the species contained in group (1) resulted in a separation of these fungi due to their basidial types.

title: CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP
abstract: The recently‐developed statistical method known as the “bootstrap” can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority‐rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.

title: The neighbor-joining method: a new method for reconstructing phylogenetic trees.
abstract: A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.

title: Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees.
abstract: Examining the pattern of nucleotide substitution for the control region of mitochondrial DNA (mtDNA) in humans and chimpanzees, we developed a new mathematical method for estimating the number of transitional and transversional substitutions per site, as well as the total number of nucleotide substitutions. In this method, excess transitions, unequal nucleotide frequencies, and variation of substitution rate among different sites are all taken into account. Application of this method to human and chimpanzee data suggested that the transition/transversion ratio for the entire control region was approximately 15 and nearly the same for the two species. The 95% confidence interval of the age of the common ancestral mtDNA was estimated to be 80,000-480,000 years in humans and 0.57-2.72 Myr in common chimpanzees.

title: MODELTEST: testing the model of DNA substitution
abstract: The program MODELTEST uses log likelihood scores to establish the model of DNA evolution that best fits the data.


AVAILABILITY
The MODELTEST package, including the source code and some documentation is available at http://bioag.byu. edu/zoology/crandall_lab/modeltest.html.

title: PERFECT STATES OF RHIZOCTONIAS ASSOCIATED WITH ORCHIDS. III
abstract: Further Rhizoctonia isolates obtained from Australian orchids have been induced to fruit in culture. The perfect states thus obtained include Tulasnella allantospora Wakefield and Pearson, T. violea (Quelet) Bourdot and Galzin, and two new species, T. cruciata and Ceratobasidium sphaerosporum. The description of Tulasnella asymmetrica is emended.

title: Rhizanthella gardneri (Orchidaceae), its rhizoctonia endophyte and close association with Melaleuca uncinata (Myrtaceae) in Western Australia
abstract: Rhizanthella gardneri R. S. Rogers, a rare subterranean achlorophyllous orchid which grows in Western Australia near plants of the Myrtaceous shrub Meialeuca uncinata R. Br. ex Alton f., is associated with an undescribed species of Rhizoctonia which forms hyphal coils within cortical cells of the orchid rhizome. The Rhizoctonia also forms ectomycorrhizas with sheath and Hartig net on Meialeuca uncinata and other ectomycorrhizal plants including Eucalyptus. 
 
Seeds of R. gardneri may germinate without the Rhizoctonia but their growth soon ceases unless the protocorm is invaded by the fungus through the protocorm hairs. Two plants of R. gardneri raised from seed in association with Rhizoctonia ectomycorrhizas on M. uncinata have flowered after 15 months. 
 
The Rhizanthella endophyte stimulated germination of seed of R. gardneri but not seed of two chlorophyllous terrestrial orchids, Prasophyllum regium R. Rogers and Microtis unifolia (Forster f.) Reichb. f. However, germination of these two orchids was stimulated by five of six other Rhizoctonias, including Tkanatephorus cucumeris (Frank) Donk and two species of Tulasnella, also isolated from soil and litter from two Rhizanthella sites.

title: A widely distributed ITS polymorphism within a biological species of the ectomycorrhizal fungus Hebeloma velutipes
abstract: The ectomycorrhizal fungus Hebeloma velutipes consists of two biological species (BSP 16 and 17). Within BSP 17 a dikaryon was found with two divergent types of the ribosomal Internal Transcribed Spacer (ITS1 and 2). The two ITS types segregated in monokaryotic progeny of that dikaryon, showing that these different ITS types represent different alleles at homologous rDNA loci in the two nuclei. RFLP analysis of a number of strains of BSP 17 showed that the polymorphism is widespread in Europe. There was no deficiency of the heterokaryotic type, demonstrating that ITS divergence in this species is not correlated with reduced intercompatibility. A strain from North America, not assigned to a biological species, showed the same polymorphism. Cladistic analysis of the two ITS sequences showed that they were not sister groups. One of the ITS types formed a monophyletic group together with the ITS type of BSP 16, the other type formed a clade with the ITS type of H. incarnatulum (BSP 18). BSP 16 and 17 showed partial intercompatibility. However, several lines of evidence suggest that the polymorphism of BSP 17 is not the result of frequent and continuing hybridisation with BSP 16. Instead, we give arguments for the hypothesis that the polymorphism evolved in allopatry and that the two types have come together relatively recently. The results of the polymorphism indicate a potential problem for molecular identification of fungal species based on ITS fingerprinting. The results also show that no generalisations are possible about the relation of speciation (the formation of BSP) and nuclear ITS divergence.

title: Tremelloscypha gelatinosa, a species of a new family sebacinaceae
abstract: Eichleriella gelatinosa Murrill, known only from the type locality in Jamaica and from Florida, is transferred to Tremelloscypha on the basis of its coriaceous, flabellate to infundibuliform basidiocarps, the relatively thick-walled, brownish hyphae lacking clamps in the trama, and the ovate to pyriform basidia lacking basal clamps. A new family, Sebacinaceae, is proposed to include Tremelloscypha, Sebacina s. str., and Tremellodendron. It is suggested that Efibulobasidium be tentatively assigned to the Sebacinaceae. This arrangement brings together several taxa that possess similar basidial and hyphal characters but whose basidiocarps vary from pustulate or incrusting to erect and branching or flabellate through infundibuliform.

title: Molecular diversity of ericoid mycorrhizal fungi
abstract: Using restriction fragment length polymorphism (RFLP) patterns from two ribosomal internal transcribed spacers (ITS) and DNA sequences from ITS2, we characterized ericoid mycorrhizal fungal isolates from culture collections.With a synoptic key to RFLP patterns, we divided 34 mycorrhizal or root-associated isolates into 16 groups. RFLP patterns were identical when fungal specific primers were used to amplify DNA from pure fungal cultures and in vitro mycorrhizae. Sequence analysis clustered 23 of 24 mycorrhizal isolates into two larger groups: the Oidiodendron group and the Hymenoscyphus group. The Oidiodendron group included genetically uniform, conidiating fungi. The Hymenoscyphus group encompassed more diversity and included other discomycetes (Leotiales) as well as sterile, unidentifiable mycorrhizal isolates from four RFLP groups. Results from our field site on Vancouver Island, British Columbia, Canada, suggest that several ericoid mycorrhizal fungi can coexist in a single root of Gaultheria shallon Pursh and that our molecular data base is not yet complete. From sixty 3-mm root sections, we cultured four genetically different fungi that formed mycorrhizae in resynthesis experiments and sequence analysis showed that one of these differed from all previously known ericoid mycorrhizal fungi.

title: Basal hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees.
abstract: • Heterobasidiomycetous species of the Sebacinaceae family, previously considered as saprophytes or parasites, are shown here to form ectomycorrhizas on temperate forest trees. • Ectomycorrhizas were collected under sebacinoid sporophores and near root systems of Neottia nidus-avis, an orchid symbiotic with sebacinoids. To identify the partners each ectomycorrhiza was submitted to amplification and sequencing of the plant and fungal internal transcribed spacer (ITS), and further investigated by light and electron microscopy whenever a sebacinoid ITS was found. • Molecular and microscopic analyses correlated well. Two sebacinoids of divergent rDNA sequences were demonstrated to form similar ectomycorrhizas, with a well-developed Hartig net and a hyphal mantle having thick-walled outer mantle hyphae. The ultrastructure of the septal pore (dolipore with imperforate caps) was typical for sebacinoids. In one case, intracellular colonization was seen. The ectomycorrhizal host range of these sebacinoids was not specific and included Betulaceae, Fagaceae and Tiliaceae. • Sebacinoids probably represent an overlooked ectomycorrhizal group and ectomycorrhizal symbiosis may be common among basal lineages of hymenomycetes.

title: Orchidaceous rhizoctonias in pot cultures of vesicular–arbuscular mycorrhizal fungi
abstract: The paper reports that previously undescribed, sterile, septate fungi (Rhizoctonia) with affinity to and attributes of orchid mycorrhizal fungi, commonly occur in pot cultures of vesicular–arbuscular (V–A) mycorrhizal fungi. Seventeen pot cultures of V–A endophytes from several sources were studied. The endophytes included unidentified organisms as well as species of Glomus, Acaulospora, and Gigaspora. A Rhizoctonia was present in every pot culture. In different cases, Rhizoctonia isolates were obtained from sporelike cells in intramatrical vesicles, extramatrical hyphae, and chlamydospores or roots of pot culture plants. In pure culture, the rhizoctonias formed pale or yellow–brown, submerged colonies composed of narrow, irregularly septate hyphae. Monilioid hyphae and terminal or intercalary, spherical chlamydospores about 12 μm in diameter developed in older mycelia. Fruiting experiments by J. H. Warcup indicated that the teleomorph of three Rhizoctonia isolates is related to Sebacina vermifera Oberwin...

title: Exidiopsis species from Devon, including the new segregate genera Ceratosebacina, Endoperplexa, Microsebacina, and Serendipita
abstract: Ten species currently referable to the tremellaceous genus Exidiopsis are described from Devon, England, together with two Sebacina species, one from the New Forest. Only those species congeneric with the type species are retained in Exidiopsis . Four new genera, Ceratosebacina, Endoperplexa, Microsebacina , and Serendipita are proposed to accommodate the rest. Three new species are described: Endoperplexa dartmorica, Serendipita orliensis , and Serendipita sigmaspora . New combinations are proposed for six British species: Ceratosebacina longispora, Endoperplexa septocystidiata, Microsebacina fugacissima, Microsebacina microbasidia, Serendipita vermifera , and Serendipita evanescens ; and for five extra-limital species: Endoperplexa enodulosa, Endoperplexa subfarinacea, Serendipita invisibilis, Serendipita inclusa , and Serendipita interna .