Considerations and consequences of allowing DNA sequence data as types of fungal taxa.

Nomenclatural type definitions are one of the most important concepts in biological nomenclature. Being physical objects that can be re-studied by other researchers, types permanently link taxonomy (an artificial agreement to classify biological diversity) with nomenclature (an artificial agreement to name biological diversity). Two proposals to amend the International Code of Nomenclature for algae, fungi, and plants (ICN), allowing DNA sequences alone (of any region and extent) to serve as types of taxon names for voucherless fungi (mainly putative taxa from environmental DNA sequences), have been submitted to be voted on at the 11th International Mycological Congress (Puerto Rico, July 2018). We consider various genetic processes affecting the distribution of alleles among taxa and find that alleles may not consistently and uniquely represent the species within which they are contained. Should the proposals be accepted, the meaning of nomenclatural types would change in a fundamental way from physical objects as sources of data to the data themselves. Such changes are conducive to irreproducible science, the potential typification on artefactual data, and massive creation of names with low information content, ultimately causing nomenclatural instability and unnecessary work for future researchers that would stall future explorations of fungal diversity. We conclude that the acceptance of DNA sequences alone as types of names of taxa, under the terms used in the current proposals, is unnecessary and would not solve the problem of naming putative taxa known only from DNA sequences in a scientifically defensible way. As an alternative, we highlight the use of formulas for naming putative taxa (candidate taxa) that do not require any modification of the ICN.

Phylogenetic and microscopic studies in the genus Lactifluus (Basidiomycota, Russulales) in West Africa, including the description of four new species.

Despite the crucial ecological role of lactarioid taxa (Lactifluus, Lactarius) as common ectomycorrhiza formers in tropical African seasonal forests, their current diversity is not yet adequately assessed. During the last few years, numerous lactarioid specimens have been sampled in various ecosystems from Togo (West Africa). We generated 48 ITS sequences and aligned them against lactarioid taxa from other tropical African ecozones (Guineo-Congolean evergreen forests, Zambezian miombo). A Maximum Likelihood phylogenetic tree was inferred from a dataset of 109 sequences. The phylogenetic placement of the specimens, combined with morpho-anatomical data, supported the description of four new species from Togo within the monophyletic genus Lactifluus: within subgen. Lactifluus (L. flavellus), subgen. Russulopsis (L. longibasidius and L. pectinatus), and subgen. Edules (L. melleus). This demonstrates that the current species richness of the genus is considerably higher than hitherto estimated for African species and, in addition, a need to redefine the subgenera and sections within it.

The genus Lactarius s. str. (Basidiomycota, Russulales) in Togo (West Africa): phylogeny and a new species described.

Lactarius s. str. represents a monophyletic group of about 40 species in tropical Africa, although the delimitation of the genus from Lactifluus is still in progress. Recent molecular phylogenetic and taxonomic revisions have led to numerous changes in names of tropical species formerly referred to Lactarius. To better circumscribe the genus Lactarius in Togo, we combined morphological data with sequence analyses and phylogeny inference of rDNA ITS sequences. Morphological and molecular data were generated from specimens sampled in various native woodlands and riverside forests; Lactarioid- and Russula sequences from public GenBank NCBI, and UNITE are included for phylogenetic analysis. The Maximum likelihood phylogeny tree inferred from aligned sequences supports the phylogenetic position of the studied samples from Togo within the subgenera Piperites, and Plinthogali. Lactarius s. str. includes about 13 species described from West Africa, of which eight were not previously known from Togo, including one new species: Lactarius subbaliophaeus identifiable by the presence of winged basidiospores, a pallisadic pileipellis with a uprapellis composed of cylindrical cells, inconspicuous pleurocystidia, and fusiform or tortuous, often tapering apex marginal cells. It can also be recognised by a transparent white latex that turns pinkish and then blackish, and a bluish reaction of the flesh context with FeSO4. These features mentioned do not match any of the morpho-anatomically most similar species, notably L. baliophaeus and L. griseogalus.

Effects of twice-ambient carbon dioxide and nitrogen amendment on biomass, nutrient contents and carbon costs of Norway spruce seedlings as influenced by mycorrhization with Piloderma croceum and Tomentellopsis submollis.

Elevated tropospheric CO(2) concentrations may increase plant carbon fixation. In ectomycorrhizal trees, a considerable portion of the synthesized carbohydrates can be used to support the mutualistic fungal root partner which in turn can benefit the tree by increased nutrient supply. In this study, Norway spruce seedlings were inoculated with either Piloderma croceum (medium distance "fringe" exploration type) or Tomentellopsis submollis (medium distance "smooth" exploration type). We studied the impact of either species regarding fungal biomass production, seedling biomass, nutrient status and nutrient use efficiency in rhizotrons under ambient and twice-ambient CO(2) concentrations. A subset was amended with ammonium nitrate to prevent nitrogen imbalances expected under growth promotion by elevated CO(2). The two fungal species exhibited considerably different influences on growth, biomass allocation as well as nutrient uptake of spruce seedlings. P. croceum increased nutrient supply and promoted plant growth more strongly than T. submollis despite considerably higher carbon costs. In contrast, seedlings with T. submollis showed higher nutrient use efficiency, i.e. produced plant biomass per received unit of nutrient, particularly for P, K and Mg, thereby promoting shoot growth and reducing the root/shoot ratio. Under the given low soil nutrient availability, P. croceum proved to be a more favourable fungal partner for seedling development than T. submollis. Additionally, plant internal allocation of nutrients was differently influenced by the two ECM fungal species, particularly evident for P in shoots and for Ca in roots. Despite slightly increased ECM length and biomass production, neither of the two species had increased its capacity of nutrient uptake in proportion to the rise of CO(2). This lead to imbalances in nutritional status with reduced nutrient concentrations, particularly in seedlings with P. croceum. The beneficial effect of P. croceum thus diminished, although the nutrient status of its host plants was still above that of plants with T. submollis. We conclude that the imbalances of nutrient status in response to elevated CO(2) at early stages of plant development are likely to prove particularly severe at nutrient-poor soils as the increased growth of ECM cannot cover the enhanced nutrient demand. Hyphal length and biomass per unit of ectomycorrhizal length as determined for the first time for P. croceum amounted to 6.9 m cm(-1) and 6.0 µg cm(-1), respectively, across all treatments.

Two sebacinoid ectomycorrhizae on Chinese pine.

Sebacinoid fungi show a broad mycorrhizal capacity; therefore, they play a very important role in natural systems. Worldwide, fungi of Sebacinales are present under different environmental conditions and associate with diverse plant hosts, however, are hitherto poorly studied in China. Two sebacinoid ectomycorrhizae (ECM), Pinirhiza multifurcata and Pinirhiza nondextrinoidea, are described in detail morphologically and anatomically in the present study. They share a plectenchymatous outer mantle with multiply ramified hyphae in a gelatinous matrix, clampless, thin, thick-walled emanating hyphae with mostly Y-shaped ramifications and triangular inflations at the point of ramification. P. multifurcata and P. nondextrinoidea can be distinguished by thick cells in mantle layers, the ramification of emanating hyphae, the presence or absence of rhizomorphs, as well as the differing color reaction in Melzer's reagent. The putative molecular phylogenetic relationships of P. multifurcata and P. nondextrinoidea were inferred by analyses of the partial large subunit nuclear rDNA (nLSU); however, an affiliation to fungal species was not possible. This is the first report of sebacinoid ECM on Chinese pine.

Description and identification of Alnus acuminata ectomycorrhizae from Argentinean alder stands.

The objective of this study was to describe the morphological and anatomical features of five unidentified ectomycorrhizal types of Alnus acuminata and to complement their identification based on ITS-rDNA sequence analysis. The combined approach of morphotyping and sequence analysis based on ITS sequence comparison with sequences contained in GenBank and the UNITE database let us assign three of the five field-collected ectomycorrhiza morphotypes to the tomentella-thelephora lineage that closely matched European and North American species. The sequencing results within Tomentella point toward alder specific clades within T. sublilacina, T. ellisii and T. stuposa sensu lato. The two other EcM morphotypes matched Lactarius omphaliiformis and a Russula sp. Better focused, concomitant fruit body surveys are needed for accurate identification of South American ectomycorrhizal fungi because of the evidence of cryptic speciation in both agaricoid and resupinate mycobionts.

Land use practices and ectomycorrhizal fungal communities from oak woodlands dominated by Quercus suber L. considering drought scenarios.

Oak woodlands in the Mediterranean basin have been traditionally converted into agro-silvo-pastoral systems and exemplified sustainable land use in Europe. In Portugal, in line with the trend of other European countries, profound changes in management options during the twentieth century have led to landscape simplification. Landscapes are dynamic and the knowledge of future management planning combining biological conservation and soil productivity is needed, especially under the actual scenarios of drought and increasing evidence of heavy oak mortality. We examined the ectomycorrhizal (ECM) fungal community associated with cork oak in managed oak woodlands (called montado) under different land use practices, during summer. ECM fungal richness and abundance were assessed in 15 stands established in nine montados located in the Alentejo region (southern Portugal), using morphotyping and ITS rDNA analysis. Parameters related to the montados landscape characteristics, land use history over the last 25 years, climatic and edaphic conditions were taken into account. Fifty-five ECM fungal taxa corresponding to the most abundant fungal symbionts were distinguished on cork oak roots. Cenococcum geophilum and the families Russulaceae and Thelephoraceae explained 56% of the whole ECM fungal community; other groups were represented among the community: Cortinariaceae, Boletaceae, Amanita, Genea, Pisolithus, Scleroderma, and Tuber. There were pronounced differences in ECM fungal community structure among the 15 montados stands: C. geophilum was the only species common to all stands, tomentelloid and russuloid species were detected in 87-93% of the stands, Cortinariaceae was detected in 60% of the stands, and the other groups were more unequally distributed. Ordination analysis revealed that ECM fungal richness was positively correlated with the silvo-pastoral exploitation regime and low mortality of cork oak, while ECM fungal abundance was positively correlated with extensive agro-silvo-pastoral exploitation under a traditional 9-year rotation cultivation system and recent soil tillage. The effects of land use on the ECM fungal community and its implications in different scenarios of landscape management options, oak mortality, and global warming are discussed.

The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses.

Aerobic sewage granules are dense, spherical biofilms, regarded as a useful and promising tool in wastewater treatment processes. Recent studies revealed that fungi can be implemented in biofilm formation. This study attempts to uncover the fungal diversity in aerobic granules by sequence analysis of the 18S and 5.8S rRNA genes and the internal transcribed spacer regions. For this purpose, appropriate PCR and sequencing primer sets were selected and an improved DNA isolation protocol was used. The sequences of 41 isolates were assigned to the taxonomic groups Pleosporaceae, Xylariales, Theleobolaceae, Claviceps, Aureobasidium, Candida boleticola, and Tremellomycetes within the fungi. It turned out that the fungal community composition in granules depended on the wastewater type and the phase of granule development.

Combresomyces cornifer gen. sp. nov., an endophytic peronosporomycete in Lepidodendron from the Carboniferous of central France.

Structurally preserved periderm of the lycophyte Lepidodendron rhodumnense from the Visean (Mississippian) of central France contains a peronosporomycete (Combresomyces cornifer gen. sp. nov.) that occurs in the form of pyriform to subglobose terminal oogonia. On the surface is a conspicuous ornamentation, which may have formed through condensation of a mucilaginous extra-oogonial wall secretion. Some oogonia contain thin-walled spherules, which may represent (walled) oospheres or spores of an endoparasitic fungus (?chytrid), whereas single, large spheres in the interior are interpreted as oospores. Antheridia adpressed to several of the specimens are clavate and paragynous. This discovery sheds light on the morphology and biology of peronosporomycetes in a terrestrial ecosystem some 330My ago. Although the organism occurs exclusively in the periderm of L. rhodumnense, it is not known whether it represents a symptomless endophyte, pathogen, or saprotroph.

Tomentella africana, a new species from Benin (West Africa) identified by morphological and molecular data.

A common resupinate thelephoroid fungus was collected in northern Guinean seasonal forests in central and north of Benin (West Africa). The species is reminiscent of Tomentella umbrinospora with respect to the color and thickness of basidiomata and rhizomorphs, the shape of basidiospores in frontal view and the size of subicular hyphae. Both species fall phylogenetically within two clades. Based on detailed anatomical comparison (mostly of rhizomorphs and basidiospores) with the holotype of T. umbrinospora and phylogenetic analyses including ITS rDNA sequences of 40 Tomentella species, T. africana is described as a new species. Genetic distance between the newly described species and T. umbrinospora is 12.1-12.9%, based on ITS rDNA sequences. T. africana is characterized anatomically by yellow-brown thick (0.3-0.8 mm) monomitic rhizomorphs that are commonly covered by irregularly shaped thin hyphae, thin- to thick-walled subicular hyphae of two size ranges, clavate and clamped basidia of 30-60 microm and light yellow to pale brown echinulate basidiospores with irregular shape in frontal view. Detailed anatomical and molecular dissimilarities between T. africana and close species are discussed. Differences between irregularly shaped surface thin hyphae and skeletal ones are highlighted. We stress the relevance of rhizomorphal structures in the discrimination of resupinate thelephoroid fungi.

Vertical distribution of an ectomycorrhizal community in upper soil horizons of a young Norway spruce (Picea abies [L.] Karst.) stand of the Bavarian Limestone Alps.

The vertical niche differentiation of genera of ectomycorrhiza (ECM) was assessed in a 17-year-old Norway spruce (Picea abies [L.] Karst.) plantation on a mountainous dolomitic site (1,050 m above sea level) of the Bavarian Limestone Alps. We determined ECM anatomotypes, recorded the abundance of corresponding ECM root tips and classified them into groups of ECM exploration types, which refer to the organisation and the extent of their extramatrical mycelia. The abundance of ECM was highest in the organic soil layers, compared to the mineral soil horizon. The ordination of the ECM communities and of the exploration types revealed segregation related to soil horizon properties. While Cenococcum geophilum preferred the organic soil layers, Lactarius spp., Tomentella spp. and Craterellus tubaeformis were generally most abundant in the mineral soil horizons. Cenococcum geophilum was the predominant species, possibly based on enhanced competitiveness under the prevailing site conditions. The short-distance exploration types (e.g. C. geophilum) preferentially colonised the organic soil layer, whereas the contact types (e.g. most of the Tomentella spp., C. tubaeformis) together with medium-distance types (e.g. Amphinema byssoides) were primarily associated with the underlying A-horizons. Therefore, the soil horizons had an important effect on the distribution of ECM and on their community structure. The spatial niche differentiation of ECM genera and exploration types is discussed in regard to specific physico-chemical properties of soil horizon and the assumed ecophysiological strategies of ECM.

Anatomical and molecular characterization of Lactarius aff. omphaliformis, Russula alnijorullensis and Cortinarius tucumanensis ectomycorrhizae on Alnus acuminata.

Ectomycorrhizae (ECM) of Lactarius aff. omphaliformis Romagn., Russula alnijorullensis (Sing.) Sing. and Cortinarius tucumanensis Mos. on Andean alder (Alnus acuminata Kunth) were characterized and identified. The identification of the fungal symbionts was achieved by morpho-anatomical observations of mycorrhizae and by comparison of ITS-RFLP patterns obtained from ECM and fruitbodies. L. aff omphaliformis ECM differed in some morphological details such as ramification and mantle type from ECM of the same species on A. glutinosa. L. aff omphaliformis ECM show an orange to ochre mantle containing latex cells, which stain with sulpho-vanillin, emanating hyphae without clamps. R. alnijorullensis ECM represent a typical Russula-type-ECM, light yellow to pinkish, the outer mantle being composed of triangular latex-filled cells staining with sulpho-vanillin, emanating hyphae without clamps. C. tucumanensis ECM exhibit a white (silvery) to yellowish brown mantle covered with soil particles, emanating hyphae with clamps.

Phylogenetic relationships of cyphelloid homobasidiomycetes.

The homobasidiomycetes includes the mushroom-forming fungi. Members of the homobasidiomycetes produce the largest, most complex fruiting bodies in the fungi, such as gilled mushrooms ("agarics"), boletes, polypores, and puffballs. The homobasidiomycetes also includes species that produce minute, cup- or tube-shaped "cyphelloid" fruiting bodies, that rarely exceed 1-2 mm diameter. The goal of this study was to estimate the phylogenetic placements of cyphelloid fungi within the homobasidiomycetes. Sequences from the nuclear large subunit (nuc-lsu) ribosomal DNA (rDNA), 5.8S rDNA, and internal transcribed spacers (ITS) 1 and 2 were obtained for 31 samples of cyphelloid fungi and 16 samples of other homobasidiomycetes, and combined with published sequences. In total, 71 sequences of cyphelloid fungi were included, representing 16 genera. Preliminary phylogenetic analyses of a 1477-sequence data set and BLAST searches using sequences of cyphelloid forms as queries were used to identify taxa that could be close relatives of cyphelloid forms. Subsequent phylogenetic analyses of one data set with 209 samples represented by nuc-lsu rDNA sequences (analyzed with parsimony) and another with 38 samples represented by nuc-lsu and 5.8S rDNA sequences (analyzed with parsimony and maximum likelihood) indicated that cyphelloid forms represent a polyphyletic assemblage of reduced agarics (euagarics clade, Agaricales). Unconstrained tree topologies suggest that there have been about 10-12 origins of cyphelloid forms, but evaluation of constrained topologies with the Shimodaira-Hasegawa test suggests that somewhat more parsimonious scenarios cannot be rejected. Whatever their number, the multiple independent origins of cyphelloid forms represent striking cases of parallel evolutionary reduction of complex fungal morphology.

Quantitative detection of agar-cultivated and rhizotron-grown Piloderma croceum Erikss. & Hjortst. by ITS1-based fluorescent PCR.

A real-time quantitative TaqMan-PCR was established for the absolute quantification of extramatrical hyphal biomass of the ectomycorrhizal fungus Piloderma croceum in pure cultures as well as in rhizotron samples with non-sterile peat substrate. After cloning and sequencing of internal transcribed spacer (ITS) sequences ITS1/ITS2 and the 5.8S rRNA gene from several fungi, including Tomentellopsis submollis, Paxillus involutus, and Cortinarius obtusus, species-specific primers and a dual-labelled fluorogenic probe were designed for Piloderma croceum. The dynamic range of the TaqMan assay spans seven orders of magnitude, producing an online-detectable fluorescence signal during the cycling run that is directly related to the starting number of ITS copies present. To test the confidence of the PCR-based quantification results, the hyphal length of Piloderma croceum was counted under the microscope to determine the recovery from two defined but different amounts of agar-cultivated mycelia. Inspection of the registered Ct values (defined as that cycle number at which a statistically significant increase in the reporter fluorescence can first be detected) in a 10-fold dilution series of template DNA represents a suitable and stringent quality control standard for exclusion of false PCR-based quantification results. The fast real-time PCR approach enables high throughput of samples, making this method well suited for quantitative analysis of ectomycorrhizal fungi in communities of natural and artificial ecosystems, so long as applicable DNA extraction protocols exist for different types of soil.