Pablo Alvarado

Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences

ABSTRACT Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.

Tubariomyces, a new genus of Inocybaceae from the Mediterranean region

The taxonomic position of Inocybe inexpectata is reconsidered on the basis of new molecular and morphological data from four published records. Up to three species can be recognized to which the name I. inexpectata has been applied. In addition to distinct morphological features present in this lineage it is proposed here as the new genus Tubariomyces. A complete taxonomical description of the genus and the species Tubariomyces inexpectatus and T. hygrophoroides sp. nov. also is provided.

Comparison between Tuber gennadii and T. oligospermum lineages reveals the existence of the new species T. cistophilum (Tuberaceae, Pezizales)

New collections of T. gennadii, T. oligospermum and T. asa (Tuberaceae, Pezizales) from the Mediterranean basin were compared on the basis of morphology and DNA sequences. Phylogenetic inference based on the internal transcribed spacer region (ITS) and the 28S nuclear large ribosomal subunit (nrLSU) indicates that T. gennadii includes at least two lineages with characteristic morphological traits. T. gennadii s. str. has small subglobose ascomata with conspicuous glebal locules that form a continuous labyrinth when mature and has a prosenchymatic peridium. T. lacunosum has medium to large, deeply lobed ascomata with small to very small isolated locules when mature and has a pseudoparenchymatic peridium. Two major clades were identified within the T. oligospermum group. On the basis of morphological, ecological and biogeographical data, these are identified with the taxa T. oligospermum (= T. asa) and T. sphaerospermum. Tuber oligospermum forms small to medium globose or subglobose, whitish ascomata, with globose to broadly ellipsoidal ascospores and is found in alkaline, sandy soils. Tuber sphaerospermum forms medium to large, irregularly globose or deeply lobed, yellowish ascomata, with regularly globose ascospores and is found in acidic soils. Finally, a new truffle species with a prosenchymatic peridium and globose to ellipsoid reticulate spores, collected in acidic argillaceous soils of Spain under Cistus ladanifer is proposed here as the new species T. cistophilum.

Atractosporocybe, Leucocybe and Rhizocybe, three new clitocyboid genera in the Tricholomatoid clade (Agaricales) with notes on Clitocybe and Lepista

A molecular multigene analysis (ITS, 18S and 28S nrLSU ribosomal DNA, tef1, rpb2) was used to support the proposition of three new genera of clitocyboid fungi. Leucocybe is proposed to accommodate the clade formed by Clitocybe connata and C. candicans. Clitocybe inornata is invested as type species of Atractosporocybe, while the new genus, Rhizocybe, is proposed for the former species of section Vernae of Clitocybe, C. vermicularis, C. pruinosa and C. rhizoides. The three lineages are related to the families Lyophyllaceae and Entolomataceae and independent from the Clitocybeae clade. Morphologically Rhizocybe is characterized by the presence of conspicuous rhizomorphs, while Atractosporocybe presents long fusiform spores. Leucocybe includes two whitish species in the former section Candicantes of Clitocybe, but no relevant shared characteristic feature was detected. Other whitish clitocyboid species, such as C. phyllophila (= C. cerussata), C. dealbata, C. rivulosa, and Singerocybe hydrogramma, are shown to be genetically related to the core lineage of the Clitocybeae.

Phylogenetic affiliation of (Choiromyces magnusii and C. venosus Tuberaceae ascomycota) from Spain.

The only two species of Choiromyces collected up to now in Spain, C. venosus and C. magnusii, were studied with both morphological and molecular methods. Phylogenetic inference of nrDNA ITS and LSU sequences showed that they should be considered independent species in the genus Choiromyces. Choiromyces venosus is a hypogeous fungus, infrequent but naturally present in the Iberian Peninsula, that is imported into Spain from central Europe without any indication of its origin or proper taxonomic identification. It is usually sold as highly-prized edible species of the genus Tuber such as T. magnatum. Choiromyces magnusii is a more frequent hypogeous fungus in Spain that is commercialized in spring on a local scale together with different species in the genus Terfezia. Additionally, the Australian species Reddellomyces westraliensis, which was introduced with Eucalyptus camaldulensis plantations, is cited here for the first time in Spain and Europe.

Inocybe flavobrunnescens, a new species in section Marginatae

The new species Inocybe flavobrunnescens is described on the basis of morphological and genetic features. It is characterized by (sub)isodiametric spores; it has a variable number of low and obtuse knobs, lageniform cystidia, and habitat preference for thermophilous oak woods on calcareous soils. It is compared with type material of Inocybe xanthomelas and I. krieglsteineri, which share with the new species a darkening flesh and small to medium sized basidiomes. ITS analysis revealed a low intraspecific variability within each Inocybe lineage, suggesting their accommodation as independent species. On the basis of this DNA region, I. xanthomelas and I. krieglsteineri are closely related. The present work suggests also updating several names in public databases to accommodate the present taxonomic conclusions. A lectotype for I. xanthomelas is designated.

Hypogeous Desert Fungi

The term ‘desert truffle’ can be applied to multiple edible hypogeous fungi growing in arid areas throughout the world. Defining what a hypogeous fungus is, and what arid lands are, is hence critical to delimit this ecological group. We choose to define ‘hypogeous’ fungi as those species with closed or ‘sequestrate’ globose fruiting bodies growing totally under soil surface or partially covered by it. The terms ‘truffle’ and ‘tuberoid’ are applied to Ascomycetes species presenting asci, while ‘false truffle’ and ‘gastroid’ are preferred for Basidiomycetes, to highlight their evolution from ‘agaricoid’ habit, regarded in some cases as an adaptation to dry environments. The most prominent genera of desert truffles are mentioned, and keys are proposed for their identification within the Ascomycetes, Basidiomycetes, and the former Glomeromycetes. The most critical morphological traits used for these keys are briefly discussed, and some images to illustrate the criteria are provided.

Amplistroma longicollis, a new species and its anamorph state described and sequenced from Europe

The new species Amplistroma longicollis is described. It is characterized by pale to dark brown stromata covered with white mucilaginous exudates and rostrate long-necked perithecia, which also present mucilaginous exudates at the base. Its relationship with other species of the genera Amplistroma in the Amplistromataceae is studied with morphological, cultural and molecular sequence data of large subunit ribosomal DNA (28S rDNA) and elongation factor 1-alpha gene (ef1-alpha).

Morchella tridentina, M. rufobrunnea, and M. kakiicolor: a study of three poorly known Mediterranean morels, with nomenclatural updates in section Distantes

A taxonomic, morphological, ecological, and molecular study of three interesting Morchella species, Morchella tridentina, M. rufobrunnea, and M. kakiicolor, is presented, based on observations and analyses of collections from Cyprus and Spain. The nomenclatural and taxonomic problems associated with the name M. tridentina and its synonyms, M. elatoides and M. frustrata, are discussed. A neotype is designated for M. quercus-ilicis to replace the lost holotype, and its synonymy with M. tridentina is proposed. The holotype of M. quercus-ilicis f. kakiicolor is confirmed as genetically distinct from the neotype of M. quercus-ilicis, and a new combination, M. kakiicolor (Clowez & L. Romero) comb. nov. is introduced. The presence of M. rufobrunnea in Europe is confirmed with molecular data for the first time. Updated descriptions of all three species, including new morphological and ecological data, as well as detailed illustrations are provided.

Amplistroma erinaceum, a new species and its anamorph from Panama

The new species Amplistroma erinaceum, collected on wood of Anacardium excelsum, is characterized by light cream-coloured, rostrate to spinose stromata, and immersed perithecia and long ostiolar necks. Its relationship to other Amplistroma species within the Amplistromataceae (incertae sedis, Ascomycota) are studied based on morphological, cultural, and molecular sequence data of the large subunit ribosomal DNA (28S rDNA).