Annemieke Verbeken

Fungal diversity notes 1–110: taxonomic and phylogenetic contributions to fungal species

This paper is a compilation of notes on 110 fungal taxa, including one new family, 10 new genera, and 76 new species, representing a wide taxonomic and geographic range. The new family, Paradictyoarthriniaceae is introduced based on its distinct lineage in Dothideomycetes and its unique morphology. The family is sister to Biatriosporaceae and Roussoellaceae. The new genera are Allophaeosphaeria (Phaeosphaeriaceae), Amphibambusa (Amphisphaeriaceae), Brunneomycosphaerella (Capnodiales genera incertae cedis), Chaetocapnodium (Capnodiaceae), Flammeascoma (Anteagloniaceae), Multiseptospora (Pleosporales genera incertae cedis), Neogaeumannomyces (Magnaporthaceae), Palmiascoma (Bambusicolaceae), Paralecia (Squamarinaceae) and Sarimanas (Melanommataceae). The newly described species are the Ascomycota Aliquandostipite manochii, Allophaeosphaeria dactylidis, A. muriformia, Alternaria cesenica, Amphibambusa bambusicola, Amphisphaeria sorbi, Annulohypoxylon thailandicum, Atrotorquata spartii, Brunneomycosphaerella laburni, Byssosphaeria musae, Camarosporium aborescentis, C. aureum, C. frutexensis, Chaetocapnodium siamensis, Chaetothyrium agathis, Colletotrichum sedi, Conicomyces pseudotransvaalensis, Cytospora berberidis, C. sibiraeae, Diaporthe thunbergiicola, Diatrype palmicola, Dictyosporium aquaticum, D. meiosporum, D. thailandicum, Didymella cirsii, Dinemasporium nelloi, Flammeascoma bambusae, Kalmusia italica, K. spartii, Keissleriella sparticola, Lauriomyces synnematicus, Leptosphaeria ebuli, Lophiostoma pseudodictyosporium, L. ravennicum, Lophiotrema eburnoides, Montagnula graminicola, Multiseptospora thailandica, Myrothecium macrosporum, Natantispora unipolaris, Neogaeumannomyces bambusicola, Neosetophoma clematidis, N. italica, Oxydothis atypica, Palmiascoma gregariascomum, Paraconiothyrium nelloi, P. thysanolaenae, Paradictyoarthrinium tectonicola, Paralecia pratorum, Paraphaeosphaeria spartii, Pestalotiopsis digitalis, P. dracontomelon, P. italiana, Phaeoisaria pseudoclematidis, Phragmocapnias philippinensis, Pseudocamarosporium cotinae, Pseudocercospora tamarindi, Pseudotrichia rubriostiolata, P. thailandica, Psiloglonium multiseptatum, Saagaromyces mangrovei, Sarimanas pseudofluviatile, S. shirakamiense, Tothia spartii, Trichomerium siamensis, Wojnowicia dactylidicola, W. dactylidis and W. lonicerae. The Basidiomycota Agaricus flavicentrus, A. hanthanaensis, A. parvibicolor, A. sodalis, Cantharellus luteostipitatus, Lactarius atrobrunneus, L. politus, Phylloporia dependens and Russula cortinarioides are also introduced. Epitypifications or reference specimens are designated for Hapalocystis berkeleyi, Meliola tamarindi, Pallidocercospora acaciigena, Phaeosphaeria musae, Plenodomus agnitus, Psiloglonium colihuae, P. sasicola and Zasmidium musae while notes and/or new sequence data are provided for Annulohypoxylon leptascum, A. nitens, A. stygium, Biscogniauxia marginata, Fasciatispora nypae, Hypoxylon fendleri, H. monticulosum, Leptosphaeria doliolum, Microsphaeropsis olivacea, Neomicrothyrium, Paraleptosphaeria nitschkei, Phoma medicaginis and Saccotheciaceae. A full description of each species is provided with light micrographs (or drawings). Molecular data is provided for 90 taxa and used to generate phylogenetic trees to establish a natural classification for species.

Ultrahigh-performance liquid chromatographic-tandem mass spectrometric multimycotoxin method for quantitating 26 mycotoxins in maize silage.

A multianalyte method was developed to identify and quantitate 26 mycotoxins simultaneously in maize silage by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The extraction and cleanup procedure consists of two extraction steps followed by purification on a Waters Oasis HLB column. The method developed was validated with the requirements of Commission Decision 2002/657/EC taken into account. The limit of detection and quantitation ranges were 5-348 and 11-695 ng/g, respectively. Apparent recovery varied between 61 and 116%, whereas repeatability and reproducibility were within the ranges of 3-45 and 5-49%, respectively. The method developed was successfully applied for maize silage samples taken at the cutting surface and 1 m behind that surface. Mainly Fusarium toxins (beauvericin, deoxynivalenol, enniatins, fumonisins, fusaric acid, and zearalenone) were detected, but postharvest toxins such as mycophenolic acid and roquefortine C were identified as well.

Genetic diversity of ectomycorrhizal Basidiomycetes from African and Indian tropical rain forests

Ectomycorrhizal (ECM) fungi have a worldwide distribution. However, the ecology of tropical ECM fungi is poorly documented, limiting our understanding of the symbiotic associations between tropical plants and fungi. ECM Basidiomycete diversity was investigated for the first time in two tropical rain forests in Africa (Western Upper Guinea) and in Asia (Western Ghats, India), using a fragment of the mitochondrial large subunit rRNA gene to type 140 sporocarps and 54 ectomycorrhizas. To evaluate taxonomic diversity, phylogenetic analyses were performed, and 40 sequences included from identified European specimens were used as taxonomic benchmarks. Five clades were recovered corresponding to six taxonomic groups: boletoids, sclerodermatoids, russuloids, thelephoroids, and a clade grouping the Amanitaceae and Tricholomataceae families. Our results revealed that the Russulaceae species display a great diversity with several putative new species, especially in Guinea. Other taxonomic issues at family/section levels are also briefly discussed. This study provides preliminary insights into taxonomic diversity, ECM status, and biogeographic patterns of ECM fungi in tropical two rain forest ecosystems, which appear to be as diverse as in temperate and boreal forests.

Lactarius volemus sensu lato (Russulales) from northern Thailand: morphological and phylogenetic species concepts explored

Lactarius volemus (Fr.: Fr.) Fr. is a well known and morphologically easily recognizable milkcap of the Northern hemisphere, forming ectomycorrhiza with both deciduous and coniferous trees. It was originally described from Europe, but is also reported in other continents. Although it is characterized by several unique macro- and micromorphological features, substantial variation in colour, lamellae spacing and changing and staining of the latex has been recorded and it is therefore considered as a putatively unresolved species complex. This study explores the concordance between morphological and phylogenetic species concepts within L. volemus sensu lato of northern Thailand, combining a critical morphological scrutiny with a multiple gene genealogy based on LSU, ITS and rpb2 nuclear sequences. Twelve strongly supported monophyletic clades and six terminal branches are discernable in all phylogenetic trees and represent 18 phylogenetic species. Six of the monophyletic clades can be morphologically distinguished and are described as new species: L. acicularis, L. crocatus, L. distantifolius, L. longipilus, L. pinguis and L. vitellinus. Five other clades also show some morphological differences, but these are too subtle and do not allow for a clear-cut species delimitation without the corroboration of molecular data. Lactarius volemus sensu lato of northern Thailand is therefore still considered as a partially cryptic species complex. Pleurolamprocystidia, pileipellis hairs and to a lesser degree also pileus colour are important diagnostic characteristics. Spore morphology, latex discoloration and pileus surface texture are less useful as diagnostic features. Whether this rich diversity is the result of in situ Pleistocene survival or post-glacial expansion and subsequent radiation, has yet to be revealed.

Lactarius ectomycorrhizae on Abies alba: morphological description, molecular characterization, and taxonomic remarks.

To date, the ectomycorrhizae formed by silver fir (Abies alba), an ecologically valuable and in- digenous tree species in many European mountain forests, have been poorly investigated. We character- ized the mycorrhizae formed by three Lactarius spe- cies (Lac. subsericatus, Lac. intermedius, Lac. salmon- icolor) on silver fir, on the basis of material originat- ing from central Italy. The identification of the fun- gal symbiont was achieved by means of morphoanatomical observations of mycorrhizae, and by comparison of ITS sequences obtained from my- corrhizae and sporocarps of putative fungal partners. Sequences also were obtained from specimens of the same species but from different geographic origin or from closely related Lactarius species. A maximum likelihood analysis of the data was performed. On the whole, the resultant tree is in good agreement with sporocarp and mycorrhiza morphology. RFLP pat- terns were calculated from sequence data. A discus- sion on the main morphoanatomical characters dis- tinguishing the Lactarius ectomycorrhizae reported in this study from those already described belonging to related species, is also included. The accuracy of different methods to identify mycorrhizae formed by closely related Lactarius species on silver fir, are dis-

Sequestrate Lactarius species from tropical Africa: L-angiocarpus sp nov and L-dolichocaulis comb. nov

Lactarius angiocarpus sp. nov. is described from miombo woodlands in Zambia. It is the third sequestrate representative of the Russulaceae described from tropical Africa. Morphological characters and DNA sequence data support its placement in Lactarius subgen. Plinthogali. Molecular kinship analyses confirm a multiple origin of sequestrate Russulaceae spp. As none of the previously sequenced sequestrate Russulaceae spp. has been assigned to the Plinthogali clade, the placement of L. angiocarpus indicates an additional point of origin of derivative sporocarp types within the Russulaceae. Within the same subgenus, another tropical African sequestrate species, L. dolichocaulis comb. nov. (syn. Arcangeliella dolichocaulis) is recognized.

Fungal diversity and community structure on Phragmites australis (Poaceae) along a salinity gradient in the Scheldt estuary (Belgium)

We examined fungal communities on stems and leaf sheaths of common reed (Phragmites australis) in four tidal marshes along a salinity gradient (mesohaline to freshwater) in the Scheldt estuary (Belgium) by direct observation. One hundred and fourteen taxa, the highest number of taxa ever recorded during a survey of phragmiticolous fungi, were found, with 56 taxa (49%) in the ascomycetes, 6 taxa (5%) in the basidiomycetes and 52 taxa (46%) of anamorphic fungi, the latter comprising 30 (26%) coelomycetes and 22 (19%) hyphomycetes. Fungal diversity in tidal marshes compared is lower than that in non-tidal reed marshes. Species distribution on reed litter, investigated by multivariate analysis, was strongly correlated with flooding frequency and salinity. Influences of salinity on the fungal community are discussed and tested by a culture experiment. Taxa were divided into three basic distributional types: taxa present only in the saline-brackish water zone, species present only in the freshwater zone, and a group of eurytopic species found over the entire salinity gradient sampled. Indicator species for each group were determined. In two sites, mesohaline brackish and freshwater, the vertical distribution of mycota was analysed. Higher similarities between the same vertical zones, especially in middle and apical parts of the culms, indicated that terrestrial fungi (no direct contact with tidal exchange water) were less influenced by site-specific characteristics. However, in both sites, a few common indicator species for standing Phragmites culms could be identified. Flooding height (and frequency) is demonstrated to influence vertical species distribution. Generally rather complicated interactions and a narrow niche differentiation among species are revealed for the phragmiticolous fungal community.

Exposing hidden diversity by concordant genealogies and morphology—a study of the Lactifluus volemus (Russulales) species complex in Sikkim Himalaya (India)

Although Sikkim belongs to one of the Global Biodiversity Hotspots, little is known about its ectomycorrhizal fungi, and even less about the main genera of Russulales, i.e. Lactarius, Lactifluus, Multifurca and Russula. Combining a multilocus genealogical and morphological study, we aimed to document the diversity within Lactifluus volemus sensu lato of Sikkim Himalaya. We compared nuclear ITS and LSU rDNA, nuclear rpb1 and rpb2 protein-coding, and mitochondrial atp6 protein-coding genealogies to determine species boundaries. Interspecific relationships were inferred from the combined dataset. Bayesian and maximum likelihood single-locus genealogies are concordant and support recognition of six species. Three of these could be identified by unique morphological characteristics and are described as new species: L. dissitus, L. leptomerus and L. versiformis.

Characterization of Lactarius tesquorum ectomycorrhizae on Cistus sp. and molecular phylogeny of related European Lactarius taxa

Lactarius is one of the larger genera of ectomycorrhizal Basidiomycota, with about 400 species recognized worldwide. The ectomycorrhizae formed by Lactarius tesquorum on Cistus sp., one of the most common and ecologically relevant shrubs in the semi-arid regions in the Mediterranean basin, are described here in terms of morphological, anatomical and molecular features. An ITS rDNA sequence-based phylogenetic analysis was performed on the related European Lactarius taxa (L. mairei, L. pubescens, L. scoticus, L. spinosulus, L. torminosulus and L. torminosus) currently classified together with L. tesquorum in the subgenus Piperites section Piperites. Piperites s.s. could be divided into two main clusters; L. mairei and especially L. spinosulus were related less closely to the other taxa. This study is part of a broader effort to extend our knowledge of the distribution, phylogeny and ectomycorrhizal biology of Lactarius species in selected ecosystems.

Tales of the unexpected: angiocarpous representatives of the Russulaceae in tropical South East Asia

Six new sequestrate Lactarius species are described from tropical forests in South East Asia. Extensive macro- and microscopical descriptions and illustrations of the main anatomical features are provided. Similarities with other sequestrate Russulales and their phylogenetic relationships are discussed. The placement of the species within Lactarius and its subgenera is confirmed by a molecular phylogeny based on ITS, LSU and rpb2 markers. A species key of the new taxa, including five other known angiocarpous species from South East Asia reported to exude milk, is given. The diversity of angiocarpous fungi in tropical areas is considered underestimated and driving evolutionary forces towards gasteromycetization are probably more diverse than generally assumed. The discovery of a large diversity of angiocarpous milkcaps on a rather local tropical scale was unexpected, and especially the fact that in Sri Lanka more angiocarpous than agaricoid Lactarius species are known now.