G.S. de Hoog

A class-wide phylogenetic assessment of Dothideomycetes

We present a comprehensive phylogeny derived from 5 genes, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, for 356 isolates and 41 families (six newly described in this volume) in Dothideomycetes. All currently accepted orders in the class are represented for the first time in addition to numerous previously unplaced lineages. Subclass Pleosporomycetidae is expanded to include the aquatic order Jahnulales. An ancestral reconstruction of basic nutritional modes supports numerous transitions from saprobic life histories to plant associated and lichenised modes and a transition from terrestrial to aquatic habitats are confirmed. Finally, a genomic comparison of 6 dothideomycete genomes with other fungi finds a high level of unique protein associated with the class, supporting its delineation as a separate taxon.

Contributions to a revision of the genus Trichosporon.

The genusTrichosporon was revised using characters of morphology, ultrastructure, physiology, ubiquinone systems, mol% G+C of DNA, DNA/DNA reassociations and 26S ribosomal RNA partial sequences. A total of 101 strains was used, including all available type and authentic cultures of previously described taxa. Nineteen taxa could be distinguished, 15 of which having Q-9 coenzyme systems and 4 having Q-10. Sixteen previously described names were reduced to synonymy. One new species was described.The genus is characterized by the presence of arthroconidia. Few species possess further diagnostic morphological characters, such as the presence of appressoria, macroconidia or meristematic conidiation. The septa of two species were found to be non-perforate, while those of the remaining species contained dolipores at variable degrees of differentiation, with or without vesicular or tubular parenthesomes. All species were able to assimilate a large number of carbon compounds; visible CO2 production was absent. The genus was found to be fairly homogeneous on the basis of a phylogenetic analysis of partial 26S rRNA sequences, with the exception ofT. pullulans which proved to be unrelated.Most taxa were found to occupy well-defined ecological niches. Within the group of taxa isolated from humans, a distinction found be made between those involved in systemic mycoses and those which mainly caused public or non-public white piedras, respectively. One species was consistently associated with animals, while others came mainly from soil or water. One species was mesophilic and another psychrophilic.

Phylogenetic lineages in the Capnodiales.

The Capnodiales incorporates plant and human pathogens, endophytes, saprobes and epiphytes, with a wide range of nutritional modes. Several species are lichenised, or occur as parasites on fungi, or animals. The aim of the present study was to use DNA sequence data of the nuclear ribosomal small and large subunit RNA genes to test the monophyly of the Capnodiales, and resolve families within the order. We designed primers to allow the amplification and sequencing of almost the complete nuclear ribosomal small and large subunit RNA genes. Other than the Capnodiaceae (sooty moulds), and the Davidiellaceae, which contains saprobes and plant pathogens, the order presently incorporates families of major plant pathological importance such as the Mycosphaerellaceae, Teratosphaeriaceae and Schizothyriaceae. The Piedraiaceae was not supported, but resolves in the Teratosphaeriaceae. The Dissoconiaceae is introduced as a new family to accommodate Dissoconium and Ramichloridium. Lichenisation, as well as the ability to be saprobic or plant pathogenic evolved more than once in several families, though the taxa in the upper clades of the tree lead us to conclude that the strictly plant pathogenic, nectrotrophic families evolved from saprobic ancestors (Capnodiaceae), which is the more primitive state.

Redefinition of Aureobasidium pullulans and its varieties

Using media with low water activity, a large numbers of aureobasidium-like black yeasts were isolated from glacial and subglacial ice of three polythermal glaciers from the coastal Arctic environment of Kongsfjorden (Svalbard, Spitsbergen), as well as from adjacent sea water, sea ice and glacial meltwaters. To characterise the genetic variability of Aureobasidium pullulans strains originating from the Arctic and strains originating pan-globally, a multilocus molecular analysis was performed, through rDNA (internal transcribed spacers, partial 28 S rDNA), and partial introns and exons of genes encoding β-tubulin (TUB), translation elongation factor (EF1α) and elongase (ELO). Two globally ubiquitous varieties were distinguished: var. pullulans, occurring particularly in slightly osmotic substrates and in the phyllosphere; and var. melanogenum, mainly isolated from watery habitats. Both varieties were commonly isolated from the sampled Arctic habitats. However, some aureobasidium-like strains from subglacial ice from three different glaciers in Kongsfjorden (Svalbard, Spitsbergen), appeared to represent a new variety of A. pullulans. A strain from dolomitic marble in Namibia was found to belong to yet another variety. No molecular support has as yet been found for the previously described var. aubasidani. A partial elongase-encoding gene was successfully used as a phylogenetic marker at the (infra-)specific level.

Spectrum of Clinically Relevant Exophiala Species in the United States

ABSTRACT Numerous members of the genus Exophiala are potential agents of human and animal mycoses. The majority of these infections are cutaneous and superficial, but also fatal systemic infections are known. We re-identified 188 clinical isolates from the United States, which had a preliminary morphological identification of Exophiala species, by sequencing internal transcribed spacer (ITS) region of the rRNA. Molecular identifications of the strains were as follows, in order of frequency: 55 E. dermatitidis (29.3%), 37 E. xenobiotica (19.7%), 35 E. oligosperma (18.6%), 13 E. lecanii-corni (6.9%), 12 E. phaeomuriformis (6.4%), 7 E. jeanselmei (3.7%), 7 E. bergeri (3.7%), 6 E. mesophila (3.2%), 5 E. spinifera (2.7%), 3 Exophiala sp. 1 (1.6%), 3 E. attenuata (1.6%), 3 Phialophora europaea (1.3%), 1 E. heteromorpha (0.5%), and 1 Exophiala sp. 2 (0.5%) strains. Exophiala strains were repeatedly isolated from deep infections (39.9%) involving lung, pleural fluid, sputum, digestive organs (stomach, intestines, bile), heart, brain, spleen, bone marrow, blood, dialysis fluid, lymph node, joint, breast, middle ear, throat, and intraocular tissues. About 38.3% of the Exophiala spp. strains were agents of cutaneous infections including skin, mucous membranes, nail, and corneal epithelium lesions. The other strains caused superficial infections (0.5%, including hair) or subcutaneous infection (12.0%, including paranasal sinusitis, mycetoma, and subcutaneous cyst). The systemic infections were preponderantly caused by E. dermatitidis, E. oligosperma, E. phaeomuriformis, E. xenobiotica, and E. lecanii-corni. Strains of E. bergeri, E. spinifera, E. jeanselmei, E. mesophila, and E. attenuata mainly induced cutaneous and subcutaneous infections. Since relatively few unknown ITS motifs were encountered, we suppose that the list of opportunistic Exophiala species in temperate climates is nearing completion, but a number of species still have to be described.

On the isolation of microcolonial fungi occurring on and in marble and other calcareous rocks

Several filamentous and microcolonial fungi (MCF) have been isolated from stone monuments and natural rock outcrops, mainly in the Mediterranean area. Most strains are characterized by melanin production. MCF proved to be meristematic; some showed dimorphic yeast-like growth. Meristematic development and melanin production is supposed to play a key role in the survival of MCF on white marble in dry and hot environments. Meristematic swelling of cells with thick cell-walls containing melanin and formation of endoconidia support their water independence and desensitivization against UV-radiation. MCF are therefore supposed to be the resident flora while filamentous fungi could only be contaminants. They are not lichenized, since no algae could be found in association with them. Optimal isolation techniques are discussed. Fungi are described after their in vitro morphology. The taxonomic status of these organisms is considered. Molecular investigations are in progress.

Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales), with standardisation of methods for Cladosporium taxonomy and diagnostics.

The Cladosporium herbarum complex comprises five species for which Davidiella teleomorphs are known. Cladosporium herbarum s. str. (D. tassiana), C. macrocarpum (D. macrocarpa) and C. bruhnei (D. allicina) are distinguishable by having conidia of different width, and by teleomorph characters. Davidiella variabile is introduced as teleomorph of C. variabile, a homothallic species occurring on Spinacia, and D. macrospora is known to be the teleomorph of C. iridis on Iris spp. The C. herbarum complex combines low molecular distance with a high degree of clonal or inbreeding diversity. Entities differ from each other by multilocus sequence data and by phenetic differences, and thus can be interpreted to represent individual taxa. Isolates of the C. herbarum complex that were formerly associated with opportunistic human infections, cluster with C. bruhnei. Several species are newly described from hypersaline water, namely C. ramotenellum, C. tenellum, C. subinflatum, and C. herbaroides. Cladosporium pseudiridis collected from Iris sp. in New Zealand, is also a member of this species complex and shown to be distinct from C. iridis that occurs on this host elsewhere in the world. A further new species from New Zealand is C. sinuosum on Fuchsia excorticata. Cladosporium antarcticum is newly described from a lichen, Caloplaca regalis, collected in Antarctica, and C. subtilissimum from grape berries in the U.S.A., while the new combination C. ossifragi, the oldest valid name of the Cladosporium known from Narthecium in Europe, is proposed. Standard protocols and media are herewith proposed to facilitate future morphological examination of Cladosporium spp. in culture, and neotypes or epitypes are proposed for all species treated.

Phylogeny and taxonomy of the family Arthrodermataceae (dermatophytes) using sequence analysis of the ribosomal ITS region

The internal transcribed spacer (ITS) region, covering the ITS1, ITS2 and 5·8S ribosomal DNA was used to evaluate phylogenetic relationships within the fungal family Arthrodermataceae. Sequences of variable length, ranging between 522 and 684 base pairs were aligned. An unrooted consensus tree based on parsimony analysis showed Trichophyton to be polyphyletic, and Microsporum to be paraphyletic. Non-monophyly of these two genera is in conflict with traditional classification. But this relation is not strongly supported by bootstrap analysis. Phylogenetic analysis showed that the two known members of the genus Epidermophyton grouped widely apart from each other. Within Trichophyton, our results suggest a separation of human pathogenic species and primarily geophilic species. Bootstrap support for these two groups is fairly high and both groups are recognized by current taxonomy. Three lineages were revealed within the T. mentagrophytes species complex. Microsporum canis, M. audouinii and M. equinum were found to be closely related. The topology of the tree was robust to various methods of analysis (parsimony and distance) and a different weighting scheme. Weighting of transversions over transitions did not improve the status of poorly supported branches of the tree.

Phylogenetic relationships of human-pathogenic Cladosporium (Xylohypha) species inferred from partial LS rRNA sequences

The controversy about the appropriate taxonomic placement of agents of subcutaneous and systemic mycoses in either Cladosporium or Xylohypha, both genera characterized by conidia being produced in dry, acropetal chains, was addressed with partial sequencing of LS ribosomal RNA. Observation of catenate anamorphs in species of Capronia (Ascomycotina, Herpotrichiellaceae), a genus which also has anamorphs in Exophiala, suggested the possibility of a close interrelationship of all human-associated taxa. To test this hypothesis, partial sequences of 43 strains of Cladosporium/Xylohypha were analysed. Human-pathogenic and saprophytic Cladosporium species were found to be phylogenetically distinct from each other and, on the basis of known teleomorph relationships, were considered to be anamorphs of Herpotrichiellaceae and Mycosphaerellaceae, respectively. They should therefore be classified in different anamorph-genera; Cladosporium being restricted to plant-associated species. A relatively large proportion of the Herpotrichiellaceae is presumed to be animal-associated. The black yeast genus Exophiala was also confirmed to be of herpotrichiellaceous relationship. The genus Xylohypha is unrelated.

A rock-inhabiting ancestor for mutualistic and pathogen-rich fungal lineages

Rock surfaces are unique terrestrial habitats in which rapid changes in the intensity of radiation, temperature, water supply and nutrient availability challenge the survival of microbes. A specialised, but diverse group of free-living, melanised fungi are amongst the persistent settlers of bare rocks. Multigene phylogenetic analyses were used to study relationships of ascomycetes from a variety of substrates, with a dataset including a broad sampling of rock dwellers from different geographical locations. Rock-inhabiting fungi appear particularly diverse in the early diverging lineages of the orders Chaetothyriales and Verrucariales. Although these orders share a most recent common ancestor, their lifestyles are strikingly different. Verrucariales are mostly lichen-forming fungi, while Chaetothyriales, by contrast, are best known as opportunistic pathogens of vertebrates (e.g. Cladophialophora bantiana and Exophiala dermatitidis, both agents of fatal brain infections) and saprophytes. The rock-dwelling habit is shown here to be key to the evolution of these two ecologically disparate orders. The most recent common ancestor of Verrucariales and Chaetothyriales is reconstructed as a non-lichenised rock-inhabitant. Ancestral state reconstructions suggest Verrucariales as one of the independent ascomycetes group where lichenisation has evolved on a hostile rock surface that might have favored this shift to a symbiotic lifestyle. Rock-inhabiting fungi are also ancestral to opportunistic pathogens, as they are found in the early diverging lineages of Chaetothyriales. In Chaetothyriales and Verrucariales, specific morphological and physiological traits (here referred to as extremotolerance) evolved in response to stresses in extreme conditions prevailing on rock surfaces. These factors facilitated colonisation of various substrates including the brains of vertebrates by opportunistic fungal pathogens, as well as helped establishment of a stable lichen symbiosis.