Beta-tubulin Sequences Research Articles

Genetic population structure and distribution of a fungal polypore,Datronia caperata(Polyporaceae), in mangrove forests of Central America

AbstractAim We examine the genetic structure of a fungal polypore,Datronia caperata(Berk.) Ryvarden (Polyporaceae), colonizing white mangrove,Laguncularia racemosa(L.) Gaertn. f. (Combretaceae), of Central America.Location Mangrove forests of Costa Rica and Panama.Methods Sequences of elongation factor alpha (EFA), beta tubulin (BTUB) and nuclear ribosomal internal transcribed spacer (ITS) regions were obtained from 54 collections ofD.caperatacollected from Caribbean and PacificL. racemosaforests in Central America. Measures of haplotype and nucleotide diversity, nested clade analyses and coalescent analyses were used to estimate the direction and extent of migration of the fungus, and the factors promoting population divergence. We also conducted phylogenetic analyses using Bayesian estimation to test whether putativeD. caperatacollected fromL. racemosawas conspecific withD. caperatacolonizing other hosts from diverse Neotropical forests.Results Our results demonstrate that there is genetic isolation betweenD. caperatapopulations from Caribbean mangroves and those from Pacific mangroves. Our data suggest that the best explanation for the observed haplotype distribution is a recent range expansion from the Caribbean to the Pacific coasts, with subsequent isolation. This is supported by the infrequent overlap of haplotypes, unidirectional migration estimates from the Caribbean to the Pacific and the older estimated age of mutations in the Caribbean low‐copy BTUB and EFA loci. In addition, our data suggest thatD. caperatafrom mangroves are not conspecific with collections from other hosts found in diverse Neotropical forests.Main conclusions The low frequency of shared haplotypes between coasts, coupled with the incomplete lineage sorting after cessation of gene flow, is consistent with isolation during the last Pleistocene glaciation. We hypothesize that the greater haplotype and nucleotide diversity in the Pacific occurs either because larger effective population sizes ofD. caperataare maintained in Pacific mangroves or becauseD. caperatapopulations underwent a significant bottleneck as a result of local extinction followed by recolonization. In addition, we found thatD. caperatafound onL. racemosawas not conspecific withD. caperatafrom non‐mangrove hosts and suggest thatD. caperatafound onL. racemosamay be a host specialist.

Partial sequence of the beta-tubulin of Histomonas meleagridis and the activity of benzimidazoles against H. meleagridis in vitro

The protozoan parasite Histomonas meleagridis is a member of the family Monocercomonadidae in the class Trichomonada. Due to food safety concerns, currently no prophylactic or therapeutic drug against the parasite is licensed in the European Union. Benzimidazoles are antiparasitic drugs, and some of them are licensed for use in food-producing animals. Benzimidazoles act on beta-tubulin, and the beta-tubulin sequence allows predictions about the efficacy of benzimidazoles. In this study, we sequenced and analyzed a part of the beta-tubulin gene of five H. meleagridis strains and of Dientamoeba fragilis. In each Histomonas strain, three to five different sequences were found. No clustering of sequences from the same strain was recognizable. A phylogenetic tree based on the amino acid sequences of trichomonal beta-tubulin genes placed the histomonal sequences on a branch with D. fragilis, separate from Monocercomonas sp. and Tritrichomonas foetus. All histomonal amino acid sequences predicted a susceptibility to benzimidazoles. However, when we tested the efficacy of five benzimidazoles, namely, albendazole, fenbendazole, flubendazole, mebendazole, and nocodazole, on H. meleagridis in vitro, all tested drugs showed no efficacy, even though the concentrations tested were higher than the concentrations found to be effective against Trichomonas vaginalis and T. foetus by other investigators.

The presence of bacteria species in semen and sperm quality

To verify the prevalence of semen bacterial contamination and whether the contamination could decrease sperm quality. Spermiogram, semen culture, and sperm transmission electron microscopy (TEM) analysis were performed. TEM data were elaborated using a mathematical formula that calculates a fertility index (FI)--able to define patients as fertile or infertile--and the percentage of sperm apoptosis, immaturity and necrosis. We aligned the amino acid sequence of beta-tubulin with protein of the most frequent species isolated from semen. Patients were divided according to the contaminating species; in each group, we observed fertile individuals, in whom the semen quality was similar to that of controls and infertile men whose sperm quality was significantly decreased, in terms of motility, FI, apoptosis and necrosis. Partial homology between beta-tubulin and bacterial proteins was observed. Sperm bacterial contamination is quite frequent and could contribute to the deterioration of the sperm quality of infertile men.

Microsporidia Evolved from Ancestral Sexual Fungi

Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested microsporidia share a common ancestor with fungi [2-7]. However, the exact nature of this phylogenetic relationship is unclear because of unusual features of microsporidial genomes, which are compact with fewer and highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, or whether microsporidia are a sister group to all fungi. Here, we present evidence addressing this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster governing sexual reproduction in which a high mobility group (HMG) transcription-factor gene is flanked by triose-phosphate transporter (TPT) and RNA helicase genes [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order. Genome-wide synteny analysis reveals multiple other loci conserved between microsporidia and zygomycetes to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor and suggest microsporidia may have an extant sexual cycle.

Genetic diversity and photobiont associations in selected taxa of the Tephromela atra group (Lecanorales, lichenised Ascomycota)

Mycobiont and photobiont genetic diversity was investigated in four taxa of the Tephromela atra complex, which differ in ecology and substratum preference (from siliceous rocks, limestone to bark), and are differently interpreted by taxonomists. Phylogenetic analyses were performed using mycobiont nuclear ITS, beta tubulin and homologous polyketide synthase gene (PKS) sequences obtained from freshly collected material sampled from the Mediterranean region to the Southern Alps. The silicicolous samples from the Alps form a basal lineage of the entire complex, and despite the morphological similarity, they do not form a monophyletic group with the Mediterranean samples. No resolution was found among the calcicolous and the silicicolous taxa from Mediterranean habitats, which are traditionally segregated at variety or species level. The epiphytic taxon, although nested with the other ecotypes, splits in two well-supported lineages. Among the four taxa, Tephromela grumosa is the only morphologically, chemically and genetically distinct taxon. However, it is also nested in the large T. atra complex. Phylogenetic analysis of photobionts ITS sequences revealed that thalli from the Mediterranean region are associated with two distinct lineages of Trebouxia, but the lineages are not correlated with substrate or mycobiont phenotype. The thalli from the Alps are exclusively associated with T. simplex, suggesting a protracted isolation from the other lineages.

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region ofPythium mercurialesp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.

Cloning and expression of the tubulin genes in barley

Tubulins are encoded by small gene families in plants. Based on the barley EST collection, cDNAs for alpha-, beta-, and gamma-tubulins were selected. Five genes for alpha-tubulin, eight newly identified beta-tubulin sequences and one gamma-tubulin gene were found to be expressed in barley. In silico analysis of relative abundance of the distinct tubulin sequences among ESTs derived from different libraries revealed that the various tubulin genes differed in their level of expression, and to some extent were tissue specific.

Evolution of host resistance in a toxin-producing bacterial–fungal alliance

The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of beta-tubulin sequences in the kingdom Fungi. A total of 49 new beta-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism--mutualism shift in insensitive fungi.

New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati

Three new species of Neosartorya and one new Aspergillus of section Fumigati are proposed using a polyphasic approach based on morphology, extrolite production and partial β-tubulin, calmodulin, and actin gene sequences. The phylogenetic analyses using the three genes clearly show that the taxa grouped separately from the known species and confirmed the phenotypic differences. Neosartorya denticulata is characterized by its unique denticulate ascospores with a prominent equatorial furrow; N. assulata by well developed flaps on the convex surface of the ascospores which in addition have two distinct equatorial crests and N. galapagensis by a funiculose colony morphology, short and narrow conidiophores and ascospores with two wide equatorial crests with a microtuberculate convex surface. Aspergillus turcosus can be distinguished by velvety, gray turquoise colonies and short, loosely columnar conidial heads. The four new taxa also have unique extrolite profiles, which contain the mycotoxins gliotoxin and viriditoxin in N. denticulate; apolar compounds provisionally named NEPS in N. assulata and gregatins in N. galapagensis. A. turcosus produced kotanins. N.denticulata sp. nov., N. assulata sp. nov., N. galapagensis sp. nov., and A. turcosus sp. nov. are described and illustrated.Electronic supplementary materialThe online version of this article (doi:10.1007/s10482-007-9183-1) contains supplementary material, which is available to authorized users.

Theissenia rogersii sp. nov. and phylogenetic position of Theissenia

Theissenia rogersii deviates from known Theissenia species primarily in having large ascospores with a thick wall layer and a unique configuration of two stromatal tissue types, one carbonaceous and the other fibrous. The carbonaceous tissue forms palisades on and beneath the perithecial layer as well as encasing individual perithecia, whereas the fibrous tissue fills the spaces between columns of the palisades as well as between encased perithecia. Phylogenetic analyses based on DNA sequences of beta-tubulin and alpha-actin genes placed Theissenia in the subfamily Hypoxyloideae among the genera that are characterized by having bipartite stromata (i.e. with the stromata differentiated into an outer dehiscing layer and an inner perithecium-bearing layer).

Micronuclear and Macronuclear Forms of β‐Tubulin Genes in the CiliateChilodonella uncinataReveal Insights into Genome Processing and Protein Evolution

Chilodonella uncinata, like all ciliates, contains two distinct nuclei in every cell: a germline micronucleus and a somatic macronucleus. During development of the macronucleus from a zygotic nucleus, the genome is processed in several ways, including elimination of internal sequences. In this study, we analyze micronuclear and macronuclear copies of beta-tubulin in C. uncinata and find at least four divergent paralogs of beta-tubulin in the macronucleus. We characterize the micronuclear version of one paralog and compare its internally eliminated sequences (IESs) with previously described IESs in this species. These comparisons reveal the presence of a conserved sequence motif within IESs. In addition, we compare the sequences of beta-tubulin from C. uncinata with other ciliates and to other alveolates in order to test the hypothesis that the mode of molecular evolution in ciliates obscures phylogenetic signal in protein-coding genes. We find that heterogeneous rates of substitution in beta-tubulin across ciliates result in unstable genealogies that are inconsistent with phylogenies based on small subunit rDNA genes and on ultrastructure. We discuss the implications of our findings for genome processing and protein evolution in ciliates.

Ophiostoma species from Protea infructescences: Four way interactions between plants, fungi, mites and beetles

Species of Ophiostoma include some of the world's best known fungal pathogens of trees. Most members are vectored by arthropods. One of the most unusual niches in which Ophiostoma species have been found is within the infructescences of Protea species in South Africa. Recent molecular phylogenetic reconstructions on Ophiostoma s.l. suggested that the three Ophiostoma spp. specifically found in Protea infructescences, form a strongly supported monophyletic lineage within Ophiostoma s.s. In this study, new collections of Ophiostoma from Protea infructescences were subjected to molecular phylogenetic reconstructions based on large subunit, ITS and beta-tubulin sequence data. Using these techniques, at least five undescribed species of Ophiostoma have been identified from these plants. Intriguingly, our results also suggest a polyphyletic origin for the Protea-associated Ophiostoma spp. This indicates multiple invasions of this unusual niche, by these fungi. Our results also revealed the first case of an Ophiostoma sp. jumping hosts between a native Protea sp. and the non-native tree genus Eucalyptus. The second aim of this study was to identify putative vectors of the Ophiostoma spp. inhabiting Protea infructescences using both molecular and direct isolation methods. The presence of reproductive propagules of Ophiostoma spp. was confirmed on four Protea-associated mite species (Oodinychus sp., two Tarsonemus spp. and Proctolaelaps vandenbergi) at high frequencies. The Oodinychus sp. mite showed significantly higher reproductive rates when fed exclusively on Ophiostoma splendens than when it was fed on various other fungi. This suggests a mutualistic association between the Oodinychus sp. and O. splendens. Long distance dispersal of these mites was restricted to vectored dispersal via Protea-infructescence inhabiting beetles (e.g. Genuchus hottentottus). Mites collected from these beetles were found to vector spores of various Ophiostoma spp. Based on these results, our view is that these mites act as primary vectors of the Ophiostoma spp. in Protea infructescences.

The new species Lecanora bicinctoidea, its position and considerations about phenotypic evolution in the Lecanora rupicola group

A phylogenetic analysis of the Lecanora rupicola group based on combined nITS rDNA and beta-tubulin sequences and a combined dataset of ITS, beta-tubulin and partial sequences of polyketide synthase genes reveals a previously unrecognized species, which here is introduced under the name Lecanora bicinctoidea. The new species is a sister group of the L. swartzii complex (including L. swartzii and L. lojkaeana), which is characterized by eucorticate ascomata, and a morphological diversity that includes also a dwarf-fruticose lineage. The preferential occurrence on vertical to overhanging siliceous rocks corresponds more closely to L. swartzii. A detailed investigation of phenotypic characters reveals that the new species differs from the superficially similar morphospecies L. bicincta in several ways, such as a thallus of comparatively small areoles and broadly sessile ascomata and the development of an amphithecial cortex devoid of algal remnants (i.e. an eucortex). L. bicinctoidea contains methyl 3alpha-hydroxy-4-O-demethylbarbatate, a chemical compound not known from other members of the L. rupicola group. We also discuss the importance of eucortex formation as one of several factors that are involved in the evolution of substrate-detached thallus structures.

Polyphasic taxonomy of Aspergillus section Candidi based on molecular, morphological and physiological data

Aspergillus section Candidi historically included a single white-spored species, A. candidus. Later studies clarified that other species may also belong to this section. In this study, we examined isolates of species tentatively assigned to section Candidi using a polyphasic approach. The characters examined include sequence analysis of partial β-tubulin, calmodulin and ITS sequences of the isolates, morphological and physiological tests, and examination of the extrolite profiles. Our data indicate that the revised section Candidi includes 4 species: A. candidus, A. campestris, A. taichungensis and A. tritici. This is strongly supported by all the morphological characteristics that are characteristic of section Candidi: slow growing colonies with globose conidial heads having white to yellowish conidia, conidiophores smooth, small conidiophores common, metulae present and covering the entire vesicle, some large Aspergillus heads with large metulae, presence of diminutive heads in all species, conidia smooth or nearly so with a subglobose to ovoid shape, and the presence of sclerotia in three species (A. candidus, A. taichungensis and A. tritici). Aspergillus tritici has been suggested to be the synonym of A. candidus previously, however, sequence data indicate that this is a valid species and includes isolates came from soil, wheat grain, flour and drums from India, Ghana, Sweden, The Netherlands and Hungary, making it a relatively widespread species. All species produce terphenyllins and candidusins and three species (A. candidus, A. campestris and A. tritici) produce chlorflavonins. Xanthoascins have only been found in A. candidus. Each of the species in section Candidi produce several other species specific extrolites, and none of these have been found in any other Aspergillus species. A. candidus has often been listed as a human pathogenic species, but this is unlikely as this species cannot grow at 37 °C. The pathogenic species may be A. tritici or white mutants of Aspergillus flavus.

Taxonomic revision of Aspergillus section Clavati based on molecular, morphological and physiological data

Aspergillus section Clavati has been revised using morphology, secondary metabolites, physiological characters and DNA sequences. Phylogenetic analysis of β-tubulin, ITS and calmodulin sequence data indicated that Aspergillus section Clavati includes 6 species, A. clavatus (synonyms: A. apicalis, A. pallidus), A. giganteus, A. rhizopodus, A. longivesica, Neocarpenteles acanthosporus and A. clavatonanicus. Neocarpenteles acanthosporus is the only known teleomorph of this section. The sister genera to Neocarpenteles are Neosartorya and Dichotomomyces based on sequence data. Species in Neosartorya and Neocarpenteles have anamorphs with green conidia and share the production of tryptoquivalins, while Dichotomomyces was found to be able to produce gliotoxin, which is also produced by some Neosartorya species, and tryptoquivalines and tryptoquivalones produced by members of both section Clavati and Fumigati. All species in section Clavati are alkalitolerant and acidotolerant and they all have clavate conidial heads. Many species are coprophilic and produce the effective antibiotic patulin. Members of section Clavati also produce antafumicin, tryptoquivalines, cytochalasins, sarcins, dehydrocarolic acid and kotanins (orlandin, desmethylkotanin and kotanin) in species specific combinations. Another species previously assigned to section Clavati, A. ingratus is considered a synonym of Hemicarpenteles paradoxus, which is phylogenetically very distantly related to Neocarpenteles and section Clavati.

Early diverging Ascomycota: phylogenetic divergence and related evolutionary enigmas

The early diverging Ascomycota lineage, detected primarily from nSSU rDNA sequence-based phylogenetic analyses, includes enigmatic key taxa important to an understanding of the phylogeny and evolution of higher fungi. At the moment six representative genera of early diverging ascomycetes (i.e. Taphrina, Protomyces, Saitoella, Schizosaccharomyces, Pneumocystis and Neolecta) have been assigned to "Archiascomycetes" sensu Nishida and Sugi ama (1994) or the subphylum "Taphrinomycotina" sensu Eriksson and Winka (1997). The group includes fungi that are ecologically and morphologically diverse, and it is difficult therefore to define the group based on common phenotypic characters. Bayesian analyses of nSSU rDNA or combined nSSU and nLSU rDNA sequences supported previously published Ascomycota frameworks that consist of three major lineages (i.e. a group of early diverging Ascomycota. [Taphrinomycotina], Saccharomycotina and Pezizomycotina); Taphrinomycotina is the sister group of Saccharomycotina and Pezizomycotina. The 50% majority rule consensus of 18000 Bayesian MCMCMC-generated trees from multilocus gene sequences of nSSU rDNA, nLSU rDNA (D1/D2), RPB2 and beta-tubulin also showed the monophyly of the three subphyla and the basal position of Taphrinomycotina in Ascomycota with significantly higher statistical support. However to answer controversial questions on the origin, monophyly and evolution of the Taphrinomycotina, additional integrated phylogenetic analyses might be necessary using sequences of more genes with broader taxon sampling from the early diverging Ascomycota.

Molecular Sequence Evidence for the Reclassification of Some Babesia Species

Taxonomic characterization of organisms in the genera Theileria and Babesia was originally based on observations of morphology and certain general phenotypic characteristics, which enabled many parasites to be unequivocally assigned to a particular genus. However, application of molecular genetic techniques, such as the polymerase chain reaction (PCR) for gene amplification, and DNA sequencing, have revealed gross inconsistencies in the assignation of some parasite genetic variants, particularly those of the B. gibsoni and B. microti complexes, to the genus Babesia. These variants cannot be assigned, on the basis of sequence information and phylogenetic analysis, to either of the genera Theileria and Babesia. The gene for which most sequence information is available for phylogenetic analysis is the small subunit ribosomal RNA (srRNA) gene. This gene allows clear distinction of the genera Theileria and Babesia (sensu stricto) and reveals that many "Babesia" variants are phylogenetically distinct from both genera. This distinction is confirmed, for some of the variants, by beta-tubulin sequence data, suggesting that the organisms should be renamed and reclassified.

Subcutaneous phaeohyphomycosis caused byPhaeoacremonium parasiticumin a renal transplant patient

A 49-year-old renally transplanted man, under a five-year course of immunosuppressive therapy with prednisone and cyclosporine A, experienced a subcutaneous phaeohyphomycosis caused by Phaeoacremonium parasiticum. The clinical presentation consisted of impressive, large, inflammatory and draining cystic tumors on the left foot that had been present for one year. A significant improvement was obtained with itraconazole plus intralesional injection with amphotericin B. Drug interaction was observed between itraconazole and cyclosporine A causing a severe hypertensive crisis and requiring a temporary sharp reduction in cyclosporine administration. Subcutaneous phaeohyphomycosis caused by P. parasiticum is uncommon among major organ transplant patients but several cases have previously been published and some patterns are emerging, e.g., limbs are generally involved but no known traumatic event has preceded lesion development. The identification of the case isolate was confirmed using a recently published online system based in part on beta-tubulin sequence comparison.

Exploiting the genetic diversity of Beauveria bassiana for improving the biological control of the coffee berry borer through the use of strain mixtures

Beauveria bassiana is an entomopathogen widely used to control the coffee berry borer in Colombia, as part of an Integrated Pest Management strategy. Traditionally, the development of fungal insect pathogens as biocontrol agents in crop pests has been oriented towards the selection and formulation of elite clonal strains. Instead, we explored the potential application of genetic diversity in B. bassiana by determining the effect of strain mixtures on coffee berry borer mortality compared to clonal isolates. Genomic DNA from 11 strains was characterized using internal transcribed spacers and beta-tubulin sequences as well as amplified fragment length polymorphism markers. Cluster analysis produced three genetic groups and confirmed the low but significant intraspecific genetic diversity present among the strains. Single strain virulence towards the coffee berry borer under laboratory conditions, using 1x10(6) conidia ml(-1), ranged between 89.9 and 57.5%. All the inoculations with mixtures resulted in coinfection events. Combinations of genetically similar strains showed no significant differences when their virulences were compared. However, mixtures of genetically different strains led to both antagonism and synergism. The lowest virulence percentage (57%) was obtained by putting together the most virulent strain of each group, contrary to the highest virulence percentage (93%) that resulted from mixing the three least virulent strains. The results indicate the promising potential of designing strain mixtures as an alternative for the biocontrol of Hypothenemus hampei and other pests and provide tools for the understanding of the ecological dynamics of entomopathogen populations under natural conditions.

Polyphasic taxonomy of Aspergillus fumigatus and related species

The variability within Aspergillus fumigatus Fresenius and related species was examined using macro-, micro-morphology, growth temperature regimes and extrolite patterns. In addition, DNA analyses including partial beta-tubulin, calmodulin and actin gene sequences were used. Detailed examination of strains, considered as A. fumigatus earlier, showed that they could be divided into four groups including A. fumigatus sensu stricto, A. lentulus and two new species. The intraspecific genetic variability within A. fumigatus sensu stricto was low, the sequence differences among 23 strains of the species was at most two bases in each partial beta-tubulin and calmodulin gene. However, intraspecific morphological diversity within the species was high and delineation of the species was equivocal. Therefore, beta-tubulin and calmodulin gene sequences could be critical determinants for the delineation of the A. fumigatus sensu stricto species. A. lentulus including isolates from clinical origin, Korean soil and from a dolphin clustered into an isolated group based on beta-tubulin, calmodulin and actin gene sequences, differing from A. fumigatus by morphological characters, growth temperature and extrolite profile. A. lentulus produces the extrolites auranthine, cyclopiazonic acid, a dimeric indole of unknown structure, neosartorin, some pyripyropens, terrein and some tryptoquivalins and tryptoquivalons. Two pair of isolates (CBS 117194, 117186 and 117520, 117519) clustered into separate groups from A. fumigatus and the other Aspergillus section Fumigati species, including the teleomorph Neosartorya, are proposed as two new species. A. fumigatiaffinis spec. nov. produces the extrolites auranthine, cycloechinulin, helvolic acid, neosartorin, palitantin, pyripyropens, tryptoquivalins and tryptoquivalons, and A. novofumigatus spec. nov. produces the extrolites cycloechinuline, helvolic acid, neosartorin, palitantin and terrein.