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The correct type species of Antennatula Fr. ex F. Strauss 1850 was quite recently considered to be Antennularia ericophila (Link) S. Hughes, not Antennatula pinophila (Nees) F. Strauss, and currently the illegitimate name Antennatula is regarded as a synonym of Metacapnodium Speg. 1918 (Metacapnodiaceae). Among Euantennaria Speg. 1918 and competing synonymous generic names Aithalomyces Woron. 1926, Phaeocapnis Cif. & Bat. 1963, Hormisciella Bat. 1956, and Hormisciomyces Bat. & Nacim. 1957, Euantennaria is adopted. As a consequence, with the change to one scientific name for pleomorphic fungi, the following 10 new combinations in Euantennaria (Euantennariaceae) for species of Antennatula sensu or used by Hughes are herein proposed: Euantennaria atra, E. cubensis, E. dingleyae, E. fisherae, E. fraserae, E. katumotoi, E. lumbricoidea, E. pinophila, E. shawiae, and E. triseptata. The respective brief notes for these species are also provided.

Four new yeast species belonging to the genus Apiotrichum and two new yeast species belonging to Cutaneotrichosporon are described for strains isolated from guano samples from bat-inhabited caves in Japan. In 2005, we reported these isolates as Trichosporon species based on sequence analyses of the D1/D2 domain of large subunit (LSU) rRNA genes according to available basidiomycetous yeast classification criteria; however, to date, they have not been officially published as new species with descriptions. Their phylogenetic positions have been reanalysed based on comparison of internal transcribed spacer (ITS) region sequences (including the 5.8S rRNA gene) and the D1/D2 domain of the LSU rRNA gene with those of known species; we confirmed clear separation from previously described species. Physiological and biochemical properties of the isolates also suggest their distinctiveness. Therefore, we describe Apiotrichum akiyoshidainum (holotype JCM 12595T), Apiotrichum chiropterorum (JCM 12594T), Apiotrichum coprophilum (JCM 12596T), Apiotrichum otae (JCM 12593T), Cutaneotrichosporon cavernicola (JCM 12590T) and Cutaneotrichosporon middelhovenii (JCM 12592T) as new species. C. cavernicola showed particularly distinctive morphology including large inflated anomalous cells on the hyphae and germination from the cells, although clear clamp connections on the hyphae were not confirmed. Further study is needed to elucidate the morph of this species.

A new species of euantennariaceous sooty mould, Antennatula katumotoi, is described and illustrated. It has a Hormisciomyces-like synasexual morph and inhabits twigs of the conifer Abies veitchii in Sugenuma (alt. ca. 1719 m), Katashina-mura, Tone-gun, Gunma Prefecture, central Honshu, Japan. The expected Euantennaria-like sexual morph linked to this genus was not seen in the specimen studied. This is the first proof that the genus Antennatula of the family Euantennariaceae also occurs in Japan. In addition, A. katumotoi also occurs with another metacapnodiaceous sooty mould that is composed of characteristic moniliform, barrel-shaped hyphae probably of a Capnobotrys-like asexual morph, but this is a minor inhabitant in the subicula.

Four new yeast species belonging to the genus

During dismantling and relocation of the Takamatsuzuka Tumulus stone chamber, many Cladophialophora and chaetothyrialean black fungi, such as Exophiala and Phialophora, were isolated from samples taken from the joints between the stone walls. However, inside the stone chamber of the Kitora Tumulus, after intermittent UV irradiation in 2009, these black fungi were also isolated from samples taken from the stone walls. Molecular phylogenetic analyses based on only nrLSU and the concatenated (nrLSU D1/D2 + ITS) sequences revealed that the 35 Takamatsuzuka and Kitora Tumuli isolates of Cladophialophora and the chaetothyrialean black fungi were divergent. Two new species of Cladophialophora are described herein: C. tumulicola from the viscous gels and various substrates on the stone walls of the Takamatsuzuka and Kitora Tumuli and C. tumbae from black substances on the plastic cover over the “thief hole,” soil and plaster pieces between the stone walls, and the exterior of the Takamatsuzuka Tumulus chamber. Also, molecular phylogenetic placements for the remaining eight Takamatsuzuka and Kitora Tumuli isolates of chaetothyrialean black fungi have been determined or suggested.

Phenotypic and genotypic characteristics of five isolates of Cephalotrichum and Sagenomella from various samples collected while dismantling the Takamatsuzuka Tumulus stone chamber were found to represent Cephalotrichum verrucisporum and Sagenomella striatispora. In addition to these, one isolate from the ceiling stone surface in the adjacent small room of the Kitora Tumulus was assignable to Sagenomella griseoviridis. Colonization on biodeteriorated plaster and stone walls as substrata or habitats of these fungi is noteworthy. Physiologically Sagenomella isolates solubilized CaCO3 and may be involved in the deterioration of the plaster walls inside the stone chamber. Cephalotrichum verrucisporum is a new addition to Japan.

Microbial outbreaks and related biodeterioration problems have affected the 1300-year-old multicolor (polychrome) mural paintings of the special historic sites Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT). Those of TT are designated as a national treasure. The microbiomes of these tumuli, both located in Asuka village, Nara, Japan, are critically reviewed as the central subject of this report. Using culture-dependent methods (conventional isolation and cultivation), we conducted polyphasic studies of the these microbial communities and identified the major microbial colonizers (Fusarium spp., Trichoderma spp., Penicillium spp., dark Acremonium spp., novel Candida yeast spp., Bacillus spp., Ochrobactrum spp., Stenotrophomonas tumulicola, and a few actinobacterial genera) and noteworthy microbial members (Kendrickiella phycomyces, Cephalotrichum verrucisporum (≡Doratomyces verrucisporus), Sagenomella striatispora, Sagenomella griseoviridis, two novel Cladophialophora spp., Burgoa anomala, one novel species Prototheca tumulicola, five novel Gluconacetobacter spp., three novel Bordetella spp., and one novel genus and species Krasilnikoviella muralis) involved in the biodeterioration of mural paintings, plaster walls, and stone chamber interiors. In addition, we generated microbial community data from TT and KT samples using culture-independent methods (molecular biological methods, including PCR-DGGE, clone libraries, and pyrosequence analysis). These data are comprehensively presented, in contrast to those derived from culture-dependent methods. Furthermore, the microbial communities detected using both methods are analytically compared, and, as a result, the complementary roles of these methods and approaches are highlighted. In related contexts, knowledge of similar biodeterioration problems affecting other prehistoric cave paintings, mainly at Lascaux in France and Altamira in Spain, are referred to and commented upon. Based on substrate preferences (or ecological grouping) and mapping (plotting detection sites of isolates), we speculate on the possible origins and invasion routes whereby the major microbial colonizers invaded the TT stone chamber interior. Finally, concluding remarks, lessons, and future perspectives based on our microbiological surveys of these ancient tumuli, and similar treasures outside of Japan, are briefly presented. A list of the microbial taxa that have been identified and fully or briefly described by us as known and novel taxa for TT and KT isolates since 2008 is presented in Supplementary Materials.

Two achlorophyllous microalgal strains were isolated from the soil and white moldy colony collected inside the stone chamber of the Takamatsuzuka Tumulus in Japan. Phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) and Dl/D2 large subunit ribosomal RNA (LSU rRNA) gene sequences, and concatenated gene sequences of the SSU and D1/D2 LSU rRNA genes indicated that our two isolates were the members of the non-photosynthetic, yeast-like microalgal Chlorellaceous genus Prototheca (Chlorellales, Trebouxiophyceae, Chlorophyta) but well distinguished from known species. Based on phenotypic and genotypic characteristics, isolates T6713-13-10T and T61213-7-11 are proposed to represent a novel species in Prototheca, P. tumulicola, with the type strain JCM 31123T (isolate T6713-13-10T).

Analysis of D1/D2 large-subunit (LSU) rRNA gene sequences predicted that 17 yeast isolates, mainly from viscous gels (biofilms) taken from the stone chamber interior of the Kitora tumulus in Nara, Japan, were placed in the Yamadazyma and Zygoascus clades. Polyphasic characterization, including morphological, physiological and chemotaxonomic characteristics, multigene sequence divergence and DNA-DNA hybridization, strongly suggested the assignment of one novel species to each of the clades; these are Yamadazyma kitorensis f.a., sp. nov., with the type strain JCM 31005T (ex-type CBS 14158T=isolate K8617-6-8T), and Zygoascus biomembranicola f.a., sp. nov., with the type strain JCM 31007T (ex-type CBS 14157T=isolate K61208-2-11T). Furthermore, the transfer of five known species of the genus Candida as novel combinations to the genera Yamadazyma and Zygoascus is proposed; these are Yamadazyma olivae f.a., comb. nov. (type strain CBS 11171T=ATCC MYA-4568T), Yamadazyma tumulicola f.a., comb. nov. (type strain JCM 15403T=ex-type CBS 10917T=isolate T6517-9-5T), Yamadazyma takamatsuzukensis f.a., comb. nov. (type strain JCM 15410T=CBS 10916T = isolate T4922-1-1T), Zygoascus polysorbophila f.a., comb. nov. (type strain NRRL Y-27161T=CBS 7317T) and Zygoascus bituminiphila f.a., comb. nov. (type strain CBS 8813T=MUCL 41424T).

During investigation of the biological contamination of the 1300-year-old mural paintings and plaster walls inside the stone chambers of the Takamatsuzuka and Kitora Tumuli (TT and KT) in Asuka-mura, Nara Prefecture, Japan, the identity of 17 bacterial isolates from blackish mouldy spots and viscous gels (biofilms) collected from both tumuli (16 isolates from TT and one from KT) during our 2005–2007 microbiological survey was systematically elucidated. One cluster of the major bacterial isolates was assigned to the genus Stenotrophomonas (class Gammaproteobacteria) by phylogenetic analysis of the 16S rRNA gene sequences. These isolates were divided into two groups A and B. Group A comprised 15 TT isolates that took a phylogenetic position near Stenotrophomonas chelatiphaga LPM-5T. Based on our analysis of the phenotypic (cultural, morphological, physiological and chemotaxonomic) characteristics and genotypic/molecular characteristics (DNA base composition, DNA–DNA relatedness, and 16S rRNA and gyrB gene sequences), the novel species name Stenotrophomonas tumulicola sp. nov. is proposed for the group A isolates with the type strain T5916-2-1bT ( = JCM 30961T = NCIMB 15009T). Group B, which contained only one TT and one KT isolate, was closely related to [Pseudomonas] geniculata, [P.] hibiscicola, [P.] beteli, Stenotrophomonas maltophilia and Stenotrophomonas pavanii. The two isolates were genotypically and phenotypically assignable to S. maltophilia.

The enigmatic basidiomycete genus Mixia includes intracellular parasites of Osmunda and Osmundastrum ferns. Here, the authors review the systematic and phylogenetic history of M. osmundae, originally known as Taphrina osmundae, and provide new data from investigations of specimens of Osmunda japonica collected in Yunnan Province, China, which we determine to be conspecific with M. osmundae. In addition, Taphrina higginsii, a parasite on fronds of Osmundastrum cinnamomeum described from Georgia, USA, was confirmed to be phenotypically identical with M. osmundae. The name T. higginsii is lectotypified with a Mix specimen. Collections examined to date document four localities for M. osmundae: Japan (Honshu and Kyushu), Taiwan (Taichung), USA (Georgia), and China (Yunnan), and host-parasite relationships with the old extant ferns Osmunda japonica and its relatives and with Osmundastrum cinnamomeum. The phylogenetic placement of M. osmundae on the fungal tree of life, its evolutionary implications, and recent advances in the phylogenomics of this fungus are briefly reviewed and discussed.

Integrated analysis of phenotypic and genotypic characteristics of 20 isolates of “black particles” (“Kuro-tsubu” in Japanese), that mainly appeared on the plaster wall of the ceiling tuff stone surface of the Kitora Tumulus stone chamber interior in Asuka village, Nara, Japan, has identified them as the bulbilliferous basidiomycetous anamorphic fungus Burgoa anomala. Our 28S rRNA gene D1/D2 (28S) and internal transcribed spacer (ITS) regions sequence-based phylogenies indicated that the 20 isolates displayed no genetic divergence and firmly clustered with the authentic strain B. anomala CBS 130.38. These isolates and B. anomala CBS 130.38 grouped together with species of the teleomorphic genus Sistotrema and its allies in the Cantharelloid clade. Colonization of biodeteriorated murals, plaster, and stone walls as the substrata or habitats by this fungus colonized, are noteworthy. Additionally, B. anomala is recorded for the first time from Japan.

Eight strains characterised as Gram-stain-positive, non-spore-forming and non-motile rods were isolated from samples collected from stone chambers of the Takamatsuzuka and Kitora tumuli in Asuka village, Nara Prefecture, Japan. Among them, one strain, T7528-3-6bT, was shown to form a novel lineage within the genus Microbacterium . The most closely phylogenetically related species to T7528-3-6bT was Microbacterium panaciterrae , with 97.8 % sequence similarity. The major isoprenoid quinones of T7528-3-6bT were MK-12, MK-13 and MK-11. The predominant cellular fatty acids for this isolate were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0. The diagnostic diamino acid of the peptidoglycan of this isolate was ornithine. Major polar lipids of the isolate were phosphatidylglycerol, diphosphatidylglycerol and an unknown glycolipid. The G+C content of the genomic DNA of this isolate was 70.1 mol%. On the basis of the results of physiological, biochemical and chemotaxonomic tests and molecular phylogenetic analysis, T7528-3-6bT is considered to represent a novel species of the genus Microbacterium , for which the name M. tumbae sp. nov. has been proposed. The type strain is T7528-3-6bT (=JCM 28836T=NCIMB 15039T). The results of comparisons of both phenotypic and genotypic (16S rRNA gene sequence) characteristics indicated that the remaining seven isolates were very closely related to Microbacterium shaanxiense . Although the sequence similarity between the two was 99.2 %, further detailed multifaceted comparisons are needed to determine their accurate taxonomic assignment.

Ten strains of Gram-stain-negative, rod-shaped, non-spore-forming bacteria were isolated from the burial mound soil collected before the dismantling and samples collected during the dismantling work on the Takamatsuzuka Tumulus in Asuka village, Nara Prefecture, Japan in 2007. On the basis of the 16S rRNA gene sequence analysis of the isolates, they were accommodated in the genus Gluconacetobacter (class Alphaproteobacteria ) and can be separated into four groups within the cluster containing the genus Gluconacetobacter . One of the groups demonstrated a phylogenetic position identical to that of Gluconacetobacter asukensis , which was isolated from small holes on plaster walls of the stone chamber interior of Kitora Tumulus in Asuka village, Nara Prefecture, Japan. The remaining three groups consisted of novel lineages within the genus Gluconacetobacter . A total of four isolates were selected from each group and carefully identified using a polyphasic approach. The isolates were characterized on the basis of their possessing Q-10 as the major ubiquinone system and C18 : 1ω7c (58.5–65.2 %) as the predominant fatty acid. A DNA–DNA hybridization test was used to determine that the three lineages represented novel species, for which the names Gluconacetobacter tumulisoli sp. nov., Gluconacetobacter takamatsuzukensis sp. nov. and Gluconacetobacter aggeris sp. nov. are proposed. The type strains are T611xx-1-4aT ( = JCM 19097T = NCIMB 14861T), T61213-20-1aT ( = JCM 19094T = NCIMB 14859T) and T6203-4-1aT ( = JCM 19092T = NCIMB 14860T), respectively.

The Kitora tumulus, which is thought to have been built around the late seventh to early eighth century, has beautiful mural paintings which were executed directly on a very thin layer of plaster in the stone chamber. When the paintings were found, the plaster was partly detached from the stone wall and the murals were therefore considered to be likely to fall off the wall with a casual touch or stimulation. Therefore, it was decided to detach the mural paintings and to store them flat and under controlled environmental conditions. This was initiated immediately after the excavation of the tumulus in 2004. However, fungal growth and biofilm development were observed within the stone chamber during the relocation work. In 2005, small holes containing black substances were observed on certain areas of the ceiling plaster, and following investigation an acetic acid bacterium, Gluconacetobacter sp., was isolated from the black substances. The bacterium was also isolated from the ceiling, floor, and ea...

A Gram-stain-positive, facultatively anaerobic actinomycete, designated strain T6220-5-2bT, was isolated from a sample taken from a mouldy spot on the surface of a mural painting (the white tiger, Byakko) inside the stone chamber of Takamatsuzuka Tumulus in Asuka village, Nara Prefecture, Japan. Based on 16S rRNA gene sequence analysis of the isolate, it was closely related to the genus Promicromonospora , but formed of a novel lineage within the family Promicromonosporaceae . The closest related species to strain T6220-5-2bT was Promicromonospora flava , with which it shared 99.1 % 16S rRNA gene sequence similarity. The isoprenoid quinone systems were menaquinones MK-9(H2), MK-9(H0) and MK-9(H4). The predominant cellular fatty acids for the isolate were anteiso-C15 : 0 and iso-C15 : 0. The peptidoglycan contained glutamic acid, aspartic acid, alanine and lysine, with the last named being the diagnostic diamino acid. The cell-wall acyl type was acetyl. The major polar lipids of the isolate were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositolmannoside, two unknown phospholipids and an unknown phosphoglycolipid. Whole-cell sugars of the isolate were galactose, glucose and ribose. The DNA G+C content of the genomic DNA was 75.2 mol%. Based on the results of phylogenetic, physiological and biochemical analyses and DNA–DNA hybridization experiments, the isolate was considered to represent a novel species of a new genus in the family Promicromonosporaceae , for which the name Krasilnikoviella muralis gen. nov., sp. nov. is proposed. The type strain of Krasilnikoviella muralis is T6220-5-2bT (=JCM 28789T=NCIMB 15040T). The reclassification of Promicromonospora flava as Krasilnikoviella flava comb. nov. is also proposed with the emended description of this species.

Using an integrated analysis of phenotypic and genotypic characterizations, a total of 18 isolates of “bristle-like” fungal colonizers of the Kitora and Takamatsuzuka Tumuli, which had been provisionally identified as Phialocephala phycomyces, were here determined to be Kendrickiella phycomyces (Auersw.) K. Jacobs & M. J. Wingf. The 18 isolates consisted of 10 from stone surfaces or viscous gels (biofilms) of the stone chamber interior and adjacent small room, and air in the adjacent small room of the Kitora Tumulus, and 8 from viscous gels on the stone surfaces of the stone chamber interior, plant roots, and soil in the adjacent space or stone wall interspaces (interstices) in the stone chamber of the Takamatsuzuka Tumulus. Plaster and stone walls in both tumuli were recorded as novel substrates of this fungus. Our 18S sequence-based phylogeny indicated that K. phycomyces and species of the leotiomycetous anamorph genera Chaetomella, Pilidium, Sphaerographium, and Synchaetomella formed a monophyletic lineage distant from the core taxa of the Leotiomycetes (Pezizomycotina, Ascomycota). The relationship between the physicochemical characteristics of these isolates on GYC agar plates, i.e., soluble brownish pigments and dissolution of calcium carbonate (CaCO3), and the biodeterioration of the plaster and plaster walls of both tumuli, are briefly discussed.

Six Gram-negative, rod-shaped, non-spore-forming bacterial strains were isolated from small holes on plaster walls of the stone chamber interior of the Kitora Tumulus in Asuka village, Nara Prefecture, Japan. These were investigated by means of a polyphasic approach. All the isolates were strictly aerobic and motile by peritrichous flagella. Phylogenetic trees generated based on 16S rRNA gene sequences identified two novel lineages (comprising five isolates and one isolate, respectively) within the genus Gluconacetobacter . The isolates were characterized by having Q-10 as the major ubiquinone system and C18 : 1ω7c (58.7–63.1 % of the total) as the predominant fatty acid. DNA–DNA hybridization experiments endorsed the species rank for the two lineages, for which the names Gluconacetobacter tumulicola sp. nov. (type strain K5929-2-1bT = JCM 17774T = NCIMB 14760T) and Gluconacetobacter asukensis sp. nov. (type strain K8617-1-1bT = JCM 17772T = NCIMB 14759T) are proposed.

Unidentified black spots (or stains) appeared on the plaster walls of the Takamatsuzuka and Kitora Tumuli in the village of Asuka, Nara Prefecture, Japan. Public attention was drawn to the biodeterioration of the colorful 1,300-year-old murals. A total of 46 isolates of Acremonium sect. Gliomastix were obtained from various samples (mainly black spots) of the Takamatsuzuka Tumulus (TT) (sampling period, May 2004–December 2006) and the Kitora Tumulus (KT) (June 2004–May 2007). These isolates were assignable to four known taxa and a new species in the ‘series Murorum’ sensu W. Gams as inferred from the integrated analysis of phenotypic and genotypic (i.e., ITS and 28S rDNA-D1/D2 sequences) characters: these were Acremonium masseei, A. murorum, A. felinum comb. nov. with the neotype designation, A. polychromum, and A. tumulicola sp. nov., which have been accommodated in the validated series Murorum in the section Gliomastix. The black spots on the murals of the TT and KT were caused mainly by A. masseei and A. murorum, respectively.

Penicillium appeared as the major dweller in the Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT) stone chambers, both located in the village of Asuka, Nara Prefecture, in relation to the biodeterioration of the 1,300-year-old mural paintings, plaster walls and ceilings. Of 662 Penicillium isolates from 373 samples of the TT (sampling period, May 2004-2007) and the KT (sampling period, June 2004-Sep 2007), 181 were phenotypically assigned as Penicillium sp. 1 which shared similar phenotypic characteristics of sect. Roqueforti in Penicillium subg. Penicillium. Fifteen representative isolates of Penicillium sp. 1, 13 from TT and 2 from KT, were selected for molecular phylogenetic analysis. The 28S rDNA D1/D2, ITS, beta-tubulin, and lys2 gene sequence-based phylogenies clearly demonstrated that the three known species P. roqueforti, P. carneum and P. paneum in sect. Roqueforti, and all TT and KT isolates grouped together. In addition to this, TT and KT isolates formed a monophyletic group with the ex-holotype strain CBS 101032 of P. paneum Frisvad with very strong bootstrap supports. So far, P. paneum has been isolated only from mouldy rye breads, other foods, and baled grass silage. Therefore, this is the first report of P. paneum isolation from samples relating to the biodeteriorated cultural properties such as mural paintings on plaster walls.

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

In an effort to clarify the cause of the deterioration of the colorfully painted murals that adorn the inner walls of the small stone chambers in the Takamatsuzuka and Kitora Tumuli in Japan, we enumerated the fungi that were isolated from moldy spots on the plaster walls collected between May 2004 and April 2005. The 262 fungal isolates from 79 samples of both tumuli were identified as approximately 100 species based on their phenotypic characters. Fusarium, Trichoderma, and Penicillium species were the predominant colonizers in the stone chamber interior and adjacent areas of both tumuli. In addition to the 28S phylogeny, neighbor-joining and Bayesian phylogenies of partial EF-1-alpha gene sequences revealed 24 genetically diverse fusaria in the Takamatsuzuka and Kitora Tumuli. Most of the fusaria were nested in clade 3 of the Fusarium solani species complex (FSSC); however, a few isolates were members of the F. oxysporum species complex (FOSC) clade or the F. avenaceum/F. tricinctum species complex clade. The FSSC isolates were compared with those detected in the Lascaux cave in France. In addition, a partial EF-1α gene phylogeny indicated that 13 Trichoderma isolates clustered in the Harzianum-Virens clade and 5 isolates in the Viride clade or Trichoderma sect. Longibrachiatum. Our analyses suggest that most of the fungi recovered from both tumuli are typically soil dwellers.

Phytoceratiomyxa osmundae, parasitic on fronds of Osmunda japonica var. sublancea in Taiwan, was described as a new genus and species in the Myxomycetes by Sawada in 1929. Our investigations on the type specimen, and related descriptions and illustrations of P. osmundae, revealed that this microorganism, originally identified as a myxomycete, was phenotypically identical with Mixia osmundae, which was transferred from the Ascomycota to the Basidiomycota based on the integrated analysis of molecules and morphology by Nishida et al. in 1995. In addition, a lectotype for Taphrina osmundae, the basionym of Mixia osmundae, is also designated. A nomenclatural proposal regarding the generic names Phytoceratiomyxa and Mixia related to this study will be published elsewhere.

During a survey of yeast strains having high conversion effi ciency to ethanol from cellobiose, ‘Ogataea pini’ ATCC 28781 and ‘Pichia pini’ NBRC 1794 were found to be distinct from any known species and from each other by a BLAST homology search using the D1/D2 LSU rRNA gene sequences. The D1/D2 phylogeny showed that ‘O. pini’ ATCC 28781 and ‘P. pini’ NBRC 1794 belonged to the Ogataea cluster, whereas a comparison of the ITS 1 and 2 regions sequences showed that the ATCC and NBRC strains each formed a species distinct from O. ganodermae, O. pini, O. henricii, and P. zsoltii, based on the D1/D2 sequence divergence. The ATCC and NBRC strains formed two to four hat-shaped ascospores and two to four, or more ones per deliquescent ascus, respectively, were negative for DBB and urease reactions, assimilated methanol slowly and nitrate not at all, and had the major ubiquinone system Q-7. These characteristics coincided basically with the defi nition of Ogataea proposed by Yamada et al. in 1994, excluding the number of ascospores. On the other hand, the ATCC and NBRC strains differed not only from each other but from relatives in various phenotypic characteristics. These differences suggest that two new yeasts of Ogataea be described as novel. The new species and their type strains are as follows: O. neopini ATCC 28781 T ; and O. corticis NBRC 1794 T . In addition, the emendation of the genus Ogataea is made; besides, we propose the transfer of P. zsoltii, P. dorogensis, and P. trehaloabstinens, which were placed in the Ogataea cluster based on the D1/D2 sequence analysis, to the genus Ogataea as O. zsoltii comb. nov., O. dorogensis comb. nov., and O. trehaloabstinens comb. nov.

Ten strains of Gram-stain-negative, non-spore-forming, non-motile coccobacilli were isolated from the plaster wall surface of 1300-year-old mural paintings inside the stone chamber of the Takamatsuzuka tumulus in Asuka village (Asuka-mura), Nara Prefecture, Japan. Based on 16S rRNA gene sequence analysis of the isolates, they belonged to the proteobacterial genusBordetella(classBetaproteobacteria) and could be separated into three groups representing novel lineages within the genusBordetella.Three isolates were selected, one from each group, and identified carefully using a polyphasic approach. The isolates were characterized by the presence of Q-8 as their major ubiquinone system and C16 : 0(30.0–41.8 %), summed feature 3 (C16 : 1ω7cand/or C16 : 1ω6c; 10.1–27.0 %) and C17 : 0cyclo (10.8–23.8 %) as the predominant fatty acids. The major hydroxy fatty acids were C12 : 02-OH and C14 : 02-OH. The DNA G+C content was 59.6–60.0 mol%. DNA–DNA hybridization tests confirmed that the isolates represented three separate novel species, for which the namesBordetella muralissp. nov. (type strain T6220-3-2bT = JCM 30931T = NCIMB 15006T),Bordetella tumulicolasp. nov. (type strain T6517-1-4bT = JCM 30935T = NCIMB 15007T) andBordetella tumbaesp. nov. (type strain T6713-1-3bT = JCM 30934T = NCIMB 15008T) are proposed. These results support previous evidence that members of the genusBordetellaexist in the environment and may be ubiquitous in soil and/or water.

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.

The Zygomycota is an ecologically heter- ogenous assemblage of nonzoosporic fungi compris- ing two classes, Zygomycetes and Trichomycetes. Phylogenetic analyses have suggested that the phylum is polyphyletic; two of four orders of Trichomycetes are related to the Mesomycetozoa (protists) that diverged near the fungal/animal split. Current circumscription of the Zygomycota includes only orders with representatives that produce zygospores. We present a molecular-based phylogeny including recognized representatives of the Zygomycetes and Trichomycetes with a combined dataset for nuclear rRNA 18S (SSU), 5.8S and 28S (LSU) genes. Tree reconstruction by Bayesian analyses suggests the Zygomycota is paraphyletic. Although 12 clades were identified only some of these correspond to the nine orders of Zygomycota currently recognized. A large superordinal clade, comprising the Dimargaritales, Harpellales, Kickxellales and Zoopagales, grouping together many symbiotic fungi, also is identified in part by a unique septal structure. Although Harpel- lales and Kickxellales are not monophyletic, these lineages are distinct from the Mucorales, Endogo- nales and Mortierellales, which appear more closely related to the Ascomycota + Basidiomycota + Glomeromycota. The final major group, the insect- associated Entomophthorales, appears to be poly- phyletic. In the present analyses Basidiobolus and Neozygites group within Zygomycota but not with the Entomophthorales. Clades are discussed with special reference to traditional classifications, mapping mor- phological characters and ecology, where possible, as a snapshot of our current phylogenetic perspective of the Zygomycota.

The phylum Ascomycota has been resolved into three major phylogenetic lineages: the subphyla Saccharomycotina (e.g., Saccharomyces, Pichia, Candida), Taphrinomycotina (e.g., Protomyces, Taphrina, Pneumocystis), and Pezizomycotina (e.g., Aspergillus, Neurospora, Peziza). We discuss the ecology, physiology, molecular biology, biotechnology, phylogeny, and systematics of Saccharomycotina and Taphrinomycotina, which represent the yeasts and yeastlike fungi of Ascomycota. Major changes in all aspects of our knowledge of these two subphyla have resulted from molecular studies, and the focus of the chapter is on these changes and their impact on present and future applications of the yeasts.

Microsporidia are intracellular parasitic microbes, which were once thought to be among early diverging eukaryotes, mainly because they lack mitochondria. However, this evolutionary scheme has been challenged by protein-based molecular phylogenetic studies, which suggest a more recent origin of the Microsporidia which clustered Microsporidia with or within ‘true’ fungal lineages. However, most of these studies did not include sequences from basal fungal phyla ( Chytridiomycota and Zygomycota ), and unrepresentative taxon sampling could have resulted in misleading relationships being suggested. To further investigate the evolutionary origin of Microsporidia relative to Fungi we performed molecular phylogenetic analyses with newly determined RPB1 (DNA-dependent RNA polymerase II largest subunit) and EF-1α (translation elongation factor I alpha) including sequences from basal fungi. Although the phylogenetic position of Microsporidia in the EF-1α tree still might be misplaced due to the unusually high rate of sequence divergence of the microsporidian EF-1α gene, the phylogenies recovered based on these two protein sequences do not provide strong evidences for a close relationship between Microsporidia and Fungi. Moreover, we have identified within the EF-1α genes a characteristic two amino acid deletion conserved in all fungal sequences currently available, whereas this deletion is absent in microsporidian sequences. These results argue against the view of Microsporidia as highly degenerate fungi, and whether Microsporidia and Fungi are sister taxa remains unresolved.

The ubiquinone (coenzyme Q) systems were determined for 176 teleomorphic isolates, 14 anamorphic isolates, and three samples of fruit-bodies of Dendrosphaera eberhardtii, which belonged to Eurotiales, Onygenales, and related taxa. In Eurotiales, Ascosphaera had Q-9, whereas Bettsia had Q-10. All isolates of Monascaceae had the Q-10 system, whereas those of four genera of Pseudeurotiaceae had the Q-10(H2) system. The Q-10(H2) system was found in genera of Trichocomaceae, except for Aspergillus, Penicillium, Paecilomyces, and their related taxa. However, Thermoascus had the Q-9 system. In Onygenales, members of Arthrodermataceae had Q-9, and those of Gymnoascaceae had Q-10(H2). Isolates of Myxotrichaceae were characterized by Q-10(H2) with few exceptions, which had Q-10. The quinones of Onygenaceae belonged to complex systems, i.e., Q-9, 0-10 and 0-10(H2), and a combination of two systems. Families Onygenaceae and Trichocomaceae are likely a phylogenetic heterogeneity. Ubiquinone analysis provides a very useful criterion of great promise for classifying eurotialean taxa and also for identifying their isolates.

A Youngia japonica strain had a group I intron that was suggested to have been transferred from Protomyces inouyei, a pathogenic fungus of Y. japonica. It was located in the miraculin homologue coding gene by reverse complementation. The deduced amino acid sequence of this miraculin homologue of Y. japonica was similar to the amino acid sequences of tobacco and tomato pathogenesis-related proteins.

Chemotaxonomic and phylogenetic characteristics of Sphingomonas strains isolated from plants of the family Gramineae were investigated. All strains contained the monosaccharide (glucuronic acid) type of glycosphingolipid (GSL-1). Most were found also to contain the oligosaccharide-type glycosphingolipids. Fatty acid and sphingosine profiles of the isolates were identical, although the ratio of the contents varies among the isolates. They all contained ubiquinone Q-10, and the G1C contents were from 66 to 68%. Phylogenetic analysis using 16S rRNA gene base sequences revealed that all the isolates were placed in the phylogenetic group of Sphingomonas paucimobilis in the alpha-4 subclass of Proteobacteria. By DNA-DNA hybridization experiments, the plant isolates were divided into five genotypic groups (groups 1 to 5). The strains of group 5 showed common physiological characteristics and formed pink-yellow colored colonies. Based on these results, Sphingomonas roseoflava sp. nov. was proposed for that homology group.

Although molecular phylogenetic studies of cyanobacteria on the basis of the 16S rRNA gene sequence have been reported, the topologies were unstable, especially in the inner branchings. Our analysis of 16S rRNA gene phylogeny by the maximum-likelihood and neighbor-joining methods combined with rate homogeneous and heterogeneous models revealed seven major evolutionary lineages of the cyanobacteria, including prochlorophycean organisms. These seven lineages are always stable on any combination of these methods and models, fundamentally corresponding to phylogenetic relationships based on other genes, e.g., psbA, rbcL, rnpB, rpoC, and tufA. Moreover, although known genotypic and phenotypic characters sometimes appear paralleled in independent lineages, many characters are not contradictory within each group. Therefore we propose seven evolutionary groups as a working hypothesis for successive taxonomic reconstruction. New 16S rRNA sequences of five unicellular cyanobacterial strains, PCC 7001, PCC 7003, PCC 73109, PCC 7117, and PCC 7335 of Synechococcus sp., were determined in this study. Although all these strains have been assigned to "marine clusters B and C," they were separated into three lineages. This suggests that the organisms classified in the genus Synechococcus evolved diversely and should be reclassified in several independent taxonomic units. Moreover, Synechococcus strains and filamentous cyanobacteria make a monophyletic group supported by a comparatively high statistical confidence value (80 to 100%) in each of the two independent lineages; therefore, these monophylies probably reflect the convergent evolution of a multicellular organization.

Molecular phylogenetic analyses of fern rusts were carried out based on 18S rDNA sequences. We sequenced the 18S rDNAs of fern rusts (Hyalopsora polypodii andUredinopsis intermedia) and non-fern rusts (Aecidium epimedii, Coleosporium asterum, Ochropsora kraunhiae, Puccinia suzutake andPhysopella ampelopsidis) and analyzed their phylogenetic relationships with other members of the Urediniomycetes. Our bootstrapped neighbor-joining tree obtained from these analyses showed that rust fungi were apparently monophyletic at high confidence level (100% bootstrap confidence). In this molecular phylogenetic tree, the two fern rusts did not occupy the basal position within the rust fungal lineage and did not form a monophyletic lineage. Two species of the Cronartiaceae (Peridermium harknessii, Cronartium ribicola) and one species of the Coleosporiaceae (Coleosporium asterum) grouped with the fern rusts. Therefore, our results suggested that the two fern rusts were not primitive. On the other hand,Mixia osmundae, which is parasitic on the primitive fernOsmunda, was phylogenetically far from the fern rusts.

Aspergillus subgenus Clavati has four recognized species: A. clavatus (the type species), A. clavatonanicus, A. giganteus, and A. longivesica. These species are strictly anamorphic (mitotic) and defined by the morphological species concept. However, their genealogical relationships remain uncertain. In this study, we examined the genetic relatedness among the four species in this section, using electrophoretic comparison of enzymes, DNA base composition, and DNA-DNA hybridization. In a dendrogram based on the calculated similarity values of four enzymes, 10 strains in section Clavati, 3 strains in the xerophilic species, a strain in section Ornati, and a strain in section Cremei were separated into nine major clusters at a 60% similarity level. A. longivesica JCM 10186(T) had Q-10 in our analysis, but Kuraishi et al. (1990) reported A. longivesica JCM 1720(T) had Q-9 (49%) and Q-10 (46%). The G+C contents of the four species of section Clavati ranged from 48 to 50 mol%. The degree of the intraspecific reassociation among the DNAs from the strains of these species ranged from 77 to 99%, whereas the degrees of interspecific relative binding among strains of the four species ranged from 30 to 59%. Our data from enzyme patterns and DNA relatedness support the validity of the three species in section Clavati, except for A. longivesica.

We examined the identity of Aspergillus penicillioides, the typical xerophilic and strictly anamorphic species, using an integrated analysis of the genotypic and phenotypic characters. Our experimental methods on two genotypic characters, i.e., DNA base composition using the HPLC method and DNA relatedness using the nitrocellulose filter hybridization technique between A. flavus, A. oryzae, and their close relations revealed a good agreement with the values by buoyant density (for DNA base composition) and spectrophotometric determination (for DNA relatedness) reported by Kurtzman et al. in 1986. On the basis of these comparisons, we examined DNA base composition and DNA relatedness of six selected strains of A. penicillioides, including IFO 8155 (originally described as A. vitricola), one strain of A. restrictus, and the respective strains from Eurotium amstelodami, E. repens, and E. rubrum. As a result, five strains within A. penicillioides, including the neotype strain NRRL 4548, had G+C contents of 46 to 49 mol%, whereas IFO 8155 had 50 mol%. A. restrictus had 52 mol%, and three Eurotium species ranged from 46 to 49 mol%. The DNA relatedness between A. penicillioides (five strains), except for IFO 8155, exhibited values greater than 70%, but the DNA complementarity between four strains and IFO 8155 in A. penicillioides revealed values of less than 40%. DNA relatedness values between three species of Eurotium were 65 to 72%. We determined 18S, 5.8S, and ITS rDNA sequences as other genotypic characters from A. penicillioides (six strains), A. restrictus, and related teleomorphic species of Eurotium. In three phylogenetic trees inferred from these sequences, five strains of A. penicillioides, including the neotype strain, were closely related to each other, whereas IFO 8155 was distantly related and grouped with other xerophilic species. Our results have suggested that A. penicillioides typified by NRRL 4548 and A. penicillioides IFO 8155 (ex holotype of A. vitricola) are not conspecific. The enzyme patterns as a genotypic character and general morphology and conidial ornamentation types as phenotypic characters supported this conclusion. Therefore the name A. vitricola Ohtsuki, typified by the holotype strain IFO 8155, should be revived. Evolutionary affinities among Aspergillus species and related teleomorphs, including the xerophilic taxa, are discussed.

Recent advances in fungal systematics are reviewed in relation to our previous studies. The usefulness of the integrated analysis of genotypic (especially 18S rRNA gene sequence comparisons) and phenotypic (especially ultrastructural and chemotaxonomic data) characters has been emphasized for the major groups and selected taxa of the fungi, and the impact to fungal systematics and evolution is discussed. Our noteworthy studies and findings are: 1) polyphyly of the chytridiomycetes and zygomycetes, 2) phylogenetic origin of the entomophthoralean fungi includingBasidiobolus, 3) detection of a major new lineage “Archiascomycetes,” comprisingTaphrina, Protomyces andSaitoella, Schizosaccharomyces, andPneumocystis, within the Ascomycota, and its phylogenetic and evolutionary significance, 4) polyphyletic origins of species in the anamorphic genusGeosmithia, and 5) phylogenetic placement ofMixia osmundae, species correctly and incorrectly assigned to the genusTaphrina, and basidiomyceotus yeasts. The newest 18S rDNA sequence-based neighbor-joining trees of the Ascomycota are demonstrated.

(2 R )-2-amino-6-hydroxy-4-hexynoic acid, (2 RS )-2-amino-4,5-hexadienoic acid, (2 S )-2-amino-4-hexynoic acid, and 2-amino-5-chloro-5-hexenoic acid were isolated and characterised from the fruit bodies of Amanita miculifera , with the first and the last mentioned amino acids being new natural products. Their structures were based on analysis of the results from elementary analysis, oxidation with KMnO 4 , determination of optical rotations, 1 H NMR-spectra, and hydrogenation over Adams or Lindlar catalysts. (2 RS )-2-Amino-6-hydroxy-4-hexynoic acid was synthesized and optically resolved by renal acylase to confirm its natural (2 R )-configuration. The configuration at C-2 of 2-amino-4,5-hexadienoic acid was also assumed from its ORD spectrum, as well as that of its hydrogenation product. The optical rotation (2-amino-5-chloro-5-hexenoic acid) was not determined because it was isolated in trace amounts; it was also prepared from 2-amino-5-hexynoic acid, although in very low yield. A possible biosynthetic route leading to the non-protein amino acids in this fungus is presented.

The archiascomycetous fungus Protomyces pachydermus has two group I introns within the nuclear small subunit (nSSU) rRNA gene. One of these introns has an internal open reading frame (ORF) that encodes a predicted protein of 228 amino acid residues. On the other hand, Protomyces macrosporus has two group I introns that insert at the same positions as P. pachydermus, which have no ORF. Each alignment was constructed with Protomyces group I introns located in the same position and other introns retrieved by the BLAST Search. Each phylogenetic tree based on the alignment shows that Protomyces introns are monophyletic but the relationships among fungal introns do not reflect on the fungal phylogeny. Therefore, it is suggested that two different horizontal transfers of group I introns occurred at the early stage of Protomyces species diversification.

SUMMARY Twenty-eight axenio planktonic cyanobacterial strains (10 Microcystis, three Oscillatoria, one Spirulina, one Aphanizomenon, 13 Anabaena) were investigated for their fatty acid composition by measurement of non-polar and hydroxy fatty acids. No 2-hydroxy fatty acids were detected in any strain, but 3-hydroxy fatty acids were detected in minor quantities in 24 strains. The highest portion of total fatty acids were non-polar fatty acids. Qualitative and quantitative analyses of 3-hydroxy fatty acids showed no taxonomic value in these strains, while the type of non-polar fatty acid composition was shown to be consistent within Microcystis and Anabaena strains, distinguishing them as type 4, characterized by the presence of 18:4, and type 2, characterized by 18:3 (α) of the Kenyon-Murata system. Two Oscillatoria agardhii Gomont strains were also included in the type 2 group due to the presence of 18: 3 (α), but the difference in characteristics of 16:2 and 16:3 between O. agardhii and Anabaena further divided type 2 into two subgroups: type 2A for Anabaena and type 2B for O. agardhii. A simplified unweighted pair group method with arithmetic averages (UPGMA) dendrogram demonstrated that the classification of 28 strains (Microcystis spp., Anabaena spp., Aphanizomenon flos-aquae (Lemmermann) Ralfs f. gracile (Lemmermann) Elenkin, O. agardhii and Spirullnasubsalsa Oersted ex Gomont based on numerical analysis of non-polar fatty acids corresponded to morphological species criteria, suggesting that non-polar fatty acid composition is a valuable chemical marker in the taxonomy of planktonic cyanobacteria. However, the fatty acid composition in Oscillatoria raciborskii is similar to that of Microcystis and very different from that of O. agardhii, suggesting its special position in Oscillatoria and the chemical diversity in the genus Oscillatoria.