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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.

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).

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.

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.

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.

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.

During a survey of the mycobiota in the stone chamber of the Takamatsu-zuka tumulus in the village of Asuka, Nara Prefecture, Japan, we isolated 19 yeast strains assigned to the genus Candida from various samples, taken mainly from mouldy spots where the colour of the murals had changed to black, white or another tone, and from viscous gels (biofilms) on plaster walls. The 26S rDNA D1/D2 domain sequence-based phylogeny clearly indicates two groups of isolates. Polyphasic characterization, including morphological, physiological and chemotaxonomic characteristics, and sequence analysis of the 26S rDNA D1/D2 domain and ITS regions suggest that each group is assignable to one of two novel species within the Candida membranifaciens clade. Proposed herein are the names Candida tumulicola sp. nov. (originally T6517-9-5T; holotype JCM 15403T; isotypes CBS 10917T, NBRC 104392T) and Candida takamatsuzukensis sp. nov. (originally T4922-1-1T; holotype JCM 15410T; isotypes CBS 10916T, NBRC 104391T). The 26S rDNA D1/D2 domain sequence divergence indicates that C. tumulicola differs from Candida friedrichii NBRC 10277T, the type strain of the nearest species, in 15 nucleotides (3 %), whereas C. takamatsuzukensis differs from Candida insectorum NBRC 10283T and Pichia mexicana NBRC 10544T, the type strains of the nearest species, in 20 nucleotides (4 %). Both novel species are also clearly distinguishable from the species closest to them by various physiological characteristics.