Abstract

The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.

Introduction

Dematiaceous fungi are usually defined as having melanin or melanin-like pigment in the wall of their hyphae and/or spores [1]. They are widely distributed in nature. The major infections caused by dematiaceous human pathogens are classified into two groups of disease: chromoblastomycosis and pheohyphomycosis [2]. Chromoblastomycosis is a chronic infection of cutaneous and subcutaneous tissues. Verrucose lesions and round, brown, and thick-walled muriform cells (sclerotic bodies) in tissues are characteristics of this infection [2,3]. The principal etiologic agents of chromoblastomycosis are Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Phialophora verrucosa, and Rhinocladiella aquaspersa[1]. Pheohyphomycosis is a primary or opportunistic infection that ranges from the superficial tissue to deep organs. The etiologic agents are present in host tissues with melanized yeast-like cells, pseudohyphae-like elements, hyphae or any combination of these forms [2]. In recent literature, 59 species of 28 genera and three classes were described as the etiologic agents of pheohyphomycosis [1]. The numbers of case reports of infections with dematiaceous fungi have increased [4–12]. Outcomes of antifungal therapies for these infections have remained poor. A high mortality rate (79%) was reported in disseminated pheohyphomycosis patients even with antifungal therapy [13]. Identification of pathogenic dematiaceous fungi is typically done by morphological and physiological procedures [14–17] however, these procedures are time-consuming, require technical expertise, and are ineffective for identification of species with poor conidia production and a wide diversity in anamorphic life cycles [18].

Genetic methods have high sensitivity and specificity for identifying microorganisms. The D1/D2 domains of the large subunit ribosomal DNA (LSUrDNA) have been reported to be useful for identification of most ascomycetous yeasts [19,20] and medically important zygomycetes [21]. Thus, the sequences of the D1/D2 domains could serve as reliable and practical criteria for identification of most known yeasts. However, no research on the D1/D2 domains of dematiaceous fungi has been done. The objective of the present study was to investigate the efficacy of these domains for identification of medically important dematiaceous fungi. We analyzed D1/D2 domain sequences for 76 strains of 46 species of fungi and related species. The sequence data were then used to study the phylogenetic relation among these organisms, and between the genus Phialophora and pathogenic Chaetothyriales.

Materials and methods

Fungi

The 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa analyzed in the present study are listed in Table 1.

Table 1

Strains examined

Species Strain Source Number of intra-species nucleotide differences Sequence length (bp) Accession number 
Alternaria alternata IFM 41348 TIMM 1289  614 AB100676 
Aureobasidium pullulans IFM 4802 ATCC 15233  614 AB104687 
Capronia hanliniana IFM 52023 CBS 588.93  603 AB100681 
Cladophialophora carrionii IFM 4808T ATCC 16264  613 AB100642 
 IFM 4805 ATCC 44535 613 AB100640 
 IFM 4810 DCU 300 613 AB100643 
 IFM 4812 DCU 302 613 AB100641 
 IFM 41446 DCU 606 613 AB100644 
 IFM 41641 BMU 237 613 AB100645 
Cladophialopora bantiana IFM 46164 CBS 364.80  613 AB100616 
 IFM 41433 DCU 607 613 AB104686 
Cladophialophora devriesii IFM 51369T CBS 147.84  613 AB100646 
Cladophialophora minourae IFM 4818 DCU 428  613 AB100647 
Cladophialophora arxii IFM 52022T CBS 306.94  613 AB100683 
Cladophialophora boppii IFM 52024T CBS 126.86  612 AB100684 
Cladophialophora emmonsii IFM 52025T CBS 979.96  613 AB100682 
Cladosporium cladosporioides IFM 41447 IFO 6368  608 AB100650 
Cladosporium colocasiae IFM 51371 CBS 386.64  608 AB100649 
Cladosporium coralloides IFM 41451 IFO 6536  608 AB100658 
Cladosporium elatum IFM 41452 IFO 6372  614 AB100652 
Cladosporium fulvum IFM 40703 IAM 5006  608 AB100653 
Cladosporium herbarum IFM 41454 TMI  614 AB100651 
Cladosporium minusculum IFM 51370 URM 721  608 AB100648 
Cladosporium sphaerospermum IFM 41453 IFO 4458  608 AB100654 
Cladosporium variabile IFM 41458 IFO 6378  608 AB100655 
Exophiala alcalophila IFM 4823T IAM 12519  616 AB100672 
Exophiala dermatitidis IFM 41479T CBS 207.35  616 AB100659 
 IFM 41818 Venezuela 616 AB100660 
 IFM 41828 soil, Brazil 616 AB100661 
 IFM 45986 tap water 616 AB100662 
Exophiala jeanselmei IFM 4852T NCMH 123  618 AB100664 
 IFM 41691 BMU 2756 618 AB100663 
 FM 45989 patient 618 AB100665 
 IFM 4974 bathroom drainpipe 618 AB100666 
Exophiala moniliae IFM 41500T CBS 520.76  618 AB100667 
 IFM 41832 Venezuela 618 AB100668 
Exophiala spinifera IFM 4883T ATCC 18218 618 AB100673 
 IFM 41504 CBS 670.76 618 AB100679 
 IFM 41505 patient 618 AB100680 
Fonsecaea pedrosoi IFM 4887T CBS 271.37  613 AB100632 
 IFM 4856 DCU 677 613 AB100631 
 IFM 4889 ATCC 44356 613 AB100633 
 IFM 4914 Venezuela 613 AB100634 
 IFM 41705 bark, China 613 AB100635 
 IFM 46410 soil, Brazil 613 AB100636 
Fonsecaea compacta IFM 4886 KUM 911 613 AB100637 
 IFM 41704 BMU 4845 613 AB100638 
 IFM 41931 MTU 613 AB100639 
Hormoconis resinae IFM 51372 IFO 8588  615 AB100657 
Hortaea werneckii IFM 4885T CBS 107.67  602 AB079584 
 IFM 51373 URM 704 602 AB100674 
 IFM 41538 patient 602 AB079586 
 IFM 41541 patient 602 AB079588 
Lecythophora hoffmannii IFM 4922 CBS 245.38  602 AB100627 
Lecythophora mutabilis IFM 4923 ATCC 26223  602 AB100628 
Phialophora verrucosa IFM 4928 ATCC 38561  613 AB100610 
 IFM 41710 corn, China 613 AB100611 
 IFM 41871 soil, Colombia 613 AB100612 
 IFM 41873 Venezuela 613 AB100613 
 IFM 41879 soil, Colombia 613 AB100614 
 IFM 41898 soil, Brazil 613 AB100615 
Phialophora alba IFM 51363 IFO 31973  615 AB100618 
Phialophora americana IFM 51361 CBS 273.37  613 AB100616 
Phialophora atrovirens IFM 51364 IFO 6793  615 AB100617 
Phialophora bubakii IFM 51365 IFO 6794  615 AB100620 
Phialophora cinerescens IFM 51366 IFO 6849  615 AB100621 
Phialophora fastigiata IFM 41577 IFO 6856  616 AB100625 
Phialophora heteromorpha IFM 41578 IFO 6878  615 AB100626 
Phialophora lagerbergii IFM 51367 IFO 8576  615 AB100622 
Phialophora melinii IFM 51362 CBS 268.33  615 AB100617 
Phialophora oxyspora IFM 51368 URM 2904  616 AB100630 
Phialophora repens IFM 4925 CBS 423.73  602 AB100623 
Phialophora richardsiae IFM 4926 KUM 1681  602 AB100624 
Phaeoacremonium parasiticum IFM 4924 KUM 1827  602 AB100629 
Rhinocladiella aquaspersa IFM 4930 CBS 313.73  617 AB100677 
Rhinocladiella atrovirens IFM 4931T CBS 317.33  617 AB100678 
Species Strain Source Number of intra-species nucleotide differences Sequence length (bp) Accession number 
Alternaria alternata IFM 41348 TIMM 1289  614 AB100676 
Aureobasidium pullulans IFM 4802 ATCC 15233  614 AB104687 
Capronia hanliniana IFM 52023 CBS 588.93  603 AB100681 
Cladophialophora carrionii IFM 4808T ATCC 16264  613 AB100642 
 IFM 4805 ATCC 44535 613 AB100640 
 IFM 4810 DCU 300 613 AB100643 
 IFM 4812 DCU 302 613 AB100641 
 IFM 41446 DCU 606 613 AB100644 
 IFM 41641 BMU 237 613 AB100645 
Cladophialopora bantiana IFM 46164 CBS 364.80  613 AB100616 
 IFM 41433 DCU 607 613 AB104686 
Cladophialophora devriesii IFM 51369T CBS 147.84  613 AB100646 
Cladophialophora minourae IFM 4818 DCU 428  613 AB100647 
Cladophialophora arxii IFM 52022T CBS 306.94  613 AB100683 
Cladophialophora boppii IFM 52024T CBS 126.86  612 AB100684 
Cladophialophora emmonsii IFM 52025T CBS 979.96  613 AB100682 
Cladosporium cladosporioides IFM 41447 IFO 6368  608 AB100650 
Cladosporium colocasiae IFM 51371 CBS 386.64  608 AB100649 
Cladosporium coralloides IFM 41451 IFO 6536  608 AB100658 
Cladosporium elatum IFM 41452 IFO 6372  614 AB100652 
Cladosporium fulvum IFM 40703 IAM 5006  608 AB100653 
Cladosporium herbarum IFM 41454 TMI  614 AB100651 
Cladosporium minusculum IFM 51370 URM 721  608 AB100648 
Cladosporium sphaerospermum IFM 41453 IFO 4458  608 AB100654 
Cladosporium variabile IFM 41458 IFO 6378  608 AB100655 
Exophiala alcalophila IFM 4823T IAM 12519  616 AB100672 
Exophiala dermatitidis IFM 41479T CBS 207.35  616 AB100659 
 IFM 41818 Venezuela 616 AB100660 
 IFM 41828 soil, Brazil 616 AB100661 
 IFM 45986 tap water 616 AB100662 
Exophiala jeanselmei IFM 4852T NCMH 123  618 AB100664 
 IFM 41691 BMU 2756 618 AB100663 
 FM 45989 patient 618 AB100665 
 IFM 4974 bathroom drainpipe 618 AB100666 
Exophiala moniliae IFM 41500T CBS 520.76  618 AB100667 
 IFM 41832 Venezuela 618 AB100668 
Exophiala spinifera IFM 4883T ATCC 18218 618 AB100673 
 IFM 41504 CBS 670.76 618 AB100679 
 IFM 41505 patient 618 AB100680 
Fonsecaea pedrosoi IFM 4887T CBS 271.37  613 AB100632 
 IFM 4856 DCU 677 613 AB100631 
 IFM 4889 ATCC 44356 613 AB100633 
 IFM 4914 Venezuela 613 AB100634 
 IFM 41705 bark, China 613 AB100635 
 IFM 46410 soil, Brazil 613 AB100636 
Fonsecaea compacta IFM 4886 KUM 911 613 AB100637 
 IFM 41704 BMU 4845 613 AB100638 
 IFM 41931 MTU 613 AB100639 
Hormoconis resinae IFM 51372 IFO 8588  615 AB100657 
Hortaea werneckii IFM 4885T CBS 107.67  602 AB079584 
 IFM 51373 URM 704 602 AB100674 
 IFM 41538 patient 602 AB079586 
 IFM 41541 patient 602 AB079588 
Lecythophora hoffmannii IFM 4922 CBS 245.38  602 AB100627 
Lecythophora mutabilis IFM 4923 ATCC 26223  602 AB100628 
Phialophora verrucosa IFM 4928 ATCC 38561  613 AB100610 
 IFM 41710 corn, China 613 AB100611 
 IFM 41871 soil, Colombia 613 AB100612 
 IFM 41873 Venezuela 613 AB100613 
 IFM 41879 soil, Colombia 613 AB100614 
 IFM 41898 soil, Brazil 613 AB100615 
Phialophora alba IFM 51363 IFO 31973  615 AB100618 
Phialophora americana IFM 51361 CBS 273.37  613 AB100616 
Phialophora atrovirens IFM 51364 IFO 6793  615 AB100617 
Phialophora bubakii IFM 51365 IFO 6794  615 AB100620 
Phialophora cinerescens IFM 51366 IFO 6849  615 AB100621 
Phialophora fastigiata IFM 41577 IFO 6856  616 AB100625 
Phialophora heteromorpha IFM 41578 IFO 6878  615 AB100626 
Phialophora lagerbergii IFM 51367 IFO 8576  615 AB100622 
Phialophora melinii IFM 51362 CBS 268.33  615 AB100617 
Phialophora oxyspora IFM 51368 URM 2904  616 AB100630 
Phialophora repens IFM 4925 CBS 423.73  602 AB100623 
Phialophora richardsiae IFM 4926 KUM 1681  602 AB100624 
Phaeoacremonium parasiticum IFM 4924 KUM 1827  602 AB100629 
Rhinocladiella aquaspersa IFM 4930 CBS 313.73  617 AB100677 
Rhinocladiella atrovirens IFM 4931T CBS 317.33  617 AB100678 

TType strain.

Used for matrix study.

Used for phylogenetic study.

ATCC, American Type Culture Collection, Rockville, MD, USA; BMU, Department of Dermatology, Beijing Medical University, Beijing, China; CBS, Central bureau voor Schimmelcultures, Baarn, The Netherlands; CUH, Department of Laboratory Medicine, School of Medicine, Chiba University, Chiba, Japan; DCU, Department of Dermatology, School of Medicine, Chiba University, Chiba, Japan; IAM, Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan; IFM, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan; IFO, Institute for Fermentation, Osaka, Japan; KUM, Department of Dermatology, School of Medicine, Kanazawa University, Kanazawa, Ishikawa, Japan; MTU, Department of Bacteriology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; NCMH, The North Carolina Memorial Hospital, University of North Carolina, Chapel Hill, NC, USA; TIMM, Research Center for Medical Mycology, Teikyo University, Tokyo, Japan; TMI, Tottori Mycological Institute, Tottori, Japan; UNEFM, Universidade Nacional Experimental Francisco de Miranda, Coro, Falcon, Venezuela; URM; Department of Mycology, Federal University of Pernambuco, Recife, PE, Brazil.

DNA extraction

DNAs were prepared as described previously [22]. Briefly, approximately 50 mg of fungal elements were suspended in 600 µl extraction buffer (200 mM Tris–HCl, pH 7.5, 25 mM EDTA, 0.5% w/v sodium dodecyl sulfate, 250 mM NaCl). The mixture was vortexed for 15 s, incubated at 100°C for 15 min, kept on ice for 60 min, and then centrifuged at 14,000×g for 15 min. Supernatants were transferred to new tubes and extracted with phenol–chloroform–isoamyl alcohol (25:24:1 v/v). Each sample DNA was precipitated with cold isopropanol (−20°C), dried, and resuspended in 100 µl distilled water.

Amplification and sequencing of D1/D2 domains

The D1/D2 domains of the LSUrDNA were amplified with primers NL-1, 5′-GCATATCAATAAGCGGAGGAAAAG-3′ and NL-4m, 5′-GGTCCGTGTTTCAAGACG-3′[23]. Polymerase chain reaction (PCR) was carried out in 50 µl reactions containing 5 µl of template DNA, 5 µl (2 pmol) each primer, 4 µl (2.5 mM) dNTP mixture (Nippon Gene, Tokyo, Japan), 0.25 µl (5 U µl−1) Taq polymerase (Nippon Gene), and 5 µl 10×reaction buffer (Nippon Gene). Amplification was performed with a PCR Thermal Cycler MP (TaKaRa Shuzo, Tokyo, Japan) under the following conditions: 1 cycle of 95°C for 4 min followed by 30 cycles of 94°C for 1 min, 55°C for 2.5 min, and 72°C for 2.5 min, with a final extension at 72°C for 10 min. The amplified products were purified with SUPREC™-02 (TaKaRa) and subjected to direct sequencing with an ABI Prism 3100 sequencer after labeling with BigDye™ Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, CA, USA). The external primers, NL-1 and NL-4m, and the internal primers, NL-2m, 5′-CTTGTGCGCTATCGGTCTC-3′ and NL-3m, 5′-GAGACCGATAGCGCACAAG-3′, were used to sequence each DNA sample.

The sequence data were aligned with CLUSTAL W (version 1.6) [24]. Phylogenetic trees were constructed with the neighbor-joining (NJ) method. The nucleotide sequences for all strains examined were registered in the DNA Data Bank of Japan (DDBJ) under the accession numbers shown in Table 1.

Results

The length of the nucleotide sequence for each strain is summarized in Table 1. They ranged from 602 bp to 618 bp. For 10 medically important species, C. carrionii, C. bantiana, E. dermatitidis, E. jeanselmei, E. moniliae, E. spinifera, F. pedrosoi, F. compacta, H. werneckii, and P. verrucosa, multiple strains were analyzed to investigate differences in sequence length and intra-species nucleotide substitutions. Variations in sequence length were not observed in any of the 10 species. These data are summarized in Table 1. To detect nucleotide differences, type strains were selected as the reference species for seven species. For the three remaining species, P. verrucosa, F. compacta, and C. bantiana, the type strains were unavailable or difficult to obtain; therefore, strains IFM 4928, IFM 4886, and IFM 46164 were used as reference strains, respectively. Intra-species nucleotide variation was not detected in C. bantiana, E. dermatitidis, E. jeanselmei, E. moniliae, E. spinifera, F. pedrosoi, and F. compacta. Single nucleotide substitutions were found in C. carrionii and H. werneckii, and changes at two nucleotides were found in P. verrucosa. Therefore, the sequences of D1/D2 domains of the medically important species examined in this study were highly conserved.

A matrix of the nucleotide differences between Cladophialophora (C.) and Cladosporium (Cl.) species is shown in Table 2. The number of nucleotide differences among members of Cladophialophora species ranged from five between C. devriesii, C. minourae, and C. arxii to 26 between C. devriesii, C. minourae and C. bantiana. These data supported the sequences of D1/D2 domains is useful for identification of Cladophialophora species. Cladosporium species showed differences ranging from zero to 134 nucleotides. Cl. coralloides and Cl. cladosporioides had identical sequences, and Cl. fulvum had two nucleotide differences from each of Cl. coralloides, Cl. cladosporioides, and Cl. colocasiae. Cl. colocasiae showed three nucleotide differences from each of Cl. coralloides and Cl. cladosporioides. For the species in which the number of nucleotide differences was less than three, the sequences of D1/D2 domains are considered to be not sufficient criteria to identify confidently each species. However, the other species can be identified by the sequence of the domains.

Table 2

Matrix of nucleotide differences in D1/D2 domains of LSUrDNA between Cladophialophora (C.) and Cladosporium (Cl.) species

Species C. carrionii C. bantiana C. devriesii C. minourae C. arxii C. boppii C. emmonsii Cl. herbarum Cl. fulvum Cl. coralloides Cl. cladosporioides Cl. colocasiae Cl. variabile Cl. sphaerospermum Cl. minusculum 
C. bantiana               
C. devriesii 21 26              
C. minourae 22 26             
C. arxii 21 23            
C. boppii 12 12 18 22 19           
C. emmonsii 25 21 12 12 11 24          
Cl. herbarum 102 102 100 100 98 98 95         
Cl. fulvum 130 132 125 120 119 117 121 118        
Cl. coralloides 129 131 121 120 120 117 121 118       
Cl. cladosporioides 128 129 117 119 119 118 121 111      
Cl. colocasiae 128 129 122 119 115 119 100 118     
Cl. variabile 127 128 121 120 118 120 119 114 113 13 13 15    
Cl. sphaerospermum 126 127 118 119 117 115 118 115 112 10 11 13   
Cl. minusculum 126 127 118 119 117 116 115 114 11 110 11 13 110 10  
Cl. elatum 134 133 130 107 126 127 125 128 108 113 11 114 113 112 111 
Species C. carrionii C. bantiana C. devriesii C. minourae C. arxii C. boppii C. emmonsii Cl. herbarum Cl. fulvum Cl. coralloides Cl. cladosporioides Cl. colocasiae Cl. variabile Cl. sphaerospermum Cl. minusculum 
C. bantiana               
C. devriesii 21 26              
C. minourae 22 26             
C. arxii 21 23            
C. boppii 12 12 18 22 19           
C. emmonsii 25 21 12 12 11 24          
Cl. herbarum 102 102 100 100 98 98 95         
Cl. fulvum 130 132 125 120 119 117 121 118        
Cl. coralloides 129 131 121 120 120 117 121 118       
Cl. cladosporioides 128 129 117 119 119 118 121 111      
Cl. colocasiae 128 129 122 119 115 119 100 118     
Cl. variabile 127 128 121 120 118 120 119 114 113 13 13 15    
Cl. sphaerospermum 126 127 118 119 117 115 118 115 112 10 11 13   
Cl. minusculum 126 127 118 119 117 116 115 114 11 110 11 13 110 10  
Cl. elatum 134 133 130 107 126 127 125 128 108 113 11 114 113 112 111 

Nucleotide differences among Phialophora species are shown as a matrix in Table 3. P. verrucosa is the type species of the genus Phialophora and is a human pathogen. P. verrucosa is morphologically and physiologically similar to P. americana, and these species are not clearly separated in the medical literature. The two species differed at five nucleotide positions, whereas they showed a large number of differences from 11 saprophytic and rare pathogenic species of this genus. In the genus Phialophora, P. lagerbergii, P. bubakii, P. atrovirens, and P. heteromorpha had identical sequences, and the sequence of P. melinii differed at only one position. P. cinerescens had four or five differences from the five species listed above. Other species, such as P. richardsiae, P. repens, P. fastigiata, and P. oxyspora, were found to have characteristic sequences that differed from each other with high numbers of nucleotide substitutions, averaging 100. With the exception of the five species with identical sequences or single nucleotide variations, the sequence of the D1/D2 domains is a useful tool for identification of both pathogenic and saprophytic species of genus Phialophora.

Table 3

Matrix of nucleotide differences in D1/D2 domains of LSUrDNA of Phialophora species

Species P. verrucosa P. americana P. alba P. cinerescens P. melinii P. lagerbergii P. bubakii P. atrovirens P. heteromorpha P. richardsiae P. repens P. fastigiata 
P. americana            
P. alba 95 94           
P. cinerescens 96 95 42          
P. melinii 95 94 41         
P. lagerbergii 96 95 40        
P. bubakii 96 98 40       
P. atrovirens 97 93 41      
P. heteromorpha 97 97 40     
P. richardsiae 121 115 110 110 114 114 114 114 114    
P. repens 122 123 113 113 117 117 117 117 117 47   
P. fastigiata 31 36 102 93 94 95 95 94 95 131 122  
P. oxyspora 63 62 97 95 94 93 93 92 93 130 125 48 
Species P. verrucosa P. americana P. alba P. cinerescens P. melinii P. lagerbergii P. bubakii P. atrovirens P. heteromorpha P. richardsiae P. repens P. fastigiata 
P. americana            
P. alba 95 94           
P. cinerescens 96 95 42          
P. melinii 95 94 41         
P. lagerbergii 96 95 40        
P. bubakii 96 98 40       
P. atrovirens 97 93 41      
P. heteromorpha 97 97 40     
P. richardsiae 121 115 110 110 114 114 114 114 114    
P. repens 122 123 113 113 117 117 117 117 117 47   
P. fastigiata 31 36 102 93 94 95 95 94 95 131 122  
P. oxyspora 63 62 97 95 94 93 93 92 93 130 125 48 

A matrix of the nucleotide differences for 10 medically important species was generated to evaluate the usefulness of these sequences for identification (Table 4). Data were obtained by comparison of D1/D2 domain sequences for the type strains of seven pathogenic species. As described earlier, IFM 4886, IFM 46164, and IFM 4928 were used as reference strains for F. compacta, C. bantiana and P. verrucosa, respectively. F. pedrosoi and its dysplastic variant, F. compacta, were found to have identical sequences as predicted. The smallest difference, three nucleotides, was found between C. bantiana and P. verrucosa, and small differences of six nucleotides were observed both between C. bantiana and C. carrionii and between E. spinifera and E. jeanselmei. The largest difference, 130 nucleotides, was observed between C. bantiana and H. werneckii. In general, the number of differences was distributed in the range of 20–30 nucleotides. These results suggest that the sequences of the D1/D2 domains might be useful for identification of these pathogenic dematiaceous species except for discrimination between C. bantiana and P. verrucosa. We also compared the data for H. werneckii, the causative agent of tinea nigra, and Exophiala species because H. werneckii was classified into the genus Exophiala prior to 1984. As shown in Table 4, there were more than 120 differences between H. werneckii and all five species of Exophiala. This strongly supports the validity of establishing Hortaea as an independent genus.

Table 4

Matrix of nucleotide differences in D1/D2 domains of LSUrDNA among medically important dematiaceous fungi

Species C. bantiana C. carrionii E. dermatitidis E. jeanselmei E. moniliae E. spinifera F. pedrosoi H. werneckii 
C. carrionii        
E. dermatitidis 35 38       
E. jeanselmei 32 31 13      
E. moniliae 32 32 15 12     
E. spinifera 30 31 15 11    
F. pedrosoi 25 19 25 17 26 21   
F. compacta 25 19 25 17 26 21  
H. werneckii 130 128 123 128 129 127 122  
P. verrucosa 37 32 34 31 24 126 
Species C. bantiana C. carrionii E. dermatitidis E. jeanselmei E. moniliae E. spinifera F. pedrosoi H. werneckii 
C. carrionii        
E. dermatitidis 35 38       
E. jeanselmei 32 31 13      
E. moniliae 32 32 15 12     
E. spinifera 30 31 15 11    
F. pedrosoi 25 19 25 17 26 21   
F. compacta 25 19 25 17 26 21  
H. werneckii 130 128 123 128 129 127 122  
P. verrucosa 37 32 34 31 24 126 

The phylogenetic trees constructed by the NJ method for Chaetothyriales and for all examined species are shown in Figs. 1 and 2, respectively. Aspergillus fumigatus was used as an outgroup. The tree of Fig. 1 shows that almost all human pathogens, including F. pedrosoi, F. compacta, P. verrucosa, P. americana, all species of Cladophialophora and Exophiala form one cluster, and also that Exophiala species are located as a monophyletic cluster separated from human pathogens described above. Polyphyletic characteristics in Phialophora species were inferred from their cluster formations; P. verrucosa and P. americana were closely related to species of the genera Fonsecaea and Cladophialophora, P. fastigiata clustered with the genus Exophiala, and P. oxyspora formed a single-membered cluster independently. Of the 13 species examined, the nine species except the four mentioned above formed a subcluster with comparatively remote distance from other species. The tree in Fig. 2 was constructed for all species examined to study phylogenetic relationships among them. The human pathogenic species analyzed in the present study are classified into the following four orders and five families of the class Euascomycetes: Chaetothyriales Herpotrichiellaceae [A], Dothideales Dothioraceae [B-1], Dothideales Mycosphaerellaceae [B-2], Sordariales Coniochaetaceae [C], and Pleosporales Pleosporaceae [D]. In Fig. 2, Chaetothyriales [A] show their phylogenetically distant relationship from the other orders Dothidiales [B-1, 2], Sordariales [C], and Pleosporales [D]. The results for Chaetothyriales in Fig. 1 were principally not affected by adding other examined species. P. richardsiae and P. repens were closely related to Phaeoacremonium and Lecythophora species of order Sordariales. The species in the order Dothideales, including Aureobasidium pullulans, H. werneckii and Cladosporium species, formed one cluster with Alternaria alternata of the order Pleosporales. Cl. herbarum was more distantly related to other Cladosporium species, with lower sequence homology.

Figure 1

NJ tree for D1/D2 domains of the genus Phialophora and pathogenic Chaetothyriales. Bootstrap values derived from 10,000 replicates are shown as percentages. The scale bar represents a difference corresponding to 0.02 (2%). For full genus and species names see Table 1.

Figure 1

NJ tree for D1/D2 domains of the genus Phialophora and pathogenic Chaetothyriales. Bootstrap values derived from 10,000 replicates are shown as percentages. The scale bar represents a difference corresponding to 0.02 (2%). For full genus and species names see Table 1.

Figure 2

NJ tree for D1/D2 domains of pathogenic dematiaceous fungi and related taxa. Bootstrap values derived from 10,000 replicates are shown as percentages. The scale bar represents a difference corresponding to 0.02 (2%). For full genus and species names see Table 1. A: Chaetothyriales Herpotrichiellaceae, B-1: Dothideales Dothioraceae, B-2: Dothideales Mycosphaerellaceae, C: Sordariales Coniochaetaceae, D: Pleosporales Pleosporaceae.

Figure 2

NJ tree for D1/D2 domains of pathogenic dematiaceous fungi and related taxa. Bootstrap values derived from 10,000 replicates are shown as percentages. The scale bar represents a difference corresponding to 0.02 (2%). For full genus and species names see Table 1. A: Chaetothyriales Herpotrichiellaceae, B-1: Dothideales Dothioraceae, B-2: Dothideales Mycosphaerellaceae, C: Sordariales Coniochaetaceae, D: Pleosporales Pleosporaceae.

Discussion

Dematiaceous fungi, including medically important species, have been typically identified by morphological and physiological characteristics. Such methods are laborious and sometimes cannot distinguish such species with polymorphic conidiogeneses or lacking conidia formation. Genetic methods such as RAPD (random amplified polymorphic DNA) and RFLP (restriction fragment length polymorphism) have been used to identify medically important dematiaceous species [25–29]; however, these methods are considered to be more appropriate for taxonomy, typing, and epidemiological investigation of fungi than identification. Recently, sequences of the internal transcribed spacer region and D1/D2 domains of rDNA have been used for identification purposes due to the higher accuracy and objectivity of such methods. Since sequence data for the D1/D2 domains of dematiaceous fungal taxa have not been reported, the present study aimed to collect the data and then to evaluate them as a criterion for identification of dematiaceous fungi, especially medical pathogens.

We examined multiple strains for each medically important species. High intra-species conservation of nucleotide sequences of the D1/D2 domains was observed in 10 pathogenic species. No intra-species nucleotide substitutions were detected in seven species described previously, and the largest number of substitutions was only two in a strain of P. verrucosa. On the other hand, high nucleotide substitutions were shown between species. F. pedrosoi and F. compacta have identical D1/D2 sequences. The two species are morphologically and physiologically similar [30,31]. RAPD and RFLP methods have been used to investigate genetic variations between these species [26,32]; however, variations were not found. It is possible that F. compacta is not an independent species but a variant of F. pedrosoi. P. verrucosa and C. bantiana have only three nucleotide substitutions and the nucleotide diversity is less than 0.5%. However, the two species have large diversity in their morphology and pathogenicity. The former is a causative agent of chromoblastomycosis, whereas the latter one is associated with cerebral pheohyphomycosis [1,15]. This is an example that morphological characteristics may have a higher contribution to discriminating two species than any genetic evidence including the D1/D2 domain sequence.

Of some saprophytic and pathogenic species of the genera Phialophora and Cladosporium, several species were found to have identical or highly homologous sequences with substitutions at only one or two positions. For such species, the nucleotide sequences are incapable of discriminating each species, and it is expected to find any genetic region with greater nucleotide variation. Four Phialophora species, P. lagerbergii, P. bubakii, P. atrovirens, and P. heteromorpha, have identical sequences in D1/D2. These data may become new evidence to discuss the necessity of their re-identification. Except for the species mentioned above, other species could be discriminated from each other by the sequence of D1/D2 domains. Especially for medically important species, except P. verrucosa and C. bantiana, the domain is applicable for identification of all species.

The tree (Fig. 1) of the species of genus Phialophora and pathogenic Chaetothyriales demonstrated the phylogenetically characteristic relationships among them: the close phylogenetic distances of human pathogens of Fonsecaea species, all Cladophialophora species, and the two species of P. verrucosa and P. americana cluster formation in all Exophiala species examined and the phylogenetic diversity of the genus Phialophora.

In the tree (Fig. 2) for all examined species, the phylogenetic results for the genus Phialophora and Chaetothyriales discussed above are principally not changed by adding other taxa. As a new finding, Rhinocladiella species are demonstrated to have a close interrelation to the genus Exophiala. The tree also includes some interesting information on phylogenetic distances among dematiaceous fungal taxa; the genus Phialophora is a polyphyletic taxon; the genus Cladosporium has a completely remote distance from the genus Cladophialophora, and should be polyphyletic; although much higher numbers of tested fungi need to be analyzed, the genera Lecythophora [C], Phaeoacremonium [C] and Alternaria [D] are supported to be accompanied in different taxa from [A] and [B]; Au. pullulans and H. werneckii, weak pathogens, might form an independent cluster in taxon [B-1]. The phylogenetic relationship of pathogenic dematiaceous fungi was defined from the trees constructed using the D1/D2 domain sequences.

In conclusion, the sequences of the D1/D2 domains were evaluated to be applicable for identification of many taxa of dematiaceous fungi, especially of human pathogenic species. On the other hand, for some pathogenic or saprophytic Phialophora and Cladosporium species which have identical sequences or an inter-species sequence diversity of less than 0.5%, the domain is not useful for their identification. For these species, morphological characteristics may be used as the prevailing criteria, because no other genetic region having a higher ability to discriminate species than D1/D2 domain has been found to date.

Acknowledgements

This study was performed as part of the program ‘Frontier Studies and International Networking of Genetic Resources in Pathogenic Fungi and Actinomycetes (FN-GRPF)’ through Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government 2003.

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