Antonie van Leeuwenhoek 68:19-33, 1995. 19 @ 1995 KluwerAcademic Publishers. Printed in the Netherlands.

Phylogenetic analysis of ten black species using nuclear small subunit rRNA gene sequences

G. Haase 1, L. Sonntag I , Y. van de Peer 2, J.M.J. Uijthof 3, A. Podbielski 1 & B. Melzer-Krick 1 1 Institute for Medical Microbiology, Klinikum RWTH Aachen, D-52057Aachen, Germany; 2 Department of Biochemistry, Universiteit Antwerpen, Universiteitsplein 1, B-2610 Antwerp, Belgium; 3 Centraalbureau voor Schimmelcultures, P.O. Box 273, NL-3740 AG Baarn, The Netherlands

Key words: black , , direct sequencing, , nuclear small subunit rRNA gene, phylogenetic analysis,

Abstract

The nuclear small subunit rRNA genes of authentic strains of the black yeasts , Wangiella dermatitidis, Sarcinomyces phaeomuriformis, , Nadsoniella nigra vat. hesuelica, Phaeoan- nellomyces elegans, Phaeococcomyces exophialae, var. jeanselmei and E. castellanii were amplified by PCR and directly sequenced. A putative secondary structure of the nuclear small subunit rRNA of Exophiala dermatitidis was predicted from the sequence data. Alignment with corresponding sequences from Neurospora crassa and was performed and a phylogenetic tree was constructed using the neighbor-joining method. The obtained topology of the tree was confirmed by bootstrap analysis. Based upon this analysis all fungi studied formed a well-supported monophyletic group clustering as a sister group to one group of the Plectomycetes (Trichocomaceae and Onygenales). The analysis confirmed the close relationship postulated between Exophiala dermatitidis, Wangiella dermatitidis and Sarcinomyces phaeomuriformis. This monophyletic clade also contains the teleomorph species Capronia mansonii thus confirming the concept of a teleomorph con- nection of the genus Exophiala to a member of the . However, Exophiala castellanii did not belong to this clade. Therefore, this species is not the anamorph of Capronia mansonii as it was postulated.

Introduction lesions frequently with lethal outcome (Matsumoto et al. 1984, 1993; Hiruma et al. 1993). Exophiala Black yeasts are dematiaceous fungi that may occur as jeanselmei is frequently involved in infections of nails, solitary cells during a part of their life cycle. Frequent- cutis, and subcutis (Schwinn et al. 1993). In addi- ly, black yeasts are synanamorphs associated with tion to their role as human pathogens, black yeasts are dematiaceous hyphomycetes. The majority of black involved in the degradation of marble and sand stone yeasts have teleomorphs in the ascomycetes, though in (Braams 1993; Wollenzien et al. 1994). different orders (de Hoog & McGinnis 1987). Conidiogenesis in black yeasts is either blastic, During the last decade there has been an increasing annellidic or phialidic. Upon changes in environmental number of reports implicating black yeasts as causative conditions (e.g., during laboratory cultivation), black agents of various kind of mycosis in humans and ani- yeasts may respond by expressing different morpho- mals (Dixon & Polak-Wyss 1990; Kwon-Chung & logical forms, thereby changing their mode of conid- Bennett 1992; Matsumoto et al. 1993). Exophiala der- iogenesis (Butterfield & Jong 1976; de Hoog 1993; de matitidis (Kano) de Hoog is now regularly isolated Hoog et al. 1994b). Since the form of conidiogenesis from sputum of patients suffering from is the leading criterion for assigning black yeasts to (Haase et al. 1991; Kusenbach et al. 1992). Systemic certain anamorph genera (de Hoog 1977; Cole 1978), phaeohyphomycotic infections may also occur as brain this variability of conidiogenesis has a negative impact 20 on successful species classification. Observing coni- et al. should represent the holomorph of E. castellanii diogenesis is further complicated by the fact that the (formerly treated as Exophiala mansonii). conditions and the media used for this analysis are not In the present paper we intend to address the ques- standardized. Dissimilar interpretations of phenome- tions outlined above by using molecular techniques. na have led to the assignment of a single isolate to In addition, we aim to show the phylogenetic relation- different genera. For example, the strain CBS 207.35 ships of black yeasts to other main groups of Ascomyc- was classified in the genus Exophiala Carmichael as E. eta. The sequence data provided can potentially be used dermatitidis based on the observed annellidic conidio- for the development of an identification system allow- genesis. In the same year McGinnis (1977) established ing quick and objective species assignment in the black the genus Wangiella based on the same strain, but yeasts. described the prevalent type of conidiogenesis as phia- lidic without collarettes. Problems of specific delim- itation arose with strain ATCC 34100, classified by Materials and methods McGinnis (1977) as Wangiella dermatitidis McGinnis but by de Hoog (1977) as Exophiala mansonii (Castell.) Strains and culture methods de Hoog. It was questioned whether E. castellanii Iwat- suet al. (new name for E. mansonii) was a separate Strains used listed in Table 1. Stock cultures were species or belonged to the dermatitidis cluster. maintained on potato-dextrose agar (Merck AG, Darm- Physiological testing used for species delimitation stadt, FRG) slants at 8 ° C and as stock cultures on and identification is tedious and time-consuming (de ceramic beads (MicrobankTM, Mast, Hamburg, FRG) Hoog & Haase 1993). Furthermore, physiological data at - 800 C. Inocula were prepared as exponential phase are incomplete or not yet available for most of the suspensions of yeast cells according to de Hoog et al. medically important black yeasts. Therefore, there is (1994b). Fungi were cultivated in a Sabouraud's broth an urgent demand for a reliable and easy-to-perform (Difco, Detroit, USA) with constant shaking at 300 C procedure for the identification of black yeasts recov- for 5 days. ered from clinical specimens. Recently, molecular techniques have become wide- DNA extraction ly utilized in fungal taxonomy (Bruns et al. 1991; McGinnis et al. 1992). Few molecular studies have DNA was prepared according to the procedure given by examined the dematiaceous fungi. Analysis of the Goodwin & Lee (1993). Purified DNA was dissolved genes coding for small subunit (SSU) ribosomal RNA in TE buffer, aliquoted and stored at - 20 o C. indicated that the sequence of this gene can provide a reliable foundation upon which taxonomic problems PCR amplification may be resolved (Bruns et al. 1991; Kurtzman 1992; Wilmotte et al. 1993). The SSU-rRNA gene was amplified using the In the present study we have sequenced > 96% of NS1/NS8 primers of White et al. (1990). The PCR the SSU-rRNA genes of Exophiala dermatitidis, of reaction was performed with 1 #g DNA and 10 ¢zl its putative synonyms and of some other Exophiala of 10 x PCR buffer (500 mM KC1, 100 mM Tris species [E. jeanselmei (Langer.) McGinnis & Pad- HC1, pH 8.3, 15 mM MgC12, 0.01% gelatin), 62.5/~M hye, E. castellanii] frequently recovered from clin- (final concentration) of deoxyribonucleotide triphos- ical specimens. Nadsoniella nigra Issatchenko var. phates [using the desoxynucleoside-triphosphate-set® hesuelica Lyakh & Ruban was studied since this (Boehringer, Mannheim, FRG)], 5 pmol each of NS1 strain was supposed to be close to E. jeanselmei and NS8 primers (for some isolates NS8 was replaced (de Hoog 1985). Since species of the genera Phaeo- by ITS2; White et al. 1990), and 2.5 units ofTaq poly- coccomyces (de Hoog) de Hoog and Phaeoannel- merase (Boehringer). Distilled deionized water was lomyces synanamorphs in the genus Exophiala we added to a final volume of 100/~1. Thermal cycling have also included the type isolates (Phaeococcomyces parameters were set at 94 o C for 1 min (denaturation), exophialae (de Hoog) de Hoog and Phaeoannel- 55 o C for 1 min (annealing), and 720 C for 1.5 min lomyces elegans (McGinnis & Schell) in our analysis. (extension) for 27 cycles. An extension step at 720 C According to Schol-Schwarz (1968), and Mtiller et al. for 7 min was performed after the final cycle. Amplifi- (1987) Capronia mansonii (Schol-Schwarz) E. Mailer 21

Table 1. Fungal strains used for sequencing of SSU-rRNA.

Species Strain-number EMBL-accession Taxon- numbers Abbreviation Exophiala dermatitidis (Kano) de Hoog CBS 207.35T X 79312-79314 E.der Wangiella dermatitidis (Kano) McGinnis ATCC 34100 X 71315-79317 W.der Wangiella dermatitidis (Kano) McGinnis KU A-0052 X 80702 Rder (= Phaeotheca synanamorphof W. dermatitidis) Sarcinomycesphaeomuriformis Matsumotoet al. CBS 131.88"r X 80710 S.pba Exophialajeanselmei (Langeron) McGinnis & Padhye ATCC 34123T X 80705 E.jea var.jeanselmei (Benedek & Spech0 de Hoog Nadsoniella nigra Issatschenkovar. hesuelica Lyakh& Ruban CBS 546.82 X 80707 N.nig Phaeoannellomyces elegans McGinnis & Schell UTMB 1286T X 80708 Rele Phaeococcomyces exophialae (de Hoog)de Hoog CBS 668.76T X 80709 Eexo Exophiala castellanii (Iwatsu et al.) CBS 158.58yr X 78480 E.man (= Exophialajeanselmei (Langeron) McGinnis & Padhye vat. castellanii) Iwatsu & Udagawa Capronia mansonii (Schol-Schwarz)E. Mfiller CBS 101.67T X 79318 C.man

cation products were purified using the 'Wizard DNA al. 1993; Van de Peer et al. 1994). Drawing of this clean up' kit® (Promega, Madison, USA). structure was performed by using the program 'CARD' (Winnepenninckx et al. 1995). Direct sequencing Sequence alignment and phylogenetic Direct sequencing was done with 10 /zl of purified tree-construction amplification products and 10 pmol primer (NS 1-NS8) using the PRISM TM Ready Reaction DyeDesoxyeTM The sequences determined in this study were aligned Terminator Cycle Sequencing Kit (Applied Biosys- with all other complete (meaning > 90% sequence tems, Weiterstadt, FRG) according to the manufactur- information of this gene) ascomycete SSU-rRNA er's instructions. Sequencing assays were analyzed on sequences (Neefs et al. 1993) so far published (Octo- an automated DNA Sequencer 373 A (Applied Biosys- ber 1994) on the basis of similarity in primary and sec- tems). In order to achieve optimal overlap of the par- ondary structures, by means of a specially developed tial sequences, two additional primers were designed: sequence editor (de Rijk & de Wachter 1993). The LS-292F (5'-ATI'CAAATTTCTGCCCTATCAAC-3') phylogenetic tree was constructed using the neighbor- and LS-825F (5'-GTTCTATITI'GTTGGTrTCT-3'). joining method (Saitou & Nei 1987), based on a dis- All primers used were synthesized with a DNA Syn- similarity matrix corrected for multiple mutations per thesizer Oligo 1000 (Beckman, Munich, FRG). Com- site (Jukes & Cantor 1969). The complete alignment pilation and analysis of sequence data were done with of all sequences was taken into account for distance the aid of the PC Gene® program (IntelliGenetics, calculation. The basidiomycete Filobasidiella neofor- Mountain View, USA). mans Kwong-Chung was chosen as an outgroup organ- ism. Confidence values for individual branches were Reconstruction of secondary structure determined by bootstrap analysis in which 500 boot- strap trees were generated from resampled characters The mature SSU-rRNA of Exophiala dermatitidis, (Felsenstein 1985). Distance calculations, tree con- CBS 207,35 was folded into the 'universal' secondary struction, and bootstrap analysis were performed with structure adopted for eukaryotic SSU-rRNA (Neefs et 22 the software package TREECON (Van de Peer & de 1992), but nearly identical to that of Endomyces fibu- Wachter 1993, 1994). liger Lindner (Wilmotte et al. 1993) and Acanthamoe- ba griffini Sawyer (Gast et al. 1994). A phylogenetic tree calculated by utilizing each Results (nearly) complete ascomycetal SSU-rRNA gene pub- lished thus far is given in Fig. 3. In this tree the ten PCR-based DNA amplification performed with the species studied cluster as a monophyletic sister-group primers NS 1/NS8 yielded products of different lengths. to the Plectomycetes (Eurotiales and Onygenales). Amplicons obtained from Exophiala (Wangiella) der- matitidis and its Phaeotheca-like synanamorph, and Sarcinomyces phaeomuriformis showed a lenght of Discussion approximately 2.7 kb, whereas those from Nadsoniella nigra var. hesuelica and Phaeoannellomyces elegans Analysis of sequence data showed a length of 2.2 kb and 2.45 kb, respectively. Amplification products of all other fungi tested, includ- Comparison of ten (nearly) entire SSU-rRNA ing Neurospora crassa Shear & Dodge, exhibited the sequences to the corresponding sequence from Neur- expected length of 1.8 kb. spora crassa revealed a remarkable variability in seg- Subsequent sequencing of the amplification prod- ments corresponding to stems of helices 12 and 13 ucts revealed the presence of two different puta- (Figs 1, 2). These regions are supposed to be high- tive introns in E. dermatitidis, the Phaeotheca-like ly conserved (Neef et al. 1993). This false assump- synanamorph, in ATCC 34100 and in S. phaeomu- tion may have arisen through the common practice of riformis. These introns were situated downstream comparing only partial sequences of selected variable of nucleotide positions 561 and 1164, respectively, regions (for example, Spatafora & Blackwell 1993), according to the SSU-rRNA sequence-numbering of N. supposing that construction of a phylogenetic tree crassa (EMBL accession number X04971). The SSU- based on entire SSU-rRNA sequences or on partial rRNA gene of N. nigra var. hesuelica harbored a dif- variable regions of the genes would lead to identi- ferent putative intron downstream of nucleotide posi- cal results (McCarrol et al. 1983). Our data indicate tion 1210 whereas the corresponding sequence from that it is advisable to sequence the entire SSU-rRNA P. elegans carried yet another intron downstream of gene when primers are designed for recognition of taxa nucleotide position 1764. Details about these introns above the level of species. Furthermore, there is a will be presented elsewhere. need to confirm the assumed conservation of target Alignment of the obtained sequences is shown in sites for 'universal' primers (White et al. 1990). This Fig. 1. The SSU-rRNA gene sequence from N. cras- sequence information is obtainable only by using addi- sa was chosen as the leading sequence. For com- tional primers. By employing the primer LS-292F we parison the SSU-rRNA sequence of a saprobic black revealed sequence diversity located at the NS3 primer yeast, Aureobasidium pullulans (de Barry) Arnaud, site (Fig. 1). was added (EMBL accession number M55639). In Sequencing of the SSU-rRNA gene amplified by Fig. 1 the locations of the primers used are marked PCR instead of rRNA-derived cDNA templates also by bold letters. Sequence differences clustered primar- revealed the occurrence of introns in the nuclear SSU- ily between the nucleotide positions 65-82, 122-187, rRNA genes of fungi (de Priest 1993; de Wachter 322-386,644-710, 752-770, 1224-1267, 1355-1378, et al. 1992). These insertions could severely affect and 1720-1747 (Fig. 1). As compared to N. crassa, hybridization assays with genomic DNA (e.g., 'ribo- most of these sequence differences occur in all species typing' experiments) or PCR-based species assign- studied, including A. pullulans. ment using restriction fragment patterns of amplifica- The predicted secondary structure of the gene prod- tion products (for example, Shen & Lanchance 1993; ucts of CBS 207.35, E. dermatitidis is given in Fig. Bemier et al. 1994). Thus, for the development of 3. Secondary structures of helices 19, 20, and 21 molecular detection or diagnostics of black yeasts are different from that of Candida krusei (Castell.) using SSU-rRNA genes, sequencing of the entire gene Berkhout (Hendriks et al. 1981), Saccharomyces cere- is recommended. visiae Meyen ex E.C. Hansen (Gutell 1993) and Usti- lago maydis (de Candolle) Corda (de Wachter et al. 23

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Phylogenetic analysis

Phylogenetic tree construction with eukaryotic SSU- rRNA gene sequences using the neighbor-joining method has been proven to be a reliable predictor of taxonomic relationships (Bruns et al. 1991; Hendriks et al. 1991). The ten strains of black yeasts studied formed a monophyletic branch (Fig. 3), thus strongly support- ing the close interrelationship of the organisms con- cerned. They are a sister-group to the Plectomycetes (Eurotiales and Onygenales). In contrast, the Aureobasidium pullulans appears unrelated. The exclusion of A. pullulans from the combined group of black yeasts and Plectomycetes indicates that Locu- loascomycetes do not form a natural, monophyletic group. The precise phylogenetic arrangement relative to the Pyrenomycetes (Ophiostomatales and Sphaeri- ales) remains unclear since these branches are not well supported. The black yeasts studied are strongly sep- arated from the white yeasts and the basidiomycetous outgroup F neoformans, so that formation of yeast cells must be a secondarily derived character. Using an ascomycetous organism as outgroup organism did not alter the topology of the tree obtained.

Taxonomy

The close interrelationship of Sarcinomyces phaeomu- riformis and Exophiala (Wangiella) dermatitidis and its purported Phaeotheca-like anamorph (Matsumoto et al. 1990) was well supported by the phylogenetic analysis. The type strain ofExophiala (Wangiella)der- matitidis and strain ATCC 34100 exhibit 9 nucleotides difference (-- 0.5%; Fig. 1). The branch between these two species is statistically not significant, indicating that these two strains belong to the same species. The four strains mentioned above are indistinguish- able from each other by exoantigen test (Matsumoto i,o

Go et al. 1986). All have two different introns (but with r-I identical sequences) inserted at the same position in I

~. : : : :~. c- Schwarz 1968; Samuels & Mtiller 1978; MiJller et o al. 1987; de Hoog et al. 1994a). Further members of the Herpotrichiellaceae, as well as of supposed related anamorphic genera (e.g., Fonsecaea Negroni, Ramichloridium Stahel ex de Hoog, Rhinocladiella 30

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u C/~ uA. o A A u ~A ~ u • u u °UG-CC EIO . 1 ~,A A~ G'C~.c u uA ~,C GG" x, A O C C U U • "CAAAA A A A u "c G.C , %~u 10 o c . A °G G. .c ° u cuucc

Fig. 2. Predicted secondary structure model of the SSU-rRNA ofExophiala dermatitidis CBS 207.35 T. Helix numbering according Neefs et al. (1993). 31 Distance 0.1

T',daromyces flavus £--- Aspergillus terreus jr--.-- Aspergillus niger ~9~ ~ Eurotium rubrum I----~ ~ Aspergillus flavus ~.~ ~ Aspergfllus nidu]ans Eurotiales 17 6~, PeniciUum notatum ~oo~..~ t~ "~ Monascus purpureus [ 6 6~ F---- Thermoascus crustaeeus 1 ~ Byssochlamys nivea [ r ~ Trichopbyton rubrum ~0x I ~.~ --- Blastomyces dermaKtidis "-"1[ !----- Histoplasma eapsulatum ~624 Eremaseus albus [~-~ Ascosphaera apis 99~ q r- -- Malbranehea gypsea Onygenales "~e~ xoo~ r-- Unclnocarpus rees~ t. Coeddioide~ immitis ~ Malbranchea albolutea .~.oe~, ~ Malbranchea filamenlosa Malbranchea dendritica Exophiala eastellanil ...... ,nn~[ ~ ph aeococcomyees ex op hi al ae I --7.~ ~S~ I I ExophJala jeanselmei var.jeanselmeJ I / ~-'~.oo~ ~ Phaeoanndlomyces elegans I f04~ ~ Nadsoniella nigra var. hesudica [ I"~k t ~.k., : I [ ~,xr------CapronJamnaso~ | ,~-aeto,n~r,a,es t..~¢~ f... Exophiala dermatiUdis | 6ex r" Wangiella dermalJfidis ATCC 34100 | ~.~ Pbaeotheea syaaaamorph ofW. dermalttidls [ Sareinomyces phaeomuriformis ! ?1Ix~r--- Alternaria bl-a~eae ...... 9~ ~--- Alternaria alternata ~O6~S4~lr Alternaria raphani [ ~ Alternaria brmsicicola t.__ Pleospora berbarum ~$x7~[------~- ~ Pleospora rvdis Pleosporales I ~9~ r---- Septoria Dodorum 100~ ~ 79~ r-- Leptosphaeria maculans | s4~-- Leptosphaeria microscopiea [ ~ Leptosphaeria doliolum l Leptosphaeria bicolor -- ~,~m~-- '--""'"-'.....'....'...... '.L...... '_.. Dothideales ~.0o~. r- Ophiostoma stenocer~ ! :.0o~ [--'-t. Ophiostoma sehenckii 5r~ l Ophiostoma ~a~ Ophiostomatales [ t -- -- Leucostoma persoonii ...... I ~.0o41 ~.o6~, Pseudallescheria boydil -~ ~ ' Mi ..... ¢i ...... Microascales i l~ -- -- Hypomyces chrvsospermus S~ 1. -- ~ Glomerella cingulala I :~*o,~ I -- -- Podospora anserina ~pnaerlales t -- ~aaetomium datum --Ss~ 97~ t Sordarla firmJcola

Neu rospora cr-assa ...... Sc] erotinia sclerollorum t ioo4,r-- Spathularla flavtda Leotiales t...- Cudonia collfusa 99~ [ , , lnermisia ~a~ Pezizales Gvromitra ec,culenta ~.o O~ {--__Waltomyces lipofer ~ [ Dip°dasc°psis uninudea~ e e~ [ .... G alactomycm geotriehum 97% Endomyees geotriehum I / DJpodascus albJdus L $~.-- • yarroma" llpolylica'" [ [ . . , Metschnikowia bicuspidala [ ' Clavispora lusitaniae ~ J ?o~, ~oo~, Pichia membrana~aciens ..... t ~-~ ~ lssatehenkJa orienlalis I [ ~ -- - Dekkera bruxellensis 1 [ r--- -- Pichia anomala 7o4 [ 64 r--- Torniaspora ddbrueckii ] [ .i ~_ ~ , . . Saccharomyce~ cerevisiae [ I ~',~---- IOuyveromyces polvsporus 77L~ 75~'~ ~ Candidaglabrata ~-[ ~ -- Zygosaccharomvces rouxi ~, t . ,~ ~ L -- IGuyveromyces lacUs. [ ~.q t Saccharomycodes ludwtg~J ~eJ~ [ [ -- -- Hansenimpora uvarum r -- Pichia angusta 0 I [ xoox, Saccharomycopsis capsulafis "~ --.'.1 I Endomyces fi buliger = 6 ~,.?-4 ~.oo~. ~. Yamadazyma guilliermondii [.~ [ ~7~ [ t- Debaryomyces han~nii s Candida albicans ~"e 2'~ [ Candida maltosa '6e4 r- Candida tropicalis $';~ Candida viswanalhii Can dida parapsilosis Neolecta vitdlina SailoeUa compDcala Pneumocystis carinii :-00~ t ...... Schizosaccharomyc~ japonicus ' Schizosaccharomyces pombe Protomvces inouve/ ~.oo~ I Taphrina wlesneri

~.00~ [ Taphrina deformans ...... Filobasidiella neoformans

Fig. 3. Rooted phylogenetic tree of ascomycetes (with more than 90% sequence information of the SSU-rRNA gene) including the ten new SSU-rRNA sequences determined in this study. The basidiomycete Filobasidiella neoformans was used as outgroup organism. The distance between two organisms or groups of organisms, measured in substitutions per nucleotides, is obtained by summing the lengths of the connecting branches along the horizontal axis, using the scale on the top. Bootstrap analysis percentage (out of 500 samples) higher than 50% are indicated alongside the branch considered. 32

Nannf.) need to be studied for a further elucidation ing region to be used for designing species-specifc of teleomorph connections of dematiaceous human oligonucleotides. pathogens. The strain CBS 158.58 was designated by de Hoog (1977) as neotype Of E. mansonii because the origi- Acknowledgements nal material described by Castellani (1905) was lost. McGinnis (1977) pointed out that this designation was The authors wish to thank T. Matsumoto and M.R. in conflict with the International Code of Botanical McGinnis for providing cultures. G.S. de Hoog is Nomenclature, since Castellani's original description acknowledged for his comments on the manuscript. (1905) of the patient's infection was in disagreement with the clinical picture caused by CBS 158.58. There- fore, Iwatsu et al. (1984) proposed the name Exophiala References castellanii for this strain. In 1990 the new combination Exophiala jeanseImei var. castellanii (Iwatsu et al.) B emier L, Hamelin RC & Ouellette GB (1994) Comparison of ribo- Iwatsu & Udagawa was proposed for this strain since somal DNA length and restriction site polymorphism in Grem- meniella and Ascocalyx isolates. Appl. Environ. Microbiol. 60: it would differ only in minor morphological characters 1279-1286 from the other varieties of E. jeanselmei. Our phylo- Braams J (1993) Ecological studies on the fungal microflora inhab- genetic analysis strongly supports a separation of E. iting historical sandstone monuments. Ph.D. Thesis, Oldenburg jeanselmei and E. castellanii. The taxon is also clear- Butterfield W & Jong SC (1976) Effect of carbon source on coni- diogenesis in Fonsecaea dermatitidis, agent of chromomycosis. ly different from E. dermatitidis (Fig. 3). Therefore, Mycopathologia 58:59-62 we recommend retention of E. castellanii as a sepa- Bruns TD, White TJ & Taylor JAW (1991) Fungal molecular sys- rate species, as proposed by de Hoog (1977), Iwatsu tematics. Ann. Rev. Ecol. Syst. 22:525-564 Castellani (1905) Tropical form of pityriasis versicolor. Br. Med. J. et al. (1984) and Matsumoto et al. (1993). Capronia 2:1271-1272 mansonii (formerly treated as Dictyotrichiella man- Cole GT (1978) Conidiogenesis in the black yeasts. PAHO Sci. Publ. sonii Schol-Schwarz) was described as the teleomorph 356:66-78 of E. mansonii (Schol-Schwarz 1968; Mtiller et al. Dixon DM & Polak-Wyss A (1990) The medically important dema- tiaceous fungi and their identification. Mycoses 34:1-18 1987). This finding is in conflict with our phylogenetic Felsenstein J (1985) Confidence limits on phylogenies: an approach analysis (Fig. 3). using the bootstrap. Evolution 39:783-791 De Hoog (1985) suggested that N. nigra var. hesuel- Gast RJ, Fuerst PA & Byers TJ (1994) Discovery of group I introns in ica was an Exophiala species close to E. jeanselmei. the nuclear small subunit ribosomal RNA genes of Acanthamoe- ba. Nucl. Acids Res. 22:592-596 Our analysis favours the maintenance of this strain Goodwin DC & Lee SB (1993) Microwave miniprep of total genomic as a separate species. Phaeococcomyces exophialae DNA from fungi, plants, pro fists and animals for PCR. BioFeed- and Phaeoannellomyces elegans were described as back 15:438-444 synanamorphs of Exophiala which could not be Gutell RR (1993) Collection of small subunit (16S- and 16S-like) ribosomal RNA structures. Nucl. Acids Res. 21: 3051-3054 assigned to any particular species (de Hoog 1977; Haase G, Skopnik H, Groten T, Kusenbach G & Posselt H-G (1991) McGinnis et al. 1985). The coherence of these taxa Long-term fungal cultures from patients with cystic fibrosis. with species of Exophiala is in good agreement with Mycoses 34:373-376 Hendfiks L, Goris A, Peer Y van de, Neefs JM, Vancanneyt M, the results of our phylogenetic analysis. Kersters K, Hennebert GL & Wachter R de (1991) Phylogenetic In conclusion, direct sequencing of nuclear SSU- analysis of five medically important Candida species as deduced rRNA genes of black yeasts and a subsequent phy- on the basis of small subunit RNA sequences. J. General Micro- logenetic analysis with the obtained data appears to biol. 137:1223-1230 Hiruma M, Kawada A, Ohata T, Ohnishi &, Takahashi H, Yamazaki be suitable for resolving taxonomic problems in this M, Ishibashi A, Hatsuse K, Kakihara M & Yoshida M (1993) pleomorphic group of fungi. For the development of a Systemic caused by Exophiala dermatitidis. reliable diagnostic system, more species of the genus Mycoses 36:1-7 Exophiala and of related genera should be examined. Hoog GS de (1977) Rhinocladiella and allied genera. Stud. Mycol. 15:1-140 Particularly intraspecific variability of the SSU-rRNA -- (1985) The taxonomic structure of Exophiala. In: Howard DH gene needs to be determined. Yet our results already (Ed) Fungi Pathogenic for Humans and Animals, BII (pp 327- indicate that the segment of dematiaceous SSU-rRNA 336) Marcel Dekker, New York genes corresponding to the stem of helix 43 (Fig. -- (1993) Evolution of black yeasts: possible adaptation to the human host. Antonie van Leeuwenhoek 63:105-109 2) of the secondary structure model (corresponding nucleotide position 1340-1389 in Fig. 1) is a promis- 33

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