Medical Mycology June 2004, 42, 223 /228

Ascospore-derived isolate of Arthroderma benhamiae with morphology suggestive of Trichophyton verrucosum

MASAKO KAWASAKI*, TAKASHI MOCHIZUKI*, HIROSHI ISHIZAKI* & MACHIKO FUJIHIRO$ *Department of Dermatology, Kanazawa Medical University, Uchinada and $Department of Dermatology, Ibi General Hospital, Ibigawa-cho, Japan

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Sixty-one ascospores were isolated from an ascocarp produced by the mating of two Arthroderma benhamiae strains, RV 26678 and KMU4169, that differed in their mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) patterns and in the sequences of their nuclear ribosomal internal transcribed spacer (ITS) regions. RV 26678 is a genetically typical A. benhamiae isolate, while KMU4169, though morphologically indistinguishable from A. benhamiae , is an isolate with a deviating ITS sequence and with a mtDNA RFLP profile identical to that of T. verrucosum . One of the 61 progeny ascospores formed a colony, KMU5-46, that was quite different from both parental isolates. KMU5-46 is a faviform colony morphologically similar to Trichophyton verrucosum , although its mtDNA RFLP patterns and ITS sequences were identical to those of A. benhamiae parental strain RV 26678. The morphological alteration manifested in KMU5-46, as well as this isolates complete loss of sexual response, indicates the possibility that the asexual T. verrucosum and the sexual A. benhamiae are conspecific. Keywords Arthroderma benhamiae , ascospore, ITS/5.8S sequence, mating, morphological variation, Trichophyton verrucosum

Introduction
Since Arthroderma benhamiae was first isolated from Japanese sources in 1999 as an apparent introduced species, more than 10 strains have been reported [1]. These isolates were identified based on their morphological characteristics and on mating tests using representatives of the Americano /European and African races of A. benhamiae as tester strains. Of the 10 Japanese isolates checked so far, nine have mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) patterns, obtained using the restriction enzyme Hae III, that are identical to the pattern shown by Trichophyton verrucosum [2] and different from that of A. benhamiae. However, while

Received 18 October 2002; Accepted 15 February 2003 Correspondence: Masako Kawasaki, Department of Dermatology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. Fax: '/81 76 286 6369; E-mail: masako-k@kanazawa-med.ac.jp

three of these Japanese isolates (KMU4136, KMU4137 and KMU4169) had the same mtDNA type as T. verrucosum , the nucleotide sequences of their nuclear ribosomal internal transcribed spacers (ITS) and 5.8S rRNA genes (together making up the ITS/5.8S region) were different from the corresponding sequences in T. verrucosum and in both races of A . benhamiae [2]. Among the three isolates themselves, sequences of the ITS/5.8S region were identical. In a phylogenic tree inferred from the ITS/5.8S sequences, these isolates, like T. verrucosum , fell between the Americano / European race and the African races of A. benhamiae , though they were more closely related to the Americano /European race. One of these anomalous isolates, KMU4169, successfully mated with a ('/) mating type tester strain of the A. benhamiae Americano /European race, RV 26678 [ 0/IHEM 3287, Belgian Coordinated Collections of Microorganisms (BCCM), Brussels, Belgium], and produced many ascocarps [3], while KMU4136 and KMU4137 mated with A. benhamiae
DOI: 10.1080/13693780310001644699

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African race mating type ('/) tester RV 30000 ( 0/IHEM 3293) [2]. In this study, 61 ascospores were isolated from an ascoma produced between KMU4169 and RV 26678, and were separately cultured. One of 28 ascosporederived isolates was morphologically quite different from both A. benhamiae parents. This isolate and parental strains of A. benhamiae were compared in morphological, physiological and molecular biological characters.

The extent of red pigmentation was assessed on the basal medium. Mating tests for KMU5-46 were performed at 258C using Americano /European race testers RV 26678 ('/) and RV 26680 ( /) ( 0/IHEM 3288), and African race RV 30000 ('/) and RV 30001 ( /) ( 0/IHEM 3298) as tester strains.

Genetic features
Mitochondrial DNA prepared from each strain was digested with the restriction enzyme Hae III, and electrophoresed on a 0.8% agarose gel. After staining had been done with ethidium bromide, mtDNA-RFLP gel banding patterns were compared as previously described [5]. Total DNA was also prepared from each strain by the method of Makimura et al . [6]. The ITS/5.8S region was amplified by the method of White et al . [7], digested with the restriction enzyme Hin f I, and electrophoresed on a 6% acrylamide gel. After staining had been done with ethidium bromide, restriction fragment length polymorphism (RFLP) banding patterns were compared. The amplified fragments were also sequenced using ABI Prism BigDyeTM Terminator Cycle Sequencing Ready Reaction Kits (PE Biosystems, Foster City, USA), and the ABI PRISMTM 310 Genetic Analyzer automated sequence-reading software (PE Biosystems). The sequences of the ITS/5.8S region were compared to each other and with the GenBank sequences.

Materials and methods

Establishment of ascospore-derived strains
Strains derived from single ascospores were established as described previously [3]. In brief, one ascoma was picked out from the ridge of ascomata between the parental colonies, RV 26678 and mating type ( /) Japanese isolate KMU4169 (JCM12202), and placed on a fresh agar plate. After removal of microconidia from around the vicinity of the ascoma, the ascoma was cracked open and a clump of ascospores was picked out and put on a new agar plate. After the cells were confirmed under a microscope at 400 )/magnification to be ascospores, they were drawn into a 5-ml syringe, suspended in Sabouraud liquid medium and planted onto a new agar plate. After 36 h standing at room temperature, an ascospore which was seen to be well isolated from other cells and which had germinated was selected and transplanted onto a new agar plate with the assistance of an inverted microscope. Sixty-one ascospores were isolated from the ascoma and numbered. One of the 28 ascospore-derived colonies obtained, KMU5-46 (JCM12203), was selected for detailed study because it was seen to be morphologically quite different from A. benhamiae .

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Results
Macromorphology on Sabouraud dextrose agar at 258C
RV 26678 grew rapidly and formed a white downy and partially powdery colony; the reverse color was reddish brown. The A. benhamiae -like Japanese parental strain KMU4169 grew rapidly and formed a white fluffy colony; its reverse color was tan. KMU5-46 grew slowly and formed a tan, glabrous, heaped and convoluted colony; the reverse color was tan.

Morphological and physiological features

The macro- and micromorphologies of the parental strains and of the anomalous ascospore-derived strain KMU5-46, were compared as seen on Sabouraud dextrose agar at 258C. Also, the growth of each strain on Sabouraud dextrose agar at 378C was compared with growth at 258C. Nutritional requirements were tested by culturing the isolates at 258C on a Sabouraud dextrose agar positive control, compared with vitamin-free basal medium [4] (0.25% casamino acid vitamin free, 4% glucose, 0.01% magnesium sulfate, 0.18% monobasic potassium phosphate, 2% agar), and with basal medium containing either thiamine, inositol or both.

Micromorphology on Sabouraud dextrose agar at 258C

RV 26678 produced abundant spherical microconidia in clusters and numerous pyriform microconidia singly along hyphae. Spirals and macroconidia were also present. KMU4169 produced abundant microconidia that were pyriform or clavate, or shaped somewhere in between, singly along hyphae. Spirals were present. Macroconidia were absent. KMU5-46 produced irregularly branched hyphae bearing variably sized chla 2004 ISHAM, Medical Mycology, 42, 223 /228

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Fig. 1 Chlamydospores of the atypical single-ascospore progeny isolate KMU5-46 on Sabouraud dextrose agar after 2 weeks at 258C. Lactophenol cotton blue; magnication )/200.

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mydospores 2.8 /4.7 mm in diameter terminally, or as single intercalary cells, or in chains, as well as many arthroconidia (1.9 /2.3 )/2.4 /3.8 mm), and rare clavate microconidia, (1.8 )/4.0 mm). Spirals and macroconidia were absent (Fig. 1).

Fig. 3 Nutritional requirements of the genetically atypical, mitochondrially Trichophyton verrucosum -like atypical Japanese parental Arthroderma benhamiae isolate KMU4169. B, Basal agar medium with vitamin free casein; '/I, basal agar medium containing inositol; '/T, basal agar medium containing thiamin; '/I'/T, basal agar medium containing inositol and thiamin. S, Sabouraud dextrose agar medium.

Nutritional requirements
RV 26678 grew on the basal medium as well as on Sabourauds dextrose agar medium, indicating that, as is normal with A. benhamiae , it had no exogenous vitamin requirements (Fig. 2). Parental strain KMU4169 showed thiamine requirements (Fig. 3). KMU5-46 grew more slowly on basal medium than on Sabouraud agar controls, and did so even when

inositol, thiamin or both were added to the basal medium. These results suggested that it KMU5-46 has one or more nutritional requirements for a factor other than thiamin or inositol (Fig. 4).

Red pigment production

RV 26678 produced a wine-red pigment in the basal medium while KMU4169 and KMU5-46 produced no red pigment.

Fig. 2 Nutritional requirements of the genetically typical Arthroderma benhamiae Americano /European parental isolate RV 26678. B, Basal agar medium with vitamin free casein; '/I, basal agar medium containing inositol; '/T, basal agar medium containing thiamin; '/I'/T, basal agar medium containing inositol and thiamin. S, Sabouraud dextrose agar medium.

Fig. 4 Nutritional requirements of the atypical isolate KMU5-46 derived from a cross of RV 26678 and KMU 4169. O, Old colony. B, Basal agar medium with vitamin free casein; '/I, basal agar medium containing inositol; '/T, basal agar medium containing thiamin; '/I'/T, basal agar medium containing inositol and thiamin. S, Sabouraud dextrose agar medium.

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Discussion
The very small possibility that KMU5-46 was contaminated by another dermatophyte strain type was eliminated by the dilution method. All the resultant colonies tested showed the same morphology and the same ITS/5.8S RFLP patterns. In this study, although both RV 26678 and KMU4169 are morphologically typical of the T. mentagrophytes species complex, one ascospore-derived isolate, KMU5-46, was morphologically dissimilar to T. mentagrophytes , demonstrating that A. benhamiae can yield morphologically different progeny phenotypes. Analyses of mtDNA and the ITS/5.8S region indicated that KMU5-46 appears to have inherited these genetic characters not from KMU4169, which has the vitamin requirements and mtDNA type of T. verrucosum , but from RV 26678, the authentic A. benhamiae parent. This also suggests that genes controlling the morphological and physiological characters tested here are not linked to rDNA and are not components of mtDNA. As all the mating tests with both races of A. benhamiae failed, the mating type of KMU5-46 is unknown. It is not possible to discern the parent from which KMU5-46s non-expressed mating genes were inherited. The incompatibility seen with both races was considered to be linked to degeneration of the sexual ability, a phenomenon usually observed with slow growing dermatophytes of faviform morphology (e.g. T. verrucosum , T. violaceum and T. concentricum ). Although the frequency of morphological and physiological variations resembling KMU5-46 among progeny strains and the potential reversibility of the changes seen remain unknown, KMU5-46, a morphological and physiological mutant or mutant-like recombinant, raises questions about the taxonomy of dermatophytes. If its origin were unknown and KMU5-46 were seen in a diagnostic laboratory, it might be identified as T. verrucosum or as another faviform dermatophyte species (e.g. white variant of T. violaceum ), rather than as A. benhamiae , based on its morphological and physiological characteristics. Such a strong taxonomic weight has been accorded to faviform growth that such identifications might very well be made even though the vitamin reactions of KMU5-46 were not precisely consistent with those of any described dermatophyte, and even though the profuse arthroconidia formed also would tend to contradict identification as a known faviform species. In current morphotaxonomy, there would be a strong tendency to classify the typical A.
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Fig. 5 Growth at 378C on Sabouraud dextrose agar medium. Growth of RV 26678, KMU4169 and KMU5-46 can be compared with 258C growth depicted in Figs. 2 /4, respectively. The other ve strains shown are not mentioned in this paper.

Growth at 378C
RV 26678 grew more rapidly at 258C than at 378C. KMU4169 grew equally rapidly at both 258C and 378C. KMU5-46 grew slowly at 258C and at the same rate or slightly faster at 378C (Fig. 5).

Mating tests
KMU5-46 failed to mate with any of the four tester strains of A. benhamiae and was not stimulated to produce infertile gymnothecia by any of the testers.

mtDNA analysis
RV 26678, KMU4169 and KMU5-46 showed the mtDNA-RFLP patterns of A. benhamiae , T. verrucosum and A. benhamiae , respectively.

ITS/5.8S analysis
RV 26678 and KMU4169 showed different ITS/5.8SRFLP patterns. The patterns of KMU5-46 were identical with those of RV 26678. The 649-bp nucleotide sequence of the ITS/5.8S region between RV 26678 and KMU4169 differed at only eight positions. The sequences of RV 26678 (GenBank accession no. AB088677) and KMU4169 (GenBank accession no. AB088678) were identical with previously described sequences of RV 26680 (Genbank accession no. AF17045) and KMU4136 (GenBank accession no. AB048192), respectively. The sequence of KMU5-46 (GenBank accession no. AB088676) was identical with that of RV 26678.

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benhamiae parent isolate and the progeny isolate /two isolates of approximately the same genotype /as members of two different species. The genetic basis underlying such conflicts between morphology and phylogenetic taxonomy may be illuminated by our discovery that A. benhamiae can have T. verrucosum like morphology. These data can be interpreted as showing that T. verrucosum and A. benhamiae are conspecific. Although the KMU5-46 cannot be definitely identified as T. verrucosum because it did not show clear thiamine or inositol requirements and was not isolated from or confirmed to grow with epidemiological competence on cattle, the existence of this isolate clearly indicates the possibility of A. benhamiae producing T. verrucosum -like progeny. In a previous paper [2], we proposed that T. verrucosum is one of the anamorphs of A. benhamiae because the sequence difference of the ITS/5.8S region in rDNA is within the range of infraspecific variation of A. benhamiae . The data on KMU5-46 obtained in this study strongly supports our proposition that T. verrucosum and A. benhamiae are conspecific. Moreover, spontaneous mating of the various combinations may be possible, as many genotypes have been found in Japan recently [1]. Considering the results of the present study and other recent studies showing that not only genetic but also morphological characteristics consistent with T. verrucosum are included within the infraspecific range of variation in A. benhamiae , the conventional criteria used in identification should be reevaluated. It seems that our knowledge of genetic, morphological and ecological variation within A. benhamiae , T. verrucosum , T. concentricum , T. erinacei and other

genetically related species is still inadequate for the determination the borderlines between species. Taxonomists faced with an isolate that does not show any typical characteristics or has characteristics of two different species tend to describe a new species [8]. This has resulted in a plethora of named asexual dermatophytes species. Moreover, the identifications given to dermatophytes may differ depending on whether morphological, biological or molecular biological methods are used. In an attempt to get around these inconsistent identifications, one proposal is that species with the same genotype should be considered conspecific [9] and another is the introduction of the term genospecies [10]. But these proposals are based on the analysis of one kind of DNA or DNA region. With the accumulation of phylogenetic analyses using many unlinked genes, the species boundary may perhaps be established on the basis of genealogical concordance phylogenetic species recognition criteria, as described by Taylor et al . [11]. A very primitive simultaneous analysis of two gene genealogies is shown in Fig. 6. Only two kinds of genetic characters were used in this analysis, and one of those, the mtDNA RFLP profile, was not based on nucleotide sequencing. Moreover, we recognize that patterns of inheritance of mitochondrial genomes may differ from patterns of nuclear inheritance and that some reticulation may be expected in analyses involving both nuclear and mitochondrial markers. Therefore, the delineation of the border between species may not be entirely reliable. But the incongruity of ITS and mitochondrial genealogies between A. benhamiae and T. verrucosum is definitely revealed, suggesting infraspecific genetic polymorphism and hence conspecificity.

Fig. 6 Simultaneous analysis of mitochondrial DNA (mtDNA) and ribosomal internal transcribed spacer (ITS) DNA sequence genealogies. The phylogeny of the mtDNA was inferred from the restriction fragment length polymorphism (RFLP) data of Mochizuki et al. [12] and Nishio et al. [5]. The phylogeny of the ITS region was based on sequences from Kawasaki et al. [2] and Summerbell et al. [13]. Numbers in parentheses are the accession numbers in GenBank. The order of the branching points is signicant but the lengths of branches are arbitrary, because the relation of branch lengths in the two trees was not investigated.

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In future, a more reliable simultaneous analysis using genealogies of many other genes is expected. Unless a definite species boundary is established, it might be better to consider the notion that T. verrucosum and similar asexual dermatophytes closely related to sexual species are still undergoing species differentiation.

Acknowledgements
We thank Richard C. Summerbell for his suggestions during the preparation of the manuscript.

References
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