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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

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 Trichophytonverrucosum, 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 morpho-

logical characteristics and on mating tests using

representatives of the Americano/European and Afri-

can races of A. benhamiae as tester strains.

Of the 10 Japanese isolates checked so far, nine have

mitochondrial DNA restriction fragment length poly-

morphism (mtDNA-RFLP) patterns, obtained using

the restriction enzyme Hae III, that are identical to the

pattern shown by Trichophytonv

errucosum [2] anddifferent from that of A. benhamiae. However, while

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 Amer-

icano/European race.One of these anomalous isolates, KMU4169, suc-

cessfully mated with a ('/) mating type tester strain of

the A. benhamiae Americano/European race,

RV 26678 [0/IHEM 3287, Belgian Coordinated Col-

lections of Microorganisms (BCCM), Brussels, Bel-

gium], and produced many ascocarps [3], while

KMU4136 and KMU4137 mated with A. benhamiae

Correspondence: Masako Kawasaki, Department of Dermatology,

Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan.

Fax: '/81 76 286 6369; E-mail: [email protected]

Received 18 October 2002; Accepted 15 February 2003

2004 ISHAM DOI: 10.1080/13693780310001644699

Medical Mycology June 2004, 42, 223/228

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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 ascospore-

derived 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.

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 placedon 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 ascomaand numbered. One of the 28 ascospore-derived

colonies obtained, KMU5-46 (JCM12203), was se-

lected for detailed study because it was seen to be

morphologically quite different from A. benhamiae.

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 phos-

phate, 2% agar), and with basal medium containing

either thiamine, inositol or both.

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 pat-

terns 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.

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.

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 irre-

gularly branched hyphae bearing variably sized chla-

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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).

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. 1 Chlamydospores of the atypical single-ascospore progeny

isolate KMU5-46 on Sabouraud dextrose agar after 2 weeks at 25 8C.

Lactophenol cotton blue; magnification)/200.

Fig. 2 Nutritional requirements of the genetically typical Arthro-

derma 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. 3 Nutritional requirements of the genetically atypical, mito-

chondrially 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.

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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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/5.8S-

RFLP patterns. The patterns of KMU5-46 were

identical with those of RV 26678. The 649-bp nucleo-

tide 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.

Discussion

The very small possibility that KMU5-46 was con-

taminated 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 andKMU4169 are morphologically typical of the T.

mentagrophytes species complex, one ascospore-de-

rived isolate, KMU5-46, was morphologically dissim-

ilar to T. mentagrophytes, demonstrating that A.

benhamiae can yield morphologically different progeny

phenotypes.

Analyses of mtDNA and the ITS/5.8S region in-

dicated 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 con-

trolling 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 phy-siological variations resembling KMU5-46 among

progeny strains and the potential reversibility of the

changes seen remain unknown, KMU5-46, a morpho-

logical and physiological mutant or mutant-like recom-

binant, 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.

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 five

strains shown are not mentioned in this paper.


226 Kawasaki et al.

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benhamiae parent isolate and the progeny isolate/two

isolates of approximately the same genotype/as mem-

bers of two different species. The genetic basis under-

lying 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 asshowing that T. verrucosum and A. benhamiae are

conspecific. Although the KMU5-46 cannot be defi-

nitely identified as T. verrucosum because it did not

show clear thiamine or inositol requirements and was

not isolated from or confirmed to grow with epidemio-

logical 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. More-

over, 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 dermato-

phytes species. Moreover, the identifications given todermatophytes may differ depending on whether mor-

phological, 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 andT. verrucosum is definitely revealed, suggesting infra-

specific genetic polymorphism and hence conspecificity.

Fig. 6 Simultaneous analysis of mitochondrial

DNA (mtDNA) and ribosomal internal transcribed

spacer (ITS) DNA sequence genealogies. The phylo-

geny of the mtDNA was inferred from the restriction

fragment length polymorphism (RFLP) data ofMochizuki 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

significant 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.

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