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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.
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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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