Bull. Natl. Mus. Nat. Sci., Ser. B, 46(3), pp. 95–100, August 21, 2020

Notes on Two New Records of Mycobiota in Japan,
Kostermansinda nana and Durella macrospora
Tsuyoshi Hosoya1, * and Michiko Yano2
1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan
2
Graduate School of Environment and Information Sciences, Yokohama National University,
79–7 Tokiwadai, Hodogaya, Yokohama 240–8501, Japan*E-mail: hosoya@kahaku.go.jp(Received 17 April 2020; accepted 24 June 2020)

Abstract Two new records of mycobiota in Japan were documented, i.e. Kostermansinda nanaand Durella macrospora. While the latter clearly belongs to Helotiales, the phylogenetic positionof the former was not clear based on BLAST search on ITS-5.8S, LSU, mtSSU, RPB1, RPB2, andEF1a.
Key words : barcode, Durella macrospora, Japan, Kostermansinda nana, mycobiota, taxonomy.

Introduction
In spite of continuous attempts in elucidationof mycobiota in Japan, a number of taxa havebeen remained unreported. In the present paper,
we documented two new records of fungi
scarcely known. To increase the value of theiroccurrence data, we also added genetic information for the future phylogenetic analysis.
Materials and Methods
For the isolation of Kostermansinda nana, single spore isolation method using Skermanʼs
manipulator (Skerman, 1968) was used. Isolateof Durella macrospora was obtained from multispores discharged onto the agar as previously
described (Itagaki et al., 2019). Isolates were
deposited in Biological Resource Center,
National Institute of Technology and Evaluation(NITE-BRC, Tokyo, Japan, https://www.nite.go.
jp/en/nbrc/cultures/index.html). Extracted DNA
were deposited to the Center for Molecular Bio© 2020 National Museum of Nature and Science

diversity Research, National Museum of Natureand Science. Coordinates followed WGS84
datum.
Extraction of DNA, PCR and sequencing followed Iatagaki et al. (2019). The following
primer sets were used. For internal transcribedspacers (ITS1 and ITS2) and 5.8S ribosomal
region (ITS-5.8S), ITS1 and ITS4 (for Kostermansinda nana) or ITS1Fand ITS4 (for Durellamacrospora) (White et al., 1990); for large subunit of ribosomal RNA, LR0R and LR5 (Vilgalysand Hester, 1990); for mitochondrial small subunit of ribosomal RNA, mrSSU1 and mrSSU3R(Zoller et al., 1999); for DNA-directed RNA
polymerase II subunit RPB1, RPB1-AF and
RPB1-CR (Castlebury et al., 2004); for partial
sequence of the second largest DNA-directed
RNA polymerase II subunit (RPB2), RPB2-P6F
and RPB2-P7R (Liu et al., 1999); for partial
sequence of elongation factor 1 (EF1a), EF1983F and EF2218R (Rehner and Buckley, 2005).
BLAST search was conducted based on theobtained sequences using the NCBI website(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PRO

96

Tsuyoshi Hosoya and Michiko Yano

GRAM=blastn&BLAST_PROGRAMS=mega
Blast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome, visitedon March 15, 2020). Only sequences matchedwith E＝0.0 and higher percent identity value
were selected for discussion.
Results and Discussion
Descriptions
1. Kostermansinda nana (J.N.Kapoor & Munjal)
G.Z.Zhao [as ʻnanumʼ], Journal of Shandong
Agricultural University 34(3): 437. 2003.
[Fig. 1]
≡Sclerographium nanum J.N.Kapoor & Munjal,
Indian Phytopath. 19(4): 352. 1967 [1966].
＝Kostermansinda minima Cabello & Aramb., in
Arambarri, Cabello & Mengascini, Mycotaxon
29: 32. 1987.
Mycelia immersed in the substrate, thick-walled, multi-septate, brown, forming synnemata.
Synnemata upright, 200–300×20–40 μm, composed of dark-brown, multi-septate, smooth,unbranched hyphae of 2–3 μm wide. Conidiophores macronematous, thick-walled. Conidiogenous cells monoblastic, integrated, terminal,
determinate. Conidia 20–30×12–15 μm, solitary, acrogenous, broadly ellipsoidal clavate,
muriform, typically composed of three rows and
3–5 levels of cells with pale to dark-brown wall,arranged on one smaller hyaline basal cell. Germination occurs while the conidia are still on theconidiophores, and detached conidia germinate
readily on artiﬁcial agar media.
Specimens examined. TNS-F-24817 (culture
FC-1894＝NBRC 112551), 24818, and 24819 on
bark of unidentiﬁed tree, Myoriki-ji, Iryuda,
Odawara-shi, Kanagawa Pref. 2009-III-20. col.
T. Hosoya; TNS-F-25226 and 25227 on unidentiﬁed log, Takizawa-en, Tanzawa, Kanagawa Pref.

Fig. 1. Kostermansinda nana (TNS-F-24817). A. Synnemata. B. Spores detached and scattered on the substrate.
C. Synnnemata connected with other synnema at the basal hyphae. D. Close up of the head apical region of
the synnema. E. Close up of the head of synnema with a cluster of conidia. F. Crush mount of the conidia producing part. Note that the conidia are produced at the end of brown hyphae. G. Conidia and conidiogenous
cells with terminal enlargement. H. Detached conidia, with globular, hyaline cell at the base. I. Juvenile
conidia and end of conidiogenous cells. J. Germinating conidium on agar mediuma (potato dextrose agar).
Scales: A–B＝100 μm, C–I＝10 μm, J＝20 μm

Kostermansinda nana and Durella macrospora(35.398717, 139.183162), Oct., 1989. col. T.
Hosoya; TNS-F-37038 (culture FC-2752＝NBRC 109679) on bark of living tree, Jinmuji,
Zushi-shi,
Kanagawa
Pref.,(33.43705,
132.637803), 2010–X–11. col. M. Yano.
Sequences registered. LC534830 (ITS-5.8S),
LC534831(LSU),
LC535799(mtSSU),
LC535800 (RPB1), LC535801 (RPB2), andLC535802 (EF1α) obtained from NBRC 112551
Notes. Kostermansinda is a relatively newly
described genus, containing small number of
species. Kostermansinda nana has been collected
several times in Japan, and previously known as
K. minima, but no description was provided. The
fungus has been known from Argentina (Arambarri et al., 1987), India (Bhat, 2006), Taiwan(Chang, 1989) and Thailand (Kodsueb et al.,2008), and this is the ﬁrst occurrence report ofKostermansinda in Japan.
The phylogenetic position of Kostermansinda
has remained unresolved, and it is disposed in“Ascomycota” in Index Fungroum (http://www.
indexfungorum.org/names/Names.asp, visited on
March 10, 2020), and inc. sedis in Wijayawardene et al. (2020). In the present study, BLAST
search based on several genes are presented asfollows (higher taxonomy followed Wijayawardene et al. 2020).
When ITS-5.8S was used for BLAST search,
the only genus speciﬁed with E＝0.0 was Entrophospora
infrequnentan(Glomeromycota)
NY349 (U94714) with Max score 662, Querycoverage 96%, and %Ident. 93.89% which is too
low to evaluate taxonomic position based on thesimilarity.
LSU provided more genera as candidates in
E＝0.0, but no genus was speciﬁed for %Ident＞90%. Below %Ident ＜90%, Funbolia (Eriomycetaceae, order unknown), Phellinocrescentia(Eriomycetaceae, order unknown), Tuckermanopsis (listed as orthologous variant Tuckermannopsis; Parmeliaceae, Lecanorales), Hypogymnia(Parmeliaceae,
Lecanorales),Satchmopsis Cochlearomycetaceae, Leotiales?),
Pleurosticta (Parmeliaceae, Lecanorales), Bryoria (Parmeliaceae), Bryocaulon (Parmeliaceae),97

and Heleiosa (Eriomycetaceae, order unknown)
were listed in this order.
For mtSSU, Anisomeridium sp. (Monoblastiaceae, Monoblastiales, Dothideomycetes) isolate
e4012523987 (MH028639; Max score 710,
Query coverage 97%, and %Ident. 83.78%), followed by Megalotremis verrucosa (Monoblastiaceae) (GU327694; 710, 86%, and 85.80%),
Trypetheliopsis
kalbi(Monoblastiaceae)(GU327703; 652, 86%, and 84.27%) were listed
in the range of E＝0.0.
For RPB1, no matches were obtained with
E＝0.0.
For
RPB2,
Wiesneriomyces
javanicus(Wiesneriomycetaceae, Tubeuﬁales) isolate YMF
1.04036 (MK267285; 726, 94%, and 84.01%),
W. javanicus isolate YMF 1.04044 (MK267286;
721, 94%, and 83.82%) were obtained.
For EF1α, many matches were obtained, but
no match was presented for more than
＞90%Ident value. The highest match showed
Dictyocheirospora (Dictyosporiaceae, Pleosporales).
The above results are a bit confusing, butallow preliminary consideration about the position of this mysterious fungus. Some of the
BLAST search result suggest the phylogenetic
relationship of K. nana with Dothideomycetes.
This suggestion is also supported by brown to
black, dictyosporous conidia often observed inthis class.
Another possible interpretation brings up a
doubt for the correct identiﬁcation of the registered sequences. BLAST search listed somelichen members (members of Lecanorales).
These sequences may have been originated fromlichenicolous Dothideomycetes. The latter possibility can be eliminated by incorporating multiple sequences resulted from multiple specimens
or examination of voucher specimen. However,
due to the paucity of the genomic data for alliedtaxa, further analysis was not conducted.
2. Durella macrospora Fuckel, Jb. nassau. Ver.
Naturk. 23–24: 281. 1870 [1869–70].
[Figs. 2, 3]

98Tsuyoshi Hosoya and Michiko Yano

Fig. 2. Durella macrospora (TNS-F-61703). A–B. Dried specimens. C. Surface of ectal excipulum in crush
mount. Note upper portion composed of undulating hyphae and lower portion composed of larger, thickerwalled cells. D. Brown epitheical structure. E. Ascus, F. Ascospore. G. Paraphyses apex, H. Ascal apex, I. Asciand undulating paraphyses in crush mount. Scales: A＝1 mm, B＝0.5 mm, C＝20 μm, D–I＝10 μm.

Apothecia superﬁcial, gregarious, sessile, turbinate to shallowly concave, totally black whenfresh, drying shallow to ﬂat, attached to theneighboring apothecia, 120–250 μm in diameter.
Ectal excipulum textura intricata, composed ofundulating hyphae with elongate cells withbrown-colored wall with thin, fewer septa
2–4 μm wide; wall becoming thicker toward themargin ending up in somewhat clavate terminal
cell; cells shorter, thicker-walled and more prismatic (10–25×5–8 μm, with 1 μm thick wall)
toward the base. Asci 60–88×7–9 μm, cylindrical clavate, arising from croziers; apex rounded;pore stained faintly in Melzerʼs reagent without
3% KOH pretreatment, greatly enhanced by 3%
KOH pretreatment. Ascospores 10–19×3–4 μm(13.47±2.24×3.37±0.44 μm in average±sd,n＝15), biseriate or biseriate only in upper
region; clavate or ellipsoid, often narrower
toward the pointed base, aseptate to three-septate. Paraphyses ﬁliform, 1.5–2 μm wide at thebase, undulate, simple or branched at the base orapical region, thicker-walled and enlarged toward
the apex, up to 5 μm wide, brown-colored at theapex; almost the same length or slightly exceeding the asci, exudating brown, resinous matter
forming epithecium.
Japanese name. Ko-kuro-sara-byotake

Specimen examined. TNS-F-61703 (culture
FC-5679＝NBRC 114601), on unidentiﬁed wood,Renkoji, Tama-shi, Tokyo (35.636111, 139.4600),
2015-II-12. col. M. Nakajima.
Sequences registered. LC534832 (ITS-5.8S)
Notes. This is the ﬁrst occurrence report of
Durella in Japan. It is previously known mainly
from Europe and America but so far no records
from Asia (GBIF, https://www.gbif.org/, visitedon April 25, 2020). Overall morphology agrees
with Durella connivens (Fr.) Rehm (Hansen andKnudsen, 2000; Medardi, 2004; Luis et al.,2017), but diﬀers in the morphology of paraphyses and MLZ positive asci, and agrees with morphology of D. macrospora (Medardi, 2004). The
BLAST search based on ITS-5.8S region shows
almost perfect match with two sequences for D.
macrospora [D. macrospora voucher G.M. 201504-05-4 (KY462813; Max score 1064, Querycoverage 100%, and %Ident. 99.32%) and D.
macrospora voucher H.B. 9974 (KY462812;
1064, 100%, 99.32%)]. When considering the
98.5% identify species limit (Nilsson et al.,2012). The present specimen matches well withthe previously reported sequences, and shows nodoubt for species identity on molecular basis.
However, some contradiction with the previous
description given by Dennis (1956) are noted.

Kostermansinda nana and Durella macrospora

99

branched, and more swollen in Dennis (1956) ascompared with the present specimen (Fig. 3D).
Nevertheless, we identiﬁed this species based on
the high similarity of the barcoding region.
Johnston et al. (2019) included D. macrospora
in their phylogenetic analysis of Leotiomycetes,and demonstated its close relationship with
Arachnopezizaceae.
Baral (2016) placed the genus Durella in
Strossmayeria lineage, next to Mollisiaceae, but
Baral (2016) also pointed out the morphologicaldiﬀerences between Mollisiaceae. We also agreewith the morphological similarity of Durella in
Strossmayeria, in particular ectal excipular structure, ascospores and paraphyses.
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Fig. 3. Durella macrospora (TNS-F-61703). A.
Marginal region of ectal excipulum in crush
mount. B. Asci. C. Lower part of ectal excipulum in crush mount. D. Paraphyses. E. Ascospores.

Dennis (1956) described the asci as non-amyloid
while our data and both data stored in NCBI
shows amyloid reaction (https://www.ncbi.nlm.
nih.gov/nucleotide/KY462813.1?report=genbank&log$=nuclalign&blast_rank=1&RID=6
V48DVJS016; https://www.ncbi.nlm.nih.gov/
nucleotide/KY462812.1?report=genbank&log$=
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accessed: 10 March 2020). Because the MLZ
reaction is so faint, it is highly possible that
Dennis (1956) overlooked the reaction. The illustrations and description of paraphyses is more

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