Bull. Natl. Mus. Nat. Sci., Ser. B, 40(4), pp. 125–131, November 22, 2014

Enumeration of Remarkable Japanese Discomycetes (8):Notes on Two Hymenoscyphus Species New to Japan
Yan-Jie Zhao and Tsuyoshi Hosoya*
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan* E-mail: hosoya@kahaku.go.jp(Received 28 July 2014; accepted 24 September 2014)

Abstract Two Hymenoscyphus species new to Japan are described and illustrated: Hymenoscyphus menthae and H. ginkgonis (Helotiaceae, Helotiales), the latter with characteristic spore pigmentation and substratal stroma.
Key words : Hymenoscyphus ginkonis, Hymenoscyphus menthae, mycobiota, stroma, taxonomy.

Introduction
This is the eighth part of the series on remarkable Japanese discomycetes following Hosoya etal. (2013) to extend the knowledge of the Japanese mycobiota. The genus Hymenoscyphus isknown to include at least 150 species (Kirk et al.,2008), and new species continue to be discovered(Zheng and Zhuang, 2013). In spite of its biodiversity, comprehensive studies have scarcely
been conducted (Lizoň, 1992), and publication islimited not only for morphology, but also for
phylogeny (Zhang and Zhuang, 2004). Currently
12 species have been recorded from Japan (Katumoto, 2010; Hosoya et al., 2012; Zhao et al.,2012) but potentially more species await documentation. In the present paper, two species are
documented for the ﬁrst time with characteristic
substratal stroma.
Materials and Methods
Observation procedures followed Hosoya andOtani (1997) and Hosoya (2004). Color codes
followed the Pantone color code adopting
CYMK system referring to a Pantone color
bridge (Anonymous, 2005). For previouslyknown distribution, the database of Global Biodiversity Information Facility (GBIF, http://data.
gbif.org/welcome.htm) was searched, and countries with occurrence of the given species areshown with an asterisk (*). Distributions known
only from the literature survey are shown withdouble asterisks (**). Distribution records fromboth sources are shown with triple asterisks(***).
Isolates were cultivated in 2 ml of 2% malt
extract for 2 weeks, and the mycelia were harvested and frozen at −80°C. About 50 mg ofmycelium was mechanically lysed by a QiagenTissueLyser, using ceramic beads. DNA extraction, PCR and sequence procedure followed
Hosoya et al. (2010). DNA was extracted usingPlant Mini Kit (Qiagen, Mississauga, ON,Canada) following the manufacturerʼs instruction. To amplify internal transcribed spacer (ITS1and ITS2) and 5.8S ribosomal regions, the primer
pair ITS1F and ITS4 (White et al., 1990) wasused. DNA vouchers are deposited in the Centerfor Molecular Biodiversity Research, National
Museum of Nature and Science, and available forcollaborative research. Isolates were deposited toBiological Resource Center, National Institute ofTechnology and Evaluation (NITE-BRC).

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To observe colony characteristics, potato dextrose agar (PDA, Nissui) plate or modiﬁed
Weitzman-Silva-Hutner agar (WSH; 10 g oatmeal, 1 g KH2PO4, 1 g MgSO4·H2O, 1 g NaNO3,20 g agar, 1,000 ml distilled water) plates were
inoculated at the center with a 1 mm agar cube
containing mycelium and incubated at 20°C.
Descriptions
1. Hymenoscyphus ginkgonis J.G.Han & H.D.
Shin, Mycotaxon 103: 192. 2008.
[Figs. 1–3]

Stroma substratal, irregular to spot-like, theformer 0.2–0.5 mm wide, developed along theleaf vein, length indeterminate; the latter mostly
0.5–1.0 mm in diameter, sometimes larger; rind
2–3 μm thick, composed of condensed dark cells.
Apothecia short to long-stalked (up to 3 mm)with disc of 0.3–0.8 mm in diameter, grayish
orange (720PC＝C0 M20 Y32 K12) color withsimilar but darker colored disc when fresh,becoming darker grayish orange (721PC＝C0M31 Y43 K2) with partially blackish tint anddisc of black with violet tint (5255PC＝C97
M95 Y15 K65). Ectal excipulum textura prismatica, of several layers of brick-shaped, thick-Fig. 1. Hymenoscyphus ginkgonis (TNS-F-11208). A. Apothecia occurring from stromata on Ginkgo biloba
leaves. B. Close up of apothecia. C. Vertical section at the base of the stipe showing the stroma. Note thin,black line (rind) surrounding the base of the stipe. D. Paraphysis. Note black stains at the apical part. E. Ascus.
F. Vertical section through the stipe. Note prismatic cells arranged parallel to the outside. G. Vertical section
through the apothecium showing the ectal excipulum showing the textura prismatica tissue. H. Ectal excipulum observed under crush mount in lactic acid. Note stains. I. Vertical section showing the basal part of thestalk, showing the occurrence of apothecium from stroma. J–M. Ascospores at the various stage of germination. J. Ascospores just after discharged. K–M. Ascospores prior to germination. Note septation at the mediumand pigmentation of the spores. Bars, A, B, 1 mm; C–I, 10 μm, J–M, 5 μm.

Enumeration of Remarkable Japanese Discomycetes (8)127Fig. 2. Camera lucida illustration of Hymenoscyphus ginkgonis (TNS-F-11208). A. Vertical section of at the margin showing the ectal excipular structure. B. Paraphyses. Note coalescences between the paraphyses. C. Asci.
One at the bottom left showing the ascospores. One at the top right showing the reaction under treatment ofMelzerʼs reagent. D. Surface view of ectal excipulum showing the hairs. E. Ascospores. Cell contents in freshmaterials mounted in water are drawn for the middle ﬁve spores. The six spores at the right shows the pigmentation prior to germination. F. Schematic drawing of the apothecial structure.

walled cells, 15–30×7–12 μm, arranged parallelto the surface, becoming thinner-walled toward
the inside; external cells sometimes elongating toform hairs. Hairs up to 100 μm long, 3–5 μm
thick, cylindrical, smooth, aseptate to few-septate, occasionally containing dark purple pigments. Asci 75–95×7–9 μm, eight-spored, cylindrical clavate; apex stained blue in Melzerʼsreagent without KOH pretreatment, plugs staineddenser as two vertical lines; arising from simple
septa. Ascospores 13–18×4–6 μm (16.4±1.5×5.3±0.52, average±SD, n＝26), subelliptic toelliptic with acute lower end, smooth, often containing two to several remarkable (larger) oilglobules, sometimes constricted at the middle,

aseptate, rarely one-septate, hyaline within theasci, becoming dark pigmented after discharged
and one-septate in 48 hours on PDA before germination; often germinated from one of the polar
region. Paraphyses cylindrical, simple orbranched at the base, multi-septate, containing
black pigment diffused into the mounting ﬂuid
when mounted in lactic acid, distributed in compartment in the cells, 2–3 μm thick.
Cultural characteristics. Colonies on PDA
plate growing very slowly, attaining a diameterof 10–12 mm (23°C, 2 weeks), convex at the center, sulcate, dark brown (476PC＝C32 M67 Y63K78); reverse concolorous to more darker (Black3PC＝C72 M46 Y56 K95). Aerial mycelium

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Yan-Jie Zhao and Tsuyoshi Hosoya

Fig. 3. Cultural characters of Hymenoscyphus
ginkgonis (NBRC 109855, culture of TNS-F11208). A. Colony on PDA (20°C, 1 mo.); right,from the surface; left, from the reverse. B. Colony on PDA (20°C, 3 mo.); right, from the surface; left, from the reverse. C, D. Vertical viewof blackened area produced in 3 month on
PDA. Bars, C, 100 μm; D, 40 μm.

white, little developed, velvety. Sectors irregular.
Zonations absent. Context tough and glutinous.
Margin irregular, mycelia submerged at the margin. Soluble pigments abundant, concolorouswith the colony or paler (4485 PC＝C24 M42
Y89 K72). Rind structure inconspicuous, no vertical line developed even in prolonged incubation
up to 1 month, only observed as two layers
developed along the upper surface and at the bottom, both composed of condensed dark hyphaeof 2.5 μm wide, forming a layer of 1.0–1.5 mmwide. No conidial structure observed.
Specimens examined. HONSHU: TNS-F11208, Fuchizawa horyo, Towada-ko machi,Aomori Pref., on Ginkgo biloba leaf, 2004-VIII25 col. Y. Harada, culture FC–1093 (NBRC109855). TNS-F-13249, Green house, Hirosaki
Univ., Hirosaki-shi, Aomori Pref., on Ginkgobiloba leaves, 2006-XII-22, col. Y. Harada. TNSF-31267, Horyo-no-icho, Towada-shi, AomoriPref., on Ginkgo biloba leaves, 2006-V-25, col.
T. Hosoya. TNS-F-31110, Horyo-no-icho, Towada-shi, Aomori Pref., on Ginkgo biloba leaves,2008-IX-1, col. Y. Harada. TNS-F-44648, Myoriki-ji, Iryuda, Odawara-shi, Kanagawa Pref., onGinkgo biloba leaves, 2011-X-2, col. K. Sakai.
TNS-F-44649, Myoriki-ji, Iryuda, Odawara-shi,
Kanagawa Pref., on Ginkgo biloba leaves, 2011X-19, col. K. Sakai., TNS-F-44650, Myoriki-ji,
Iryuda, Odawara-shi, Kanagawa Pref., on Ginkgo
biloba leaves, 2011-X-21 col. K. Sakai.
Known distribution. Korea**
Japanese name: Icho-nise-byotake (newly proposed)
Notes. The morphological features well agreed
with the previous description of H. ginkgonis,
including the dark purple pigments in hairs andparaphyses (Han and Shin, 2008). The ITS
sequence obtained from NBRC 109855(AB705226) matched for 100% with previously
obtained sequence (NR 119669, from Koreanmaterial), and clearly demonstrated to be conspeciﬁc.
The pigmentation of ascospores at germination
was observed for the ﬁrst time. This pigmentation, together with a prominent substratal stroma
is a diagnostic character of Lambertella (Rutstroemiaceae, Helotiales), but our previous work
showed that Lambertella is polyphyletic, andphylogenetically unrelated clade with Lambertella is being suggested in Hymenoscyphus as aresult of convergent evolution (Zhao et al.,2012).
2. Hymenoscyphus menthae (W.Phillips) Baral,
Baral & Krieglsteiner, Beih. Z. Mykol. 6: 131.
1985.
[Figs. 4, 5]
Helotium menthae W.Phillips, Elv. Brit: no. 188.
1877.
Hymenoscyphus scutula var. menthae W.Phillips,
Man. Brit. Discomyc. (London): p. 137. 1887.

Enumeration of Remarkable Japanese Discomycetes (8)129Fig. 4. Hymenoscyphus menthae (TNS-F-40052). A. Fresh apothecia on fallen fruits of Hydrangea sp. B. Vertical section of ectal excipulum. C. Close up of ectal excipulum at the margin. D. Close up of ectal excipulum inﬂank showing the layered structure. E. Structure in medullary excipulum. F. Asci. G. Reaction of ascal apex toMelzerʼs reagent. H. Paraphyses. I. Ascospores. Bars, B 200 μm; C–F, H–I 20 μm; G 5 μm.

Phialea scutula var. menthae (W.Phillips) Sacc.,
Syll. Fung. (Abellini) 8: 266. 1889.
Helotium scutula var. menthae (W.Phillips) Boud.,Hist. Class. Discom. Eur. (Paris) p. 114. 1907.
Helotium scutula var. solani P.Karst in Not.
Sallsk. Faun. Flor. Fenn. 11: 234. 1870.

Fig. 5. Camera lucida illustration of Hymenoscyphus menthae (TNS-F-40052). A. Vertical section of an apothecium through the margin showing the ectal excipulum. B. Asci. C. Paraphyses.
D. Reaction of ascal apex to Melzerʼs reagent.
E. Ascospores.

Apothecia stipitate, occurring on fallen fruitsof Hydrangea sp.; disc ﬂat to cupulate, 0.2–
0.6 mm in diameter in dried specimen; hymenium whitish yellow when fresh, becoming yellowish (1215PC＝C0 M8 Y48 K0) to pale brownwhen dry; receptacle smooth, concolorous withhymenium or brown stipe concolorous with thereceptacle or slightly paler than receptacle, 0.2–0.7 mm long when dry, smooth. Ectal excipulum
50–85 μm thick, two layered: outer layer textura
porrecta or textura prismatica, composed ofsmooth, hyaline or subhyaline, thin-walled orslightly thick-walled, brick-shaped cells, 4–5.5 μm wide, outmost layer with slightly pale
brown; inner layer composed of slightly thickwalled, hyaline, smooth, separate hypha of
2.5–3 μm wide. Medullary excipulum textura

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Yan-Jie Zhao and Tsuyoshi Hosoya

Fig. 6. Cultural characters of Hymenoscyphus
menthae (NBRC 109865, Culture of TNS-F40052). A. Colony on PDA (20°C, 1 month);right, from the surface; left, from the reverse. B.
Colony on PDA (20°C, 3 months); right, fromthe surface; left, from the reverse. C. Colony on
WSH (20°C, 3 months; right, from the surface;
left, from the reverse. D. Colony on PDA(20°C, 6 months), showing the blackened line
formed in PDA slant. E. Vertical view of blackened line produced in 6 months on PDA. Bars,E 40 μm.

intricata, hyaline, smooth, thin-walled, separate
hypha of ca. 2.5 μm wide. Asci 80–95×5–9 μm,cylindrical-clavate, eight-spored, arising fromcroziers but obscure; apex conical, 2–3 μm thick;pore stained blue in Melzerʼs reagent without
KOH pretreatment. Ascospores 15–21.5×3–4.5 μm (18±2.1×3.8±0.4 μm on average±SD,n＝20), uniseriate or irregularly biseriate, clavate-ellipsoid or clavate-fusoid, rounded at theproximal end and pointed towards the distal end,
with 0–2, globose or irregular guttules, non-septate. Paraphyses ﬁliform, apex obtuse, septate,hyaline, enlarged at the apex up to 2.5–4 μmwide.
Cultural characteristics. Colonies on PDA
grows slowly, attaining a diameter of ca. 43 mm
after 4 weeks incubation at 23°C, irregular, cottony, grayish to pale brown, dark in the central.
Aerial mycelium gray, little developed. Margins
undulate, pallid. Reverse pale brown in the central, with black pigmentation.
Specimens examined. HOKKAIDO: TNS-F40052 (Culture FC-2800＝NBRC 109865),
on fallen fruits of Hydrangea sp. Fukkonomori,
Shikotsu
lake,
Chitose-shi,
Hokkaido(42°45′35.10″N, 141°26′23.10″E, elev. 243 m),
2011-IX-14, col. Y.-J. Zhao.
Known distribution. Germany*, Netherlands**, UK**
Japanese name: Ajisai-nise-byoutake (newlyproposed)
Notes. This is the ﬁrst report of Hymenoscyphus menthae from Japan. It is also of the ﬁrst
time report occurring on Hydrangea. Hymenoscyphus menthae is distinguished from other
Hymenoscyphus spp. by its whitish yellow apothecia and small ascospores (Baral and Krieglsteiner, 1985). Hymenoscyphus caudatus (P.
Karst.) Dennis is similar with H. menthae inmany aspects of microscopic characters, but theformer has the apically hooked ascospores(White, 1943; Lizoň, 1992). The ITS sequenced
obtained from TNS-F-40052 (AB926063)
matched for 100% with previously obtained
sequence (AY348588) though the latter labeled“Hymenoscyphus cf. menthae”. The present taxon
has been known from various hosts (Dennis,1956; Hengstmengel, 1996). Clear substratal
stroma delimited by rind structure was not produced neither on specimen nor under culture, but
a black, linear portion of the colony composed ofdark hyphae was produced in prolonged incubation, and the apothecia occurred on blackened

Enumeration of Remarkable Japanese Discomycetes (8)portion of the substrate.
Acknowledgments
The authors wish to thank Dr. Ryan Kepler,
Systematic Mycology and Microbiology Laboratory, United States Department of AgricultureAgriculture Research Service for kindly reviewing the manuscript.
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