Bull. Natl. Mus. Nat. Sci., Ser. B, 38(4), pp. 139–146, November 22, 2012

Enumeration of Remarkable Japanese Discomycetes (6):Notes on Two Inoperculate Discomycetes new toJapan and One Operculate Discomycete
Tsuyoshi Hosoya1,*, Yan-Jie Zhao2, Jae-Gu Han3, Yukiko Saito4 and Makoto Kakishima2
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
2
Faculty of Life and Environmental Science, Tsukuba University,
Tennodai 1–1–1, Tsukuba, Ibaraki 305–8522, Japan3
Mushroom Research Division, National Institute of Horticultural and Herbal Science,
Rural Development Administration, Suwon 441–707, Korea
4Center for Molecular Biodiversity Research, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan* E-mail: hosoya@kahaku.go.jp(Received 31 July 2012; accepted 26 September 2012)

Abstract Three remarkable discomycetes (two inoperculate and one operculate) are describedand illustrated: Hymenoscyphus immutabilis (Helotiaceae, Helotiales), Lachnum rachidicola(Lachnaceae, Helotiales) and Sphaerosporella brunnea (Pyronemataceae, Pezizales). The ﬁrst twospecies are documented for the ﬁrst time in Japan. Detailed microscopic description is provided for
Sphaerosporella brunnea for the ﬁrst time for Japanese material.
Key words : Hymenoscyphus immutabilis, Lachnum rachidicola, mycobiota, Sphaerosporella
brunnea, taxonomy.

Introduction
This is the sixth part of the series on remarkable
Japanese discomycetes following Hosoya et al.
(2011) to contribute the knowledge of mycobiota inJapan. Two inoperculate discomycetes with minuteapothecia are documented for the ﬁrst time fromJapan. Microscopic examination for the conﬁrmation on identiﬁcation is carried out for Sphaerosporella brunnea and description is provided.
Materials and Methods
Collection and observation procedures followed Hosoya and Otani (1997) and Hosoya(2004). Color codes followed the Pantone color
code adopting RGB system referring to a Pantone color bridge (Anonymous, 2005). For previously known distribution, the database of Global
Biodiversity Information Facility (GBIF, http://data.gbif.org/welcome.htm) was searched, andcountries with occurrence of the given species
are shown with an asterisk (*). Distributions
known only in literature are shown with double
asterisks (**). Those with both information are
shown with triple asterisks (***). To obtain
sequence of L. rachidicola, cultivation, DNA
extraction, PCR and sequence procedure followed Hosoya et al. (2010). The obtained
extracted DNA samples are deposited in the Center for Molecular Biodiversity Research,
National Museum of Nature and Science, andavailable for collaborative research.
Descriptions
1. Hymenoscyphus immutabilis (Fuckel) Dennis, Persoonia 3: 76. 1964.
[Figs. 1, 2]
Basionym: Helotium immutabilis Fuckel, Jb.

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Tsuyoshi Hosoya et al.

Fig. 1. Hymenoscyphus immutabilis (TNS-F-44242). A. Fresh apothecia. B. Section of an apothecium showing
the ectal and medullary excipulum. C. Close up of the hymenium and the margin slightly elavated. D. Close
up of ectal excipulum at the margin, showing textura prismatica tissue type. E. Close up of medullary excipulum. F. Close up of ectal excipulum near the stipe. Note the ticker-walled, globose cells. G. Ascal apex showing MLZ＋ reaction in Melzerʼs reagent. H. Asci. I. Ascopores. J. Crozier at the base of ascus (arrow). K.
Fragments of paraphyses showing the upper part. B–F, I–H, mounted in lactic acid. G, mounted in Melzerʼsreagent. Scales. B, 200 μm; C, 100 mm; D–I, H, 20 μm; J. 10 μm.

Nassau. Ver. Naturk. 25–26: 50. 1871.
See Lizoň (1992) for synonyms.
Apothecia scattered, occurring on decaying
leaves, especially on the veins, short-stipitate tosubsessile; disc ﬂat to slightly convex, 0.6–
1.5 mm in diameter when dry; hymenium whitewhen fresh, still white or becoming dark yellow
to pale brown (Pantone 721PC＝C0 M31 Y43K2) in drying; receptacle smooth, white; stipe
concolorous with the receptacle, 0.2–0.6 mm
long when dry, with smooth surface. Ectal
excipulum of textura prismatica to textura globulosa, composed of subhyaline to pale-brown,thin-walled, prismatic cells of 4.0–11.0×3.0–
7.0 μm, with slightly refractive, 7.0–11.0 μm,
globulose cells mixed in the receptacle and stipe.
Medullary excipulum of textura intricata, composed of hyaline, smooth, loosely interwoven
hyphae of 2.0–5.0 μm wide. Asci 67.5–77.5×
5.0–7.0 μm, clavate, 8-spored, croziers obscureand observed only for immature asci; apex

rounded, slightly thickened, pore slightly blue inMelzerʼs reagent even with KOH pretreatment.
Ascospores 9.0–13.0×3.5–4.0 (11.0±1.2×3.9±0.2, n＝20) μm, uniseriate or irregularly
biseriate, fusoid to subellipsoid, non-septate,eguttulate. Paraphyses ﬁliform, septate, hyaline,
simple or branched near the base, occasionallyslightly expanded at the apex of 2.0–3.0 μm wide.
Speciemens examined. HONSHU: TNS-F16614, Mizunokizawa, Yozuku, Yamakita-cho,Ashigarakami-gun, Kanagawa Pref., 3-VII-2005.
col. R. Sasagawa (culture FC-2176); TNS-F16628, Oaza-Hara, Kikuchi-shi, Kumamoto
Pref., 10-X-2005. col. R. Sasagawa; TNS-F44242, Iryuda, Odawara, Kanagawa Pref. (57 malt., 35°14′30.02″N, 139°7′11.87″E), 12-XI-2011.
col. T. Hosoya (culture FC-2837); TNS-F-37001,Fukiage gyoen, Kokyo, Chiyoda-ku, Tokyo(23 m alt., 35°41′9.73″N, 139°45′3.79″E), 5-X2010. col. T. Hosoya; TNS-F-44253, Fukiage
gyoen, Kokyo, Chiyoda-ku, Tokyo (13 m alt.,35°41′9.64″N, 139°45′3.13″E), 21-XI-2011. col.

Enumeration of Remarkable Japanese Discomycetes (6)141Fig. 2. Camera lucida illustration of Hymenoscyphus immutabilis (TNS-F-44242). A. Vertical section of an apothecium through the margin showing the ectal excipulum of textura prismatica. B. Asci. Two at the right showing the ascospores. C. Ascal apex showing the reaction in Melzerʼs reagent. D. Ascospores. E. Paraphyses.
Line drawings prepared in lactic acid mount except for C in Melzerʼs reagent. Scales. A–E, 10 μm.

T. Hosoya (culture FC-2840). All the specimens
occurring on Zelkova serrata leaf.
Previously known distribution. Denmark*,
Germany*, North America***, Spain*, Sweden*,
UK*** (White, 1943; Dennis, 1956; Seaver,1961).
Japanese name: Ochiba-shiro-byoutake
Notes. The genus Hymenoscyphus Graybelongs to the family Helotiaceae, order Helotiales with 155 species commonly accepted (Kirk etal., 2008). The generic characteristics are: sessile
to stipitate, light colored, white to yellow apothecia, ectal excipulum of textura porrecta to prismatica (rarely textura angularis to textura globulosa), medullary excipulum of textura intricata,
cylindric-clavate, 8-spored, asci with rounded orconical apex; ellipsoid to fusoid or turbinate,hyaline ascospores with no septa or 1–2-septa atmaturity (Dennis, 1956, 1981; Dumont, 1981;Lizoň, 1992). Nine species have been known

from Japan (Otani, 1966, 1987; Otani et al.,
1991; Tubaki, 1966; Katumoto, 2010).
Hymenoscyphus immutabilis is widely distributed in Europe, mainly occuring on fallen leavesof broad-leaved trees, such as Alnus, Betula,Carpinus, Fagus, Populus, Quercus, Robinia,
Salix and Ulmus (White, 1943; Dennis, 1956;Lizoň, 1992). Zelkova serrata is a new host to
this species. The specimens examined in thepresent study shared most morphological characteristics with previous descriptions (White, 1943;Dennis, 1956; Lizoň, 1992), except for smaller
asci (cf. 80.0–105.0×8.0–10.0 μm in European
and North American specimens) (White, 1943;Dennis, 1956; Dumont, 1981; Lizoň, 1992)
which may have been caused by the different
mounting ﬂuid. As a previous record from Japan,a specimen (CUP-JA 201, conserved in the Herbarium of Cornell University) collected fromKyoto in 1957 is known (Lizoň, 1902). However,142

Tsuyoshi Hosoya et al.

Lizoň (1992) indicated CUP-JA 201 had ellipsoid to ovoid, small sized ascospores (7.2–8.4×4.0 μm). CUP-JA 201 also seems to be different from the specimens examined for thepresent study, and may represent other taxa closeto H. immutabilis. So the present paper is the ﬁrst
documentation of H. immutabilis in Japan.
The distinguishing characteristics of H. immutabilis are the fusoid to ellipsoid ascospores andthe globose cells mixed in the receptacle andstipe section. The most closely related species isHymenoscyphus epiphyllus (Pers.) Rehm ex
Kauffman (White, 1943; Dumont, 1981). Bothspecies can be found on fallen leaves of broadleaved trees and share common hosts, and bothwith the globose cells mixed in ectal excipulum(White, 1943; Dennis, 1956; Dumont, 1981).

However, the hymenium color of H. epiphyllus isbright yellow to orange both in fresh and dry,while H. immutabilis is white when fresh andbecoming dark colored when dry. Hymenoscyphus epiphyllus have somewhat oblong-fusoid
and larger (15.0–23.0×3.0–5.0 μm) ascospores.
The present fungus is classiﬁed in Helotiaceae,
Helotiales.
2. Lachnum rachdicola J. G. Han, Raitv. & H.
D. Shin, Mycotaxon 107: 456. 2009.
[Figs. 3, 4]
Basionym: Lachnum rachidicola J. G. Han,Raitv. & H. D. Shin, Mycotaxon 107: 456.
2009.
No other synonyms.

Fig. 3. Lachnum rachidicola (A. TNS-F-35017; B–I. TNS-F-41104). A. Fresh apothecia. Note reddish part inwounded part. B. Dried apothecia. Note more strongly reddish part in the apothecia. C. Hairs and ectal excipular cells in external view. D. Vertical section through the margin showing the hairs and ectal excipulum. E.
Vertical section through the middle receptacle showing short hair-like protrusions at the bottom. F. Asci. G.
Paraphyses exceeding the asci. H. Ascal base showing the simple septa. I. Ascospores. J. Ascal apex stained
blue in Melzerʼs reagent. Scales A, B, 1 μm; C–J＝10 μm. C–I, mounted in lactic acid, J, mounted in Melzerʼsreagent.

Enumeration of Remarkable Japanese Discomycetes (6)143Fig. 4. Camera lucida illustration of Lachnum rachidicola (TNS-F-41104). A. Paraphyses. B. Asci. One at theright contains the ascospores, showing the MLZ＋apex. C. Ascospores. D. Vertical section through the margin, showing the hairs, ectal excipulum, external protrusions with granulations. E. Hairs. Scales. 10 μm.

Apothecia gregarious, cupulate to shallowcupulate, pure white when fresh, slightly reddening if wounded, pale brown (171PC＝C0 M61
Y70 K0) when dry with partially prominent reddish tint; disc 0.5–2 mm in diameter, white topale cream-colored when fresh, yellow to reddishbrown (Pantone 113PC＝C0 M4 Y71 K0 to
1655PC＝C0 M68 Y90 K0) when dry. Hairs
cylindrical with slightly clavate apex, hyaline,2–3(-4)-septate, thin-walled, granulate, with nocrystals, 35–55×4 μm, occasionally apicallyswollen up to 5 μm wide. Ectal excipulum textura prismatica composed of thin-walled cells,
8–15×3–5 μm. Asci 34–38×4–5 μm, cylindricalclavate, arising from simple septa; apical pore
blue in MLZ. Ascospores 6–7×1.5–2 μm, elliptic to cylindrical clavate, straight to slightly
curved, hyaline, aseptate, rarely containing bipo-lar guttules. Paraphyses narrowly lanceolate tolanceolate, exceeding the asci by 5–15 μm, up to
6 μm at the widest point.
Specimens examined. HONSHU: TNS-F16645 (culture FC-2241), TNS-F-16647 (cultureFC-2242), TNS-F-16648 (culture FC-2276), andTNS-F-16649 (culture FC-2238), Hananomakishi, Iwate Pref. collected on 23-V-2006. col. R.
Sasagawa. TNS-F-16801 (culture FC-2333),
TNS-F-16830 (culture FC-2348), TNS-F-16832(culture FC-2343), TNS-F-35017 (cultureFC-2592), and TNS-F-41104 (culture FC-2742),
Tsukuba University Sugadaira Montane ResearchCenter, Ueda-shi, Nagano Pref. (1340 m alt.,
36°31′29.4″N, 138°20′57″E) collected on 7-VI2007, 10-VI-2007, 10-VI-2007, 31-V-2010,
1-VII-2011, respectively. col. T. Hosoya. All thespecimens were collected on pinnately com-

144Tsuyoshi Hosoya et al.

pound leaves of Juglans mandshurica.
Previously known distribution. Korea** (Hanet al., 2009).
Japanese name: Kurumi-shirohinano-chawantake

Notes. Lachum radicicola was describedrecently (Han et al., 2009). It is characterized byit host, absence of crosiers and reddening apothecia. The seven sequences obtained from FC-2241,2242, 2276, 2238, 2333, 2348, 2343, and twoFig. 5. Sphaerosporella brunnea (TNS-F-46870). A. Fresh apothecia occurring on burnt ground. B. Section of anapothecium showing the ectal and medullary excipulum. C. Close up of ectal excipulum at the margin. D.
Paraphyses. E. Upper portion of asci. F–G. Ascospores. Note thick wall and air bubble and oil drops containedin the ascospore. H. Ascus observed in Melzerʼs reagent. Note ascal content stained brown in Melzerʼs reagent.
I. Hairs at the bottom of ectal excipulum. J. Hairs near the margin. Note acicular, solidiﬁed apices. Scales. A.
1 μm. B, 20 μm; C, 100 μm; D–J, 10 μm. B–G, I–J, mounted in lactic acid, H, mounted in Melzerʼs reagent.

Enumeration of Remarkable Japanese Discomycetes (6)sequences from Korea (JGH52152, and JGH52679,provided by one of the author (JGH)) shared highsimilarity (＞99%), and supported our identiﬁcation. The speciﬁc concept seemed to be welldeﬁned. The present fungus is classiﬁed in Lachnaceae, Helotiales.
3. Sphaerosporella brunnea (Alb. & Schwein.) Svrček & Kubička, Česká Mykol. 15: 65.
1961.
[Fig. 5]
Basionym: Peziza brunnea Alb. & Schwein.,
Consp. fung. (Leipzig). p. 317. 1805.
See Rifai (1968) for synonyms.
Apothecia gregarious, sessile, saucer shapedor shallow cup shaped, externally pubescent tospiny due to hairs, 3–6 mm in diameter whenfresh; disc concave, reddish brown (173PC＝C0M80 Y94 K1) or paler when fresh, becoming
darker colored. Hairs at the margin or upper
ectal excipulum acicular, thin walled, 2–3 celled,with pointed, frequently solidiﬁed (thick-walled)apex, 37–63 μm in length, 5–10 μm at the base;hairs on middle or lower ectal excipulum becoming longer, cylindrical with rounded apex,
62–75 μm in length, 5–7.5 μm at the base. Ectal
excipulum textura angularis, composed of thinwalled, elongate cells of 15–30×32–50 μm,
arranged with their long axis almost perpendicular to the surface. Medullary excipulum textura
intricata, composed of hyphae of 5–10 μm wide.
Asci ca 220×15–20 μm, cylindrical, thin-walled,
MLZ- with or without 3% KOH pretreatment,
arising from crosiers; when observed in MLZ,
the contents of asci stained brown. Ascospores(14–)15(–16.5) (15±0.42 in average±SD,n＝20) μm in diameter, globose, one-celled,
moderately thick (1 μm thick) and smoothwalled, often containing a bubble when observedin lactic acid, uniseriate in the asci. Paraphyses
cylindrical, slender, septate, simple, ﬁlled withbrownish content stained brown by MLZ,
2.5–3 μm below, up to 5 μm at the apex.
Specimens examined. HONSHU: TNS-F46870, Ogashiwa, Moriya-shi, Ibaraki Pref. on

145burnt ground. 2012-VI-19. col. K. Imamura.
Previously known distribution. Australia**,Austria*, Canada*, Denmark*, Finland*, Germany*, Ireland*, Norway*, Poland*, Spain*,
Sweden*, USA*, UK* (Rifai, 1968).
Japanese name: Yakeato-marumi-chawantake
Notes. Rifai (1968) provided a detailed linedrawings for the microscopic structure, so we do
not provide line-drawings here. He also mentioned the possible anamorph (Botrytis-like)obtained by other researchers. However, obtaining a culture was not tried. We could not ﬁnd any
Botrytis-like structure on the substrate, either.
Although S. brunnea is previously known inJapan (Otani, 1989; Imazeki et al., 2011.), nodescription with microscopic features were provided before for Japanese material. The authorstherefore conﬁrmed its identiﬁcation based on
microscopic characteristics, and here provide thedescription for Japanese material. The present
fungus is classiﬁed in Pyronemataceae, Pezizales.
Acknowledgments
The author wish to thank Dr. R. P. Korf, Prof.
Emetitus, Cornell University for kindly reviewing the manuscript. The study was funded, inpart, by Grant-in-Aid for Scientiﬁc Research (C)22570102.
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