Bull. Natl. Mus. Nat. Sci., Ser. B, 47(2), pp. 51–57, May 21, 2021

Crassitunica tubakii gen. et sp. nov, a New Taxonin the Family Sclerotiniaceae s. lat.
Yan-Jie Zhao1 and Tsuyoshi Hosoya2, *1

Department of Life and Environmental Sciences, University of Tsukuba,
1–1–1 Tennodai, Tsukuba, Ibaraki 305-0005, Japan2
Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan*E-mail: hosoya@kahaku.go.jp(Received 14 January 2021; accepted 24 March 2021)

Abstract Crassitunica tubakii gen. et sp. nov. is proposed to accommodate a single species previously recognized under incorrectly identiﬁed name, Lambertella brunneola. Recent molecularphylogeny revealed it is no longer a member of Lambertella, but appropriate taxonomy for thisfungus has not been proposed. Here, we propose a new genus to accommodate the fungus for its
unique morphological features, supported by a molecular phylogenetic analysis based on ITS
rDNA sequence.
Keywords : Aucuba japonica, ITS rDNA, Lambertella brunneola, Rutstroemiaceae, stroma,taxonomy.

IntroductionThe genus Lambertella had been an intriguing,
heterogenous genus containing c. 60 species, being
the largest genus in the family Rutstroemiaceae
until Zhao et al. (2016) proposed a strict genericcircumscription based on morphology and molecular phylogenetic data. The genus Lambertella is
now deﬁned by having textura prismatica ectal
excipulum composed of brown colored cells, andascospores turning brown in the ascus before discharge. As a result of their revision, some species
previously treated in Lambertella need to be disposed to other genera.
The fungus misidentiﬁed as Lambertella
brunneola (Pat.) Le Gal. by Korf (1958) was one
such species. The ﬁrst specimen was collected
and identiﬁed in Japan by R. P. Korf and isolated
by K. Tubaki (Korf, 1958) and the culture
was deposited to the Institute for fermentation,© 2021 National Museum of Nature and Science

Osaka (currently Biological Resource Center,
National Institute of Technology and Evaluation(Tokyo, Japan, https://www.nite.go.jp/en/nbrc/cultures/index.html). The specimen was later
examined by Dumont (1971) who revised thegenus Lambertella. Dumont (1971) concluded
that the specimen was not L. brunneola, but he
did not dispose the fungus to any known taxa.
Therefore, the taxonomic position of the fungus
remained uncertain. Zhao et al. (2016) repeatedlycollected this fungus, and provided morphological and molecular phylogenetic information. It
was treated as “Lambertella sp. 2” in their analysis (Zhao et al., 2016). They clearly demonstrated that the fungus in question is distincct
from Lambertella s. str., but they hesitated toestablish a new taxon because there was no species with close phylogenetic or morphologicalaﬃnity. In spite of ﬁve years of our continuous
survey of Helotiales since then, we still have notfound appropriate genus for the present fungus.
Meanwhile, Tubakiʼs isolate of the fungus has

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Yan-Jie Zhao and Tsuyoshi Hosoya

been used in applied science studies and referred
to incorrectly as Lambertella brunneola (Tayone,2013). If the fungus is left misidentiﬁed orunidentiﬁed, further confusion may be brought
up in future research projects. To avoid this, weestablish a new genus and species for the isolate
misidentiﬁed as L. brunneola.
Materials and Methods
Methods for isolation, culture, and observation
followed Zhao et al. (2016). Description of thecolor codes followed the Pantone color code
adopting CYMK system referring to a Pantone
color bridge (Anonymous, 2005). Culture forDNA extraction to sequence procedure followed
Zhao et al. (2016), and obtained sequences weredeposited to DDBJ. Sequences for internal transcribed spacer (ITS) and 5.8S ribosome sequenceswere obtained using ITS1F and ITS4 (White etal., 1990) as primers in polymerase chain reaction.
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
Biodiversity Research, National Museum ofNature and Science. Coordinates followed
WGS84 datum.
Descriptions
Crassitunica tubakii Yan J.Zhao & Hosoya
gen. et sp. nov.
[Figs. 1–3]
Holotype. TNS-F-44021 (ex-type culture NBRC114959), on decaying leaves of Aucuba japonica
Thunb. Meijijingu Shrine, Yoyogi, Shibuya-ku,Tokyo, Japan (35.673767, 139.700775), 2011-XI8, col. T. Hosoya.
Mycobank number for the genus: MB838477
Mycobank number for the species: MB838478
Stroma substratal, visible as blackened zones
on leaf veins and petioles of host. Apothecia
superﬁcial, solitary or gregarious, short-stipitate,occurring mainly on leaves, sometimes on petioles and branches of the host; disc ﬂat to convex,
0.5–2.5 mm in diameter when dry; hymenium
beige to pale brown (Pantone 7502PC＝C0 M8
Y33 K10) when fresh, becoming paler when dry;
receptacle cream to oﬀ white (Pantone warm
gray 2C＝R213 G210 B202), ﬂoccose; stipe concolorous with receptacle, becoming black
towards the base, furfuraceous. Ectal excipulum
textura prismatica, pale brown, composed ofthick-walled, brick-shaped cells, 3–6×6–10 μm,
tightly packed and arranged almost perpendicular
to the surface, ending up to hyphal protrusions(hairs) in the outermost layer; at the upper portion of the margin, cells becoming thinner
walled, arranged almost parallel to the surface.
Medullary excipulum textura intricata, hyaline,
smooth, loosely interwoven, separate hyphae of
2.5–3.5 μm wide. Hairs mostly cylindrical, sometimes tapered to the apex, thin-walled, 1–2 septate, 4 μm thick at the base, 6–15 μm long; apicalcells mostly blunt, sometimes irregularly protruding. Asci 85–132.5×8.8–12 μm, cylindricalclavate, 8-spored, arising from croziers; apex
ﬂattened, thickened; pore faintly stained by Melzerʼs reagent, becoming more clearly stained
after KOH pretreatment. Ascospores 11.3–
13.8×4.5–6.3 μm (12.2±0.7×5.4±0.5 μm on
average±SD, n＝33), uniseriate or irregularly
biseriate, ellipsoid, aseptate, mostly with 1–2
large guttules; becoming light brown, 1-septate
and thick walled when germinated, one of theascosporous cell sometimes darker than theother, or one cell remaining hyaline; membranaceous structure sometimes observed around thegerminating ascospores. Paraphyses equal to orslightly exceeding the asci by 7.5–15 μm, ﬁliform, septate, simple or branched near the base,frequently expanded at the apex of 2.5–3.5 μm
wide, sometimes equipped with granulate resinous matter.
Cultural characteristics: Colonies on PDA fastgrowing, covering the whole plate after 4 weeks
incubation, circular, ﬂocculent, initially beige,
becoming grayish to pale brown in age; aerial
hyphae gray, cottony, partly developed; margin

Crassitunica tubakii gen. et sp. nov.

53Fig. 1. Crassitunica tubakii (TNS-F-44021). A. Fresh apothecia on decaying leaves of Aucuba japonica. B. Vertical section of an apothecium. C. Close up of ectal exipulum. D. Close up of medullary excipulum. E. Closeup of ectal excipulum at the margin. F. Ascus. G. Paraphyses. H. Reaction of ascal apex to Melzerʼs reagent. I:Ascospores. J: Germinating ascospores. Scales: B＝200 μm, C–J＝20 μm.

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Yan-Jie Zhao and Tsuyoshi Hosoya

Fig. 2. Crassitunica tubakii. A. Young ascospores. B. Ascospores tuning brown, one-septate before germination.
The dotted line shows membranaceous structure sometimes observed on germination. C. Asci and ascospores.
One at the right shows the continuous corzier formation at the base. D. Paraphyses. Resinous materials
observed at the apices. E. Vertical section of the apothecium at the margin. The ectal excipulum composed ofthick-walled cells with short protrusion. F. Lower ectal excipulum. Thick-walled cells at the outside and innertissue composed of sparsely intricated cells. G. Schematic drawing showing the outline of the apothecial structure. The black lines showing the developed stroma.

entire. Reverse brown, ﬁlamentous; rind becoming distinct after 8 weeks incubation, delimiting
irregular portions of the agar, composed of a single layer of cells with walls pigmented to various

extent, epidermoid in face view.
Etymology. The generic name refers to thethick-walled ectal excipulum. The speciﬁc epithet refers to the name of a Japanese researcher

Crassitunica tubakii gen. et sp. nov.

55

Fig. 3. Cultural characters of Crassitunica tubakii (NBRC 114959). A. Colony on potato dextrose agar (PDA,20°C, 1 month). B. Colony on PDA (20°C, 3 month). C. Rind on PDA (20°C, 6 month). D. Vertical view ofrind. E: Surface view of rind showing the epidermoid cells. Scales D–E＝20 μm.

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Yan-Jie Zhao and Tsuyoshi Hosoya

who ﬁrstly found this species.
Other specimens examined. All except for
TNS-F-36994 were on hanging decaying leavesof Aucuba japonica. Unless noted, collector isTH.
TNS-F-17633, Kikuchi Gauge, Kumamoto
Pref., 2005-X-10; TNS-F-18101, Maruyama,Iryuda, Odawara-shi, Kanagawa Pref., 2002-XI8, col. Yousuke Degawa; TNS-F-30018 (CultureNBRC 114958), The Fukiage Gardens in theImperial Palace Grounds, Chiyoda-ku, Tokyo(35.686306, 139.752222), 2009-X-7; TNS-F31044, Kazusa-kamatari, Kisarazu, Chiba Pref.,2003-VII-18; TNS-F-31157 (Culture NBRC110760), Iryuda, Odawara-shi, Kanagawa Pref.
(35.243305, 139.11711), 2005-VIII-18, col. Y.
Degawa; TNS-F-36994 (culture NBRC 110762),
on Fatsia japonica petiole, The Fukiage Gardens
in the Imperial Palace Grounds, Chiyoda-ku,Tokyo (35.686036, 139.751053), 2010-X-5; TNSF-40099, Meijijingu Shrine, Yoyogi, Shibuyaku, Tokyo (35.6697222, 139.705556), 2011-XI24, col. Y. Zhao; TNS-F-40126 (Culture NBRC109885), The Fukiage Gardens in the Imperial
Palace Grounds, Chiyoda-ku, Tokyo (35.685914,139.751219), 2012-VI-20; TNS-F-40315 andTNS-F-40319, Institute of Nature Study,
Meguro, Tokyo, 2016-XI-15; TNS-F-44244(Culture NBRC 114960), Iryuda, Odawara,Kanagawa Pref. (35.241672, 139.119964), 2011XI-12; TNS-F-44262 (Culture NBRC 114961),
The Fukiage Gardens in the Imperial Palace
Grounds, Chiyoda-ku, Tokyo (35.687317,139.748667), 2011-XI-21; TNS-F-57191, Daiomatsu, Tsukuba, Ibaraki Pref., 1997-VII-2; TNSF-190417, Government Forest Station, Asakawa,Tokyo, Honshu, 1957-XII-8, col. K. Aoshima, K.
Tubaki et al.
Japanese name. Aoki-ba-nise-kinkaku-byotake
Sequences registered. AB705246 (NBRC110760), AB705247 (NBRC 114958), AB705245(NBRC 110762), LC597363 (NBRC 114959),
LC425046 (NBRC 114960), LC597362 (NBRC114961) AB926080 (NBRC 109885); allsequences are for ITS-5.8S ribosomal RNA.

AB926208 (NBRC 109885, RPB2). AB926163(NBRC 109885, partial sequence for large subunit ribosomal RNA).
Notes. As Dumont (1971) suggested, the mostcharacteristsic morphological feature of Crassitunica tubakii is the structure of ectal excipulum,
composed of narrow, thick-walled cells verticallyoriented to the surface and tightly compressed,
having short protrusions. Such a structure isclearly diﬀerent from Lamberella s. str., supporting the unique position of the present fungus.
In the maximum likelihood analysis using
combined LSU and RPB2 by Zhao et al. (2016),
C. tubakii (cited as Lambertella sp. 2 in theirpaper) was situated at the base of strongly supported group composed of Rutstroemia ﬁrma(Pers.) P.Karst., Poculum pseudosydowianum
Yan J.Zhao & Hosoya, Lanzia pruni-serotinae(Whetzel & W.L.White) M.P.Sharma &R.M.Sharma with unidentiﬁed species referred toLanzia and Moellerodiscus. None of these genera
showed resemblance to accommodate C. tubakii,
and it was thought to be more appropriate toestablish a new genus. This clade was clearlyapart from Lambertella s. str.
All the sequences obtained in the present study
were identical in barcoding region, indicating thespecies is well-deﬁned in molecular aspect.
Crassitunica tubakii belongs to Rutstoremiaceae, a paraphyletic family recognized by Zhao etal. (2016), characterized by the presence of substratal stroma. However, in the more recent molecular analysis based on wider range of fungi andmultiple genes (Johnston et al, 2019), the circumscription of Rutstroemiaceae is more vague andshould be treated in Sclerotiniaceae s. lat.
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