全 文 ： 菌物学报
jwxt@im.ac.cn 15 November 2012, 31(6): 821-826
Http://journals.im.ac.cn Mycosystema ISSN1672-6472 CN11-5180/Q © 2012 IMCAS, all rights reserved.
Supported by Special Funds for Basic Research from National Science and Technology Ministry (No. 2006FY120100) and Knowledge
Innovation Program of the Chinese Academy of Sciences (No. KSCX2-EW-J-6)
*Corresponding author. E-mail: zhuangwy@im.ac.cn
Received: 26-03-2012, accepted: 24-04-2012
A new species of Hypomyces (Hypocreales) on Schizophyllum
sp. from China
ZHUANG Wen-Ying1* CHEN Shuang-Lin2 ZENG Zhao-Qing1 ZHENG Huan-Di1
1State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
2College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210046, China
Abstract: A new species of Hypomyces from the Tianma Nature Reserve, Jinzhai County, Anhui Province is described. The
fungus is characterized by orange perithecia immersed to partially immersed in subiculum and turning purple in KOH
aqueous solution, fusoid ascospores relatively small, and occurring on Schizophyllum sp. Distinctions between the new
species and its related fungi were discussed. Sequence analysis was also carried out, which supports the morphological
observation and recognition of the new species.
Key words: Hypomyces sinicus, morphology, sequence analysis, taxonomy, new species
寄生于裂褶菌上的菌寄生属一新种
庄文颖 1* 陈双林 2 曾昭清 1 郑焕娣 1
1中国科学院微生物研究所真菌学国家重点实验室 北京 100101
2南京师范大学生命科学院 江苏 南京 210046
摘 要：安徽天马自然保护区一种植物的树干被裂褶菌 Schizophyllum sp. 寄生，而裂褶菌的子实体又被另一真菌寄
生。这一重寄生真菌的子囊壳为橙黄色，埋生或者半埋生于橙黄色的菌丝层中，菌丝层在 3% KOH 水溶液中变为紫
色；该种的子囊孢子近梭形，具一个分隔，表面被疣状纹饰，13–16.5×3.2–4µm。它与近似种在形态学和 rDNA ITS
片段的序列上差异显著，是菌寄生属的一个新种，被命名为中国菌寄生 Hypomyces sinicus。
关键词：中国菌寄生，形态学，序列分析，分类，新种
DOI:10.13346/j.mycosystema.2012.06.018
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The early records of Hypomyces (Fr.) Tul. &
C. Tul. from China date back to 1934 when H.
aurantius (Pers.) Fuckel, H. chrysospermus Tul. &
C. Tul., and H. hyalinus (Schwein.) Tul. & C. Tul.
on fruitbodies of polypores and the agaricaceous
fungi from the south of China were reported (Teng
1934). As a continuation of the previous studies
and in connection with our current work on the
Chinese Fungus Flora (Hypocreaceae), the
specimens on deposit in the Mycological
Herbarium, Institute of Microbiology, Chinese
Academy of Sciences (HMAS) filed under
Hypomyces were re-examined, and nine taxa have
been recognized (Luo & Zhuang 2012). Among
them, five species are found from China. In our
recent field excursion to the Tianma Nature
Reserve in Anhui Province, an additional
Hypomyces collection was found on fruitbodies of
Schizophyllum sp. on an unidentified tree.
Morphological observation and DNA sequence
analysis were carried out. The results indicate that
this fungus is new to the genus.
1 MATERIALS AND METHODS
For morphological study, longitudinal sections
through ascomata were made with a freezing
microtome (YD-1508-III, Yidi Medical Appliance
Factory, Jinhua, Zhejiang) at a thickness of 8 –10μm.
Measurements were from mounts in lactophenol
cotton blue solution. Photographs were taken with a
Canon G5 (Tokyo, Japan) digital camera connected
to a Zeiss Axioskop 2 plus microscope (Göttingen,
Germany). Characters in culture were examined on
PDA and MEA. Specimens examined are deposited
in the Mycological Herbarium, Institute of
Microbiology, Chinese Academy of Sciences
(HMAS), and cultures are kept in the State Key
Laboratory of Mycology, Institute of Microbiology,
Chinese Academy of Sciences.
To confirm the morphological observation,
sequence analysis of rDNA ITS region was also
carried out. Genomic DNA was extracted from
mycelium harvested from colony on PDA after
2 weeks (Wang & Zhuang 2004). ITS sequence was
amplified using primer pairs ITS5-ITS4 (White et al.
1990). PCR was performed with an ABI 2720
Thermal Cycler (Gene Co. Ltd., Foster City,
California, USA) using a 25μL reaction system
consisting of 2.5μL 10× PCR buffer, 1.5μL MgCl2
(25mmol/L), 1.25μL each primer (10μmol/L), 0.5μL
dNTP (10mmol/L each), 1.25μL template DNA,
0.25μL Taq polymerase (5U/μL) and 16.5μL ddH2O.
PCR conditions were an initial step of 5min at 95℃,
30 cycles of 30s at 94℃, 30s at 52℃, and 30s at
72℃, followed by 10min at 72℃. PCR products
were purified with the PCR Product Purification Kit
(Biocolor BioScience & Technology Co., Shanghai,
China) and sequencing was carried out in both
directions with an ABI 3730 XL DNA Sequencer
(Genewiz Inc. Beijing, China). DNA sequences of
reference taxa along with their collection numbers
and GenBank accession numbers are listed in Table
1. All sequences were aligned using ClustalX 1.8
(Thompson et al. 1997), and the alignments were
visually adjusted by BioEdit 7.0.5 (Hall 1999) when
necessary. A neighbor-joining tree was generated
using MEGA 4.10 (Tamura et al. 2007) based on the
ITS sequences with Hypocrea rufa as the outgroup
taxon. The Kimura 2-parameter was selected as the
nucleotide substitution model, and gaps or missing
data were pairwise deleted. Bootstrap analyses were
performed with 1,000 replicates to test phylogeny
branch support.
ZHUANG Wen-Ying et al. / A new species of Hypomyces (Hypocreales) on Schizophyllum sp. from China
菌物学报
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Table 1 Materials used in this study
Species Collection number ITS nrDNA GenBank no.
Hypocrea rufa (Pers.) Fr. CBMAI1351 JN903557
GJS 74-69 FJ442642
Hypomyces aurantius (Pers.) Fuckel
MUCL 30376 Y17087
TFC 99-95 FN859427
H. australasiaticus K. Põldmaa
GJS 8431 FN859426
H. australis (Mont.) Höhn. dd08066 FJ810168
H. chlorinigenus Rogerson & Samuels SXY1 FJ803360
SXY2 FJ624396
H. chrysospermus Tul. & C. Tul.
dd08035 FJ810150
H. dactylarioides G.R.W. Arnold CBS 141.78 FN859429
H. lactifluorum (Schwein.) Tul. & C. Tul. TAAM 170476 FN859432
H. luteovirens (Fr.) Tul. & C. Tul. OSC 61916 EU837207
SR1b AY344801
H. microspermus Rogerson & Samuels
OZ1a AY344800
H. rosellus (Alb. & Schwein.) Tul. & C. Tul TFC 01-25 FN859442
GAi1716 FN859447
H. samuelsii K. Põldmaa
InBio 3-233 FN859450
H. sinicus HMAS 251317 JQ814696
H. subiculosus (Berk. & M.A. Curtis) Höhn. TFC97-166 FN859452
GAi1899 FN859453
H. virescens G.R.W. Arnold & K. Põldmaa
GAi1906 FN859454
Hypomyces sp. TFC 201334 FN859457
Hypomyces sp. GAm715k FN859455
2 TAXONOMY
Hypomyces sinicus W.Y. Zhuang, S.L. Chen, Z.Q.
Zeng & H.D. Zheng, sp. nov. Figs. 1, 2
Fungal Name FN570016
Perithecia obpyriform, 203–317µm high,
152–220µm in diam.; asci 8-spored, 75–104×5–6µm;
ascospores fusiform with a short apiculus at each end,
(0–)1-septate, surface warted, 13–16.5×3.2–4µm.
Subiculum thin on substrate and somewhat
dense surrounding perithecia, like a crater, reddish
orange when fresh and upon drying on the nature
substrate, turning purple in 3% KOH aqueous
solution; hyphae subhyaline to pale yellow, 1.5–4µm
wide, walls refractive. Perithecia gregarious to
solitary, immersed to partially immersed in
subiculum, reddish orange when fresh, reddish
orange to brownish orange when dry, and reddish in
culture (MEA), obpyriform, with an obtuse apex,
203–317µm high, 152–220µm in diam.; apical
portion 46–63µm wide at apex, ca. 76–85µm wide
at base, 50–120µm high; perithecial wall one-
layered except for the very apical portion of which
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Fig. 1 Hypomyces sinicus (from holotype). A: Perithecia on
Schizophyllum sp.; B: Perithecia produced in MEA culture; C, D:
Colony on PDA, surface view (C) and the reverse (D); E, F:
Colony on MEA, surface view (E) and the reverse (F).
two-layers may be recognized, of textura prismatica,
very faintly changing color in KOH aqueous
solution, 10–18µm thick, cells 6–22×3.5–8µm. Asci
inoperculate, 8-spored, subcylindrical, 75–104×
5–6µm. Ascospores fusiform with a short apiculus at
each end, (0–)1-septate, surface warted, containing
two large guttules and several small ones, uniseriate,
13–16.5×3.2–4µm; warts ca. 0.4µm diam.; apiculi
0.5–2µm high, 0.81µm wide at the widest, with a
blunt or slightly pointed tip.
In culture, colony on PDA white, with the
center very pale yellow and reverse white with the
center light orange-yellow; colony on MEA orange
with a reddish tint, reverse reddish orange; color of
perithecia produced on MEA somewhat different
from that of perithecia on nature substrate; conidia
(MEA) cylindrical-ellipsoid, hyaline, uniseptate,
8–16.7×2.7–5.5µm; clamydospores subglobose to
globose, 6.8–11.3µm in diam.
Holotype: China. Anhui, Jinzhai, Tianma
Nature Reserve, Tiantangzhai, alt. 700900m, on
fruitbodies of Schizophyllum sp. on an unidentified
tree, 22 Aug 2011, S.L. Chen, W.Y. Zhuang, H.D.
Zheng & Z.Q. Zeng 7763 (HMAS 251317).
3 RESULTS AND DISCUSSION
The closely related species to Hypomyces
sinicus were carefully compared. Morphologically,
the new species is most similar to H. subiculosus
(Berk. & M.A. Curtis) Höhn. occurring on
fruitbodies of Aphyllophorales (Samuels 1976;
Põldmaa & Samuels 2004). They are similar in
perithecial gross morphology, positive reaction to
KOH aqueous solution, ascospore shape and size;
while the latter fungus slightly differs in perithecia
being orange to brownish orange when dry, wider
ascospores [(11.7–)13.3–16.3(–18)(3.5–)4.5–5.2
(–6)µm], and colony color (H. subiculosus: first
white, turning deep orange, or remaining colorless
but the reverse turning pale yellow).
Hypomyces aurantius (Pers.) Tul. is similar to
H. sinicus in perithecial color, but differs obviously
in larger asci (100–1406–7µm), larger ascospores
[(13–)20–25(–27)(3–)4–6(–7.5)µm], and perithecia
turning violet to purplish-red in KOH (Rogerson &
Samuels 1993).
Hypomyces virescens is similar to H. sinicus in
occurring on Schizophyllum. However, the former
possesses purplish red perithecia which are
flask-shaped and 380–460280–350µm, asci are
much larger 160–180×7.0–8.5μm, and ascospores
are (22–)26(–30)×(5–)5.9 (–7)μm (Põldmaa 2011).
ZHUANG Wen-Ying et al. / A new species of Hypomyces (Hypocreales) on Schizophyllum sp. from China
菌物学报
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Fig. 2 Hypomyces sinicus (from holotype). A: Section of perithecia from nature substrate; B: Structure of a perithecium; C, D: An ascus with
ascospores; E: Ascospores; F: Conidia; G: Clamydospores.
Sequence analysis of rDNA ITS (the fungal
universal barcode region, see Schoch et al. 2012) of
some related Hypomyces species was carried out
with Hypocrea rufa as outgroup. Our results
revealed that two clades were recognized within the
genus. Hypomyces sinicus and its closely related
species were in a same clade with 80% bootstrap
support (Fig. 3). Hypomyces subiculosus and H.
lactifluorum turned out to be the closely related
species to H. sinicus with 99% bootstrap value.
Hypomyces subiculosus is most closely related to H.
sinicus (with 99% bootstrap support). However,
these two species share only 91% sequence
similarity and are of 55bp divergence. Hypomyces
aurantius having similar perithecial color is with
88% similarity. Hypomyces virescens occurring on a
similar or the same host fungus are not closely
related. The results recall our morphological
observation. Our molecular data support the
separation of H. sinicus from the related fungi.
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Fig. 3 Neighbor-joining tree based on rDNA ITS sequences, showing the relationship among Hypomyces species.
[REFERENCES]
Hall TA, 1999. BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows
95/98/NT. Nucleic Acids Symposium Series, 41: 95-98
Luo J, Zhuang WY, 2012. Re-examinations of the Hypomyces
specimens on deposit in HMAS. Mycosystema, 31: 784-788
Põldmaa K, 2011. Tropical species of Cladobotryum and
Hypomyces producing red pigments. Studies in Mycology, 68:
1-34
Põldmaa K, Samuels GJ, 2004. Fungicolous Hypocreaceae
(Ascomycota: Hypocreales) from KhaoYai National Park,
Thailand. Sydowia, 56: 79-130
Rogerson CT, Samuels GJ, 1993. Polyporicolous species of
Hypomyces. Mycologia, 85: 231-272
Samuels GJ, 1976. A revision of the fungi formerly classified as
Nectria subgenus Hyphonectria. Memoirs of the New York
Botanical Garden, 26(3): 1-126
Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL,
Levesque CA, Chen W, Fungal Barcoding Consortium, 2012.
The internal transcribed spacer as a universal DNA barcode
marker for Fungi. Proceedings of the National Academy of
Sciences USA, 109: 5907-6354
Tamura K, Dudley J, Nei M, Kumar S, 2007. MEGA4: molecular
evolutionary genetics analysis (MEGA) software version 4.0.
Molecular Biology and Evolution, 24: 1596-1599
Teng SC, 1934. Notes on Hypocreales from China. Sinensia, 4:
269-298
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgin DG,
1997. The ClustalX windows interface: flexible strategies for
multiple sequences alignment aided by quality analysis tools.
Nucleic Acids Research, 25: 4876-4883
Wang L, Zhuang WY, 2004. Designing primer sets for
amplification of partial calmodulin genes from penicillia.
Mycosystema, 23: 466-473
White TJ, Bruns TD, Lee SB, Taylor JW, 1990. Amplification and
direct sequencing of fungal ribosomal RNA genes for
phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White
TJ (eds.) PCR protocols: a guide to methods and applications.
Academic Press, New York. 315-222