Research Article |
Corresponding author: Wen-Ying Zhuang ( zhuangwy@im.ac.cn ) Academic editor: Nalin Wijayawardene
© 2019 Huan-Di Zheng, Wen-Ying Zhuang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zheng H-D, Zhuang W-Y (2019) Three new species of Dicephalospora from China as revealed by morphological and molecular evidences. MycoKeys 55: 87-99. https://doi.org/10.3897/mycokeys.55.33859
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Three new species of Dicephalospora are introduced based on morphological characters and DNA sequence analyses (maximum parsimony and neighbor-joining methods), viz. D. albolutea, D. shennongjiana, and D. yunnanica. All of them lack mucilaginous caps at ascospore poles. Dicephalospora albolutea is distinguished by cream to yellowish white apothecia and slightly curved ascospores. Dicephalospora shennongjiana is characterized by yellow apothecia, elliptical-fusoid ascospores 19−22 × 7−8.8 μm, and J+ asci 130−150 × 14−16.5 μm. Dicephalospora yunnanica is distinguished by orange apothecia and fusoid ascospores 16.5−25.3 × 3.3−3.5 μm. Descriptions and illustrations of the new species as well as a key to the known species in the genus are provided.
Morphology, phylogeny, species diversity, taxonomy
Dicephalospora Spooner is a small genus established by
During the examinations of helotialean fungi from China, three species fit well with the emended generic concept of Dicephalospora (
Specimens were collected, recorded, and photographed by a Canon PowerShot G16 digital camera in the field. Descriptions of gross morphology and substrate were according to field notes and photos. Dried apothecia were rehydrated with distilled water and sectioned at a thickness of 15−20 μm with a Yidi YD-1508A freezing microtome (Jinhua, China). Measurements were taken from longitudinal sections and squash mounts in lacto-phenol cotton blue solution using an Olympus BH-2 microscope (Tokyo, Japan). Iodine reactions of ascal apparatus were tested with or without 3% KOH solution pretreatment in Melzer’s reagent and Lugol’s solution (
Pure cultures were obtained from some specimens following the method provided by
Genomic DNA was extracted from dried apothecia or pure culture, using Plant Genomic DNA Kit (TIANGEN Biotech. Co., Beijing, China). ITS region was amplified and sequenced using the primer pair ITS1/ITS4 (
Newly generated sequences were assembled and edited using BioEdit 7.0.5.3 (
Species | Specimen/strain | ITS |
---|---|---|
Chlorosplenium chlora (Schwein.) M.A. Curtis | HMAS 266518 | MK425599 |
HMAS 279692 | MK425600 | |
Ciboria batschiana (Zopf) N.F. Buchw. | CBS 312.37 | KF859931 |
Ciborinia foliicola (E.K. Cash & R.W. Davidson) Whetzel | 1932.H | Z80892 |
Dicephalospora albolutea H.D. Zheng & W.Y. Zhuang | HMAS 279693 | MK425601 |
Dicephalospora aurantiaca (W.Y. Zhuang) W.Y. Zhuang & Z.Q. Zeng | HMAS 61850 | DQ986486 |
Dicephalospora chrysotricha (Berk.) Verkley | ICMP:19950 | KF727410 |
ICMP:19952 | KF727411 | |
Dicephalospora dentata Xiao X. Liu & W.Y. Zhuang | HMAS 266694 | KP204263 |
Dicephalospora huangshanica (W.Y. Zhuang) W.Y. Zhuang & Z.Q. Zeng | HMAS 74836 | DQ986485 |
HMAS 81364 | DQ986484 | |
HMAS 279694 | MK425602 | |
Dicephalospora rufocornea (Berk. & Broome) Spooner | HMAS 75518 | DQ986480 |
10106 | KU668565 | |
HMAS 279695 | MK425603 | |
HMAS 279696 | MK425604 | |
HMAS 279697 | MK425605 | |
Dicephalospora shennongjiana H.D. Zheng & W.Y. Zhuang | HMAS 279698 | MK425606 |
Dicephalospora yunnanica H.D. Zheng & W.Y. Zhuang | HMAS 279699 | MK425607 |
HMAS 279700 | MK425608 | |
HMAS 279701 | MK425609 | |
Hymenoscyphus fructigenus (Bull.) Gray | CBS650.92 | GU586933 |
HMAS 75893 | JX977144 | |
Lachnum pygmaeum (Fr.) Bres. | ARON 2924.S | AJ430215 |
Lachnum spartinae S.A. Cantrel | SAP 138 | AF422970 |
Lambertella corni-maris Höhn. | CLX 3892 | KC958560 |
CLX 4075 | KC958562 | |
Lanzia allantospora (Dennis) Spooner | PRJ D804 | AY755334 |
Lanzia luteovirescens (Roberge ex Desm.) Dumont & Korf | 1823 | KC533545 |
Moellerodiscus lentus (Berk. & Broome) Dumont | 7818 | KU668564 |
10544 | KU668566 | |
Monilinia fructicola (G. Winter) Honey | MO-3D | JN001480 |
RS10 | JF325841 | |
Rutstroemia firma (Pers.) P. Karst. | 2089.1 | Z80893 |
2089 | KC533547 | |
Sclerotinia sclerotiorum (Lib.) de Bary | 2 | KF148605 |
6 | KF148609 |
The ITS dataset included 37 sequences from eight Dicephalospora species, 11 related fungi and two outgroup taxa. The final alignment resulted in 634 characters including gaps, of which 252 were parsimony-informative, 38 were variable and parsimony-uninformative, and 344 were constant. In the MP analysis, eight most parsimonious trees were generated (tree length = 790, consistency index = 0.5899, homoplasy index = 0.4101, retention index = 0.8126, rescaled consistency index = 0.4793) and one of them was shown in Figure
From topology of the phylogenetic tree (Fig.
The specific epithet refers to the color of apothecia.
CHINA. Yunnan Province, Binchuan County, Jizu Mountain, alt. 2500 m, on rotten leaf veins, 21 September 2017, H.D. Zheng, X.C. Wang, Y.B. Zhang & Y. Zhang 11613 (HMAS 279693, ITS GenBank accession number: MK425601).
Apothecia scattered, discoid, stipitate, with even margin, 1−2.5 mm in diameter; hymenium surface cream to yellowish white; receptacle surface concolorous. Ectal excipulum of textura prismatica, 20−70 μm thick, cells somewhat thick- and glassy-walled, 16.5−40 × 5.5−11 μm. Medullary excipulum of textura porrecta and textura intricata, 25−275 μm thick, hyphae hyaline, thin-walled, 2.5−5 μm wide. Subhymenium not distinguishable. Hymenium 165−175 μm thick. Asci unitunicate, arising from simple septa, 8-spored, cylindric-clavate, J+ in Melzer’s reagent and Lugol’s solution without KOH pretreatment, visible as two blue lines, 140−156 × 9.5−10.5 μm. Ascospores sausage-shaped to subfusoid, with anterior end rounded and posterior end narrower, slightly curved, aseptate, hyaline, smooth, lacking a gel cap at each end, multiguttulate, with a dark-stained area when mounted in cotton blue solution, biseriate, 26−31 × 3.8−5.0 μm. Paraphyses filiform, straight, slightly enlarged at apex, hyaline, septate, 3−3.5 μm broad at upper portion and 1.5−2 μm below, equal to or very slightly exceeding the asci.
Dicephalospora albolutea (HMAS 279693, holotype). a fresh apothecia on natural substrate b longitudinal section of apothecium c structure of margin and hymenium d structure of flank e asci f IKI reaction of apical rings g ascospores. Mouting media: b–e, g lacto-phenol cotton f lugol’s solution. Scale bars: 5 mm (a); 200 μm (b); 20 μm (c, d); 10 μm (e, f); 5 μm (g).
The diagnostic features of D. albolutea are cream to yellowish white apothecia and sausage-shaped ascospores. The apothecial color of earlier known Dicephalospora species varied from yellow, orange, red to dark, but never as pale as that in D. albolutea. Dicephalospora calochroa (Syd. & P. Syd.) Spooner is somewhat similar in length of asci and ascospores, but differs by vivid orange apothecia, wider asci (125−150 × 12−15 μm) and ascospores (20−25 × 6−8 μm), which are pointed at both ends (
The specific epithet refers to the type locality of the fungus.
CHINA. Hubei Province, Shennongjia, Shennongyuan, alt. 2250 m, on stromatized dead vine, 15 Sept 2014, H.D. Zheng, Z.Q. Zeng, W.T. Qin & K. Chen 9589 (HMAS 279698, ITS GenBank accession number: MK425606).
Apothecia scattered, discoid to flat, stipitate, with even margin, 0.5−0.8 mm in diameter; hymenium surface greenish yellow; receptacle surface slightly darker. Ectal excipulum of textura prismatica, 15−40 μm thick, cells hyaline to pale brownish, somewhat thick- and glassy-walled, 10−20 × 4−11 μm. Medullary excipulum of textura intricata, 25−110 μm thick, hyphae hyaline, thin-walled, 2−4 μm wide. Subhymenium about 15 μm thick. Hymenium 170−180 μm thick. Asci arising from simple septa, unitunicate, 8-spored, clavate, J+ in Melzer’s reagent and Lugol’s solution without KOH pretreatment, visible as two blue lines, 130−150 × 14−16.5 μm. Ascospores elliptical-subfusoid, aseptate, hyaline, smooth, lacking a gel cap at each end, multiguttulate, with a dark-stained area when mounted in cotton blue solution, uniseriate, 19−22 × 7−8.8 μm. Paraphyses filiform, slightly enlarged at apex, hyaline, septate, branched and tangled near apex, 3−3.5 μm broad at upper portion and 1.5−2 μm below, exceeding the asci by 10−20 μm.
Dicephalospora shennongjiana (HMAS 279698, holotype) a fresh apothecia on natural substrate b dried apothecia c longitudinal section of apothecium d structure of margin, flank and hymenium e asci f IKI reaction of apical rings g ascospores in an ascus h ascospores. Mouting media: c–e, g, h lacto-phenol cotton f fugol’s solution. Scale bars: 2 mm (a); 0.4 mm (b); 200 μm (c); 20 μm (d); 10 μm (e–h).
The new species can be distinguished from other species by shape of ascospores and tangled paraphyses apices. Dicephalospora damingshanica has a similarly shaped ascospore, but larger (22−32 × 9−12.7 μm), and with a hyaline mucilaginous cap at both ends (
The specific epithet refers to the type locality of the fungus.
CHINA. Yunnan Province, Maguan County, Dabao Village, alt. 1565 m, on rotten leaf rachis, 13 August 2016, X.H. Wang, S.H. Li, H.D. Zheng & S.C. Li YN16-108 (HMAS279699, ITS GenBank accession number: MK425607).
Apothecia scattered, discoid, stipitate, with even margin, 0.8−2.0 mm in diameter; hymenium surface bright yellow to orange; receptacle surface paler. Ectal excipulum of textura prismatica, 22−60 μm thick, cells hyaline, somewhat thick- and glassy-walled, 7−20 × 5−7 μm. Medullary excipulum of textura intricata, 30−230 μm thick, hyphae thin-walled, 2−5 μm wide. Subhymenium not distinguishable. Hymenium 100−115 μm thick. Asci arising from simple septa, unitunicate, 8-spored, cylindric-clavate, J+ in Melzer’s reagent and Lugol’s solution without KOH pretreatment, visible as two faint blue lines, 85−100 × 7.5−8.5 μm. Ascospores fusoid, aseptate, with one side very slightly flattened and pointed at ends, hyaline, smooth, lacking a gel cap at each end, multiguttulate, biseriate, 16.5−25.3 × 3.3−3.5 μm. Paraphyses filiform, slightly enlarged at apex, straight or sometimes slightly curved at the apical portion, hyaline, septate, 2.5−4 μm broad at upper portion and 1.5−2 μm below, slightly exceeding the asci by about 5 μm.
Dicephalospora yunnanica (HMAS 279699, holotype) a fresh apothecia on natural substrate b dried apothecia c longitudinal section of apothecium d structure of margin, flank and hymenium e, f asci g IKI reaction of apical rings h ascospores. Mouting media: c–e, h lacto-phenol cotton f, g lugol’s solution. Scale bars: 5 mm (a); 2 mm (b); 200 μm (c); 20 μm (d); 10 μm (e–g); 5 μm (h).
CHINA. Yunnan Province, Maguan County, Xiaobaozi Town, alt. 1550 m, on rotten leaf rachis, 13 August 2016, X.H. Wang, S.H. Li, H.D. Zheng & S.C. Li YN16-135 (HMAS 279700); Maguan County, Pojiao Village, alt. 1450 m, on rotten leaf rachis, 14 August 2016, X.H. Wang, S.H. Li, H.D. Zheng & S.C. Li YN16-165 (HMAS 279700).
Dicephalospora yunnanica shares similar gross morphology with and appeared to be sister of D. aurantiaca in the phylogenetic tree (Fig.
1 | Receptacle surface covered with hairs | D. chrysotricha |
– | Receptacle surface without hairs | 2 |
2 | Apothecial margin dentate | D. dentata |
– | Apothecial margin even | 3 |
3 | Hymenium surface cream to yellowish white when fresh | D. albolutea |
– | Hymenium surface darker in color | 4 |
4 | Hymenium surface red or dark red | D. huangshanica |
– | Hymenium surface lacking of a red tint | 5 |
5 | Paraphyses with darkly pigmented contents | D. phaeoparaphysis |
– | Paraphyses without darkly pigmented contents | 6 |
6 | Ascospores with a gel cap at each end | 7 |
– | Ascospores lacking of a gel cap at each end | 10 |
7 | Ascospores 9−12.7 μm wide | D. damingshanica |
– | Ascospores less than 9 μm wide | 8 |
8 | Asci J−, ascospores 20−28 × 4.5−5.7 μm | D. pinglongshanica |
– | Asci J+ | 9 |
9 | Ascospores 23−27(−29) × 6.5−7.5 μm | D. calochroa |
– | Ascospores (27−)32−39 × 4−5.5(−6) μm | D. rufocornea |
10 | Ascospores constricted in the middle, 20−27 × 4−5 μm | D. contracta |
– | Ascospores not constricted in the middle | 11 |
11 | Ascospores 19−22 × 7−8.8 μm | D. shennongjiana |
– | Ascospores less than 7 μm wide | 12 |
12 | Ascospores 16.5−25.3 × 3.3−3.5 μm, paraphyses straight | D. yunnanica |
– | Ascospores 21−26 × 4−4.8 μm, paraphyses curved at apex | D. aurantiaca |
Identification of Dicephalospora species is mainly based on morphological features, such as color of apothecia, anatomic structure, and characteristics of asci and ascospores. DNA sequence data are sometimes considered, which play an important role in the delineation of fungal species (
In the phylogenetic analyses, only some species possessing fusoid to sausage-shaped and elliptic-subfusoid ascospores were involved due to limitation of the available sequences. The ITS barcodes seem to be useful for distinguishing Dicephalospora species, as they grouped as well-separated clades (Fig.
As to the phylogenetic position of Dicephalospora, Figure
This work was supported by the National Natural Science Foundation of China (nos. 31770019, 31570018). The authors thank all co-collectors of specimens examined in this study for their invaluable help during the field work.