A four-locus phylogeny of rib-stiped cupulate species of Helvella (Helvellaceae, Pezizales) with discovery of three new species

Xin-Cun Wang; Tie-Zhi Liu; Shuang-Lin Chen; Yi Li; Wen-Ying Zhuang

doi:10.3897/mycokeys.60.38186

Research Article

A four-locus phylogeny of rib-stiped cupulate species of Helvella (Helvellaceae, Pezizales) with discovery of three new species

Xin-Cun Wang^‡, Tie-Zhi Liu^§, Shuang-Lin Chen^|, Yi Li^¶, Wen-Ying Zhuang^‡

‡ Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

§ Chifeng University, Chifeng, China

| Nanjing Normal University, Nanjing, China

¶ Yangzhou University, Yangzhou, China

Corresponding author: Wen-Ying Zhuang ( zhuangwy@im.ac.cn )

Academic editor: Thorsten Lumbsch

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: Wang X-C, Liu T-Z, Chen S-L, Li Y, Zhuang W-Y (2019) A four-locus phylogeny of rib-stiped cupulate species of Helvella (Helvellaceae, Pezizales) with discovery of three new species. MycoKeys 60: 45-67. https://doi.org/10.3897/mycokeys.60.38186

Abstract

Helvella species are ascomycetous macrofungi with saddle-shaped or cupulate apothecia. They are distributed worldwide and play an important ecological role as ectomycorrhizal symbionts. A recent multi-locus phylogenetic study of the genus suggested that the cupulate group of Helvella was in need of comprehensive revision. In this study, all the specimens of cupulate Helvella sensu lato with ribbed stipes deposited in HMAS were examined morphologically and molecularly. A four-locus phylogeny was reconstructed using partial sequences of the heat shock protein 90, nuclear rDNA internal transcribed spacer region 2, nuclear large subunit ribosomal DNA and translation elongation factor 1-α genes. Three clades were revealed in Helvella sensu stricto. Twenty species were included in the analysis, of which 13 are distributed in China. Three new species, H. acetabuloides, H. sichuanensis and H. tianshanensis, are described and illustrated in detail. A neotype was designated for H. taiyuanensis. Helvella calycina is a new record for China, while Dissingia leucomelaena should be excluded from Chinese mycota. Hsp90 and ITS2 are recommended as useful supplementary barcodes for species identifications of the genus.

Keywords

Ascomycota, DNA barcode, phylogeny, taxonomy, typification

Introduction

The genus Helvella L. contains a group of ascomycetous macrofungi with saddle-shaped or cupulate apothecia. Helvella species are distributed worldwide, especially in temperate regions (Dissing 1966, Abbott and Currah 1997). Some of them are edible, for example, H. bachu Q. Zhao, Zhu L. Yang & K.D. Hyde (Zhao et al. 2016a) and H. taiyuanensis B. Liu, Du & J.Z. Cao (Liu et al. 1985), and some are medicinal, for example, H. lacunosa Afzel. (Shameem et al. 2016). They are also important as ectomycorrhizal symbionts (Tedersoo et al. 2006, Healy et al. 2013, Hwang et al. 2015).

Helvella was established in 1753 and more than 400 names attributable to the genus have been recorded in the databases of Index Fungorum and MycoBank. Several taxonomic treatments were proposed, based on morphological characters (Table 1). Seven sections were established by Dissing (1966): sections Acetabulum, Crispae, Elasticae, Ephippium, Lacunosae, Leucomelaenae and Macropodes. Amongst them, the sections Acetabulum and Leucomelaenae included the species having cup-shaped apothecia with ribbed stipes. Similarly, six to eight infrageneric groups (sections or subgenus) were recognised by different authors (Weber 1972, Häffner 1987, Abbott and Currah 1997). Meanwhile, many additional species were added to the genus (Weber 1975, Harmaja 1976, 1977a, b, 1978, 1979, Abbott and Currah 1988). A checklist of cupulate Helvella species having ribbed stipes and their infrageneric positions are summarised in Table 2. Recently, Helvella sensu stricto was found to be associated with Balsamia Vittad., Dissingia K. Hansen, X.H. Wang & T. Schumach., Midotis Fr., Pindara Velen. and Underwoodia Peck in Helvellaceae (Hansen and Pfister 2006; Hansen et al. 2019). Amongst them, Dissingia was proposed to accommodate the species formerly placed in Helvella section Leucomelaenae (Hansen et al. 2019).

Table 1.

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Comparison of the taxonomic systems established in Helvella.

Dissing (1966)	Weber (1972)	Häffner (1987)	Abbott and Currah (1997)	Hansen et al. (2019)
Section Leucomelaenae Dissing	Section Leucomelaenae Dissing	Section Leucomelaenae Dissing	Subgenus Leucomelaenae (Dissing) S.P. Abbott	Dissingia K. Hansen, X.H. Wang & T. Schumach.
Section Leucomelaenae Dissing	Section Leucomelaenae Dissing	Section Solitariae Häffner		Helvella L.
Section Acetabulum Dissing	Section Acetabulum Dissing	Section Acetabulum Dissing
Section Crispae Dissing	Section Helvella L.	Section Helvella L.	Subgenus Helvella L.
Section Lacunosae Dissing	Section Lacunosae Dissing	Section Lacunosae Dissing	Subgenus Helvella L.
Section Elasticae Dissing	Section Elasticae Dissing	Section Elasticae Dissing	Subgenus Elasticae (Dissing) S.P. Abbott
Section Ephippium Dissing	Section Ephippium Dissing	Section Ephippium Dissing	Subgenus Elasticae (Dissing) S.P. Abbott
Section Macropodes Dissing	Section Macropodes Dissing	Section Macropodes Dissing	Subgenus Macropodes (Dissing) S.P. Abbott
			Subgenus Cupuliformes S.P. Abbott
			Subgenus Silvicolae (S.P. Abbott) S.P. Abbott	Midotis Fr.

With the development of molecular phylogenetics, the taxonomy of Helvella has been re-evaluated. Sequences of nuclear large and small subunit ribosomal DNA (LSU and SSU) were adopted for phylogenetic inference of Helvella sensu lato and its allied genera (Hansen and Pfister 2006, Tedersoo et al. 2006, Laessoe and Hansen 2007). Protein-coding genes, RNA polymerase II the largest subunit (RPB1), the second largest subunit (RPB2) and translation elongation factor 1-α (TEF1) were also applied (Bonito et al. 2013, Hansen et al. 2013). Nguyen et al. (2013) explored Helvella phylogeny using large-scale sequence analysis of LSU and the nuclear rDNA internal transcribed spacer region (ITS) and reported two new species from North America based on molecular and morphological evidence. On the basis of examinations of the type specimens and LSU sequence analysis, Landeros et al. (2012, 2015) concluded that the sections Elasticae, Helvella, Lacunosae and Leucomelaenae were monophyletic. Skrede et al. (2017) studied molecular characteristics of 55 European species, described seven new species based on the sequence divergences of LSU, RPB2, TEF1 and heat shock protein 90 gene (Hsp90), and designated neotypes and epitypes for 30 of them. Five clades and 18 lineages were distinguished according to the phylogeny inferred from the combined Hsp90 and RPB2 datasets. The above work provides background information for understanding the species concept of Helvella. In their updated study, Hansen et al. (2019) defined Helvella s. s., treated the cupulate H. leucomelaena (Pers.) Nannf. lacking crozier at the ascus base as a separate genus Dissingia, retrieved the generic name Pindara, and transferred H. aestivalis (R. Heim & L. Rémy) Dissing & Raitv. to Balsamia. Brief comparisons amongst different taxonomic treatments are shown in Table 1.

In China, Teng (1963) recorded 11 species of Helvella and Tai (1979) listed 15 taxa. Liu, Cao and their collaborators (Liu et al. 1985, Liu and Cao 1988, Cao and Liu 1990, Cao et al. 1990) published nine species, new to the genus. With the additional investigations, our knowledge of the group accumulated (Zhuang 1989, 1995, 1996, 1997, 1998, Zhuang and Wang 1998a, 1998b, Yu et al. 2000, Wang and Chen 2002, Xu 2002, Zhuang 2004, Zhuang and Yang 2008). Zhuang et al. (2018) provided a checklist of 37 Helvella species occurring in China up to 2013. Recently, Zhao and his collaborators (Ariyawansa et al. 2015, Zhao et al. 2015, Hyde et al. 2016, Wang et al. 2016, Zhao et al. 2016a, 2016b, Tibpromma et al. 2017) described 12 new species with two bearing cupulate apothecia (Table 2), as well as two new Chinese records, H. subglabra N.S. Weber and H. ulvinenii Harmaja. There are about 51 species currently known from the country.

The present study is aimed at exploring species diversity of the cupulate Helvella species with ribbed stipes.

Table 2.

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A checklist of cupulate Helvella species sensu lato with ribbed stipes.

Species	Section Acetabulum	Section Leucomelaenae	Section Solitariae	Section Macropodes	Subgenus Leucomelaenae	Remark
Acetabula calyx Sacc., 1873	–	Syn. of H. solitaria (Dissing 1966); Syn. of H. leucomelaena (Harmaja 1977a)	–	–	Syn. of H. leucomelaena (Abbott and Currah 1997)	Syn. of H. leucomelaena (Landeros et al. 2015)
Balsamia aestivalis (R. Heim & L. Rémy) K. Hansen, Skrede & T. Schumach, 2019	–	Häffner 1987	–	–	Abbott and Currah 1997	as Helvella aestivalis
Dissingia crassitunicata (N.S. Weber) T. Schumach & Skrede, 2019	–	Weber 1975, Häffner 1987	–	–	Abbott and Currah 1997	as Helvella crassitunicata
Dissingia confusa (Harmaja) K. Hansen & X.H. Wang, 2019	–	Harmaja 1977a, Häffner 1987	–	–	Syn. of H. leucomelaena (Abbott and Currah 1997)	as Helvella confusa
Dissingia leucomelaena (Pers.) K. Hansen & X.H. Wang, 2019	–	Dissing 1966, Weber 1975, Häffner 1987	–	–	Abbott and Currah 1997	as Helvella leucomelaena
Dissingia oblongispora (Harmaja) T. Schumachand Skrede, 2019	–	Harmaja 1978, Häffner 1987	–	–	Abbott and Currah 1997	as Helvella oblongispora
Helvella acetabulum (L.) Quél, 1874	Dissing 1966, Weber 1972, Häffner 1987	–	–	–	Abbott and Currah 1997	Valid species
Helvella arctoalpina Harmaja, 1977	Harmaja 1977b, Häffner 1987	–	–	–	Syn. of H. verruculosa (Abbott and Currah 1997)	Valid species
Helvella calycina Skrede, T.A. Carlsen & T. Schumach, 2017	–	–	–	–	–	Valid species
Helvella costata Schwein, 1822	–	–	–	–	Syn. of H. acetabulum (Abbott and Currah 1997)	Valid species
Helvella costifera Nannf, 1953	Dissing 1966, Häffner 1987	–	–	–	Abbott and Currah 1997	Valid species
Helvella dryadophila Harmaja, 1977	Harmaja 1977b, Häffner 1987	–	–	–	Syn. of H. verruculosa (Abbott and Currah 1997)	Valid species
Helvella floriforma Q. Zhao & K.D. Hyde, 2016^*	–	–	–	–	–	Valid species
Helvella griseoalba N.S. Weber, 1972	Weber 1972, Häffner 1987	–	–	–	Syn. of H. costifera (Abbott and Currah 1997)	Valid species
Helvella helvellula (Durieu) Dissing, 1966	–	Dissing 1966	–	–	–	Member of lasunosa clade (Skrede et al. 2017)
Helvella hyperborea Harmaja, 1978	Harmaja 1978, Häffner 1987	–	–	–	Abbott and Currah 1997	Valid species
Helvella jiaohensis J.Z. Cao, L. Fan & B. Liu, 1990^*	–	–	–	–	–	Holotype lost
Helvella jilinensis J.Z. Cao, L. Fan & B. Liu, 1990^*	–	–	–	–	–	Holotype lost
Helvella pedunculata Harmaja, 1978	–	Harmaja 1978, Häffner 1987	–	–	Syn. of H. leucomelaena (Abbott and Currah 1997)	?Syn. of H. costifera (Skrede et al. 2017)
Helvella pocillum Harmaja, 1976	Häffner 1987	Harmaja 1976	–	–	–	Syn. of B. aestivalis (Hansen et al. 2019)
Helvella queletii Bres, 1882	–	Syn. of H. solitaria (Harmaja 1977a, Häffner 1987)	–	Dissing 1966, Weber 1972	Syn. of H. solitaria (Abbott and Currah 1997)	Syn. of H. solitaria (Landeros et al. 2012)
Helvella robusta S.P. Abbott, 1988	Abbott and Currah 1988	–	–	–	Abbott and Currah 1997	Valid species
Helvella solitaria P. Karst, 1871	–	Dissing 1966	Häffner 1987	–	Abbott and Currah 1997	Valid species
Helvella taiyuanensis B. Liu, Du & J.Z. Cao, 1985^*	–	–	–	–	–	Neotypification here
Helvella tinta Q. Zhao, B. Feng & K.D. Hyde, 2016^*	–	–	–	–	–	Valid species
Helvella ulvinenii Harmaja, 1979	Harmaja 1979	–	Häffner 1987	–	Abbott and Currah 1997	Syn. of H. solitaria (Landeros et al. 2015)
Helvella unicolor (Boud.) Dissing, 1966	Dissing 1966, Häffner 1987	–	–	–	Abbott and Currah 1997	In need of reassessment (Skrede et al. 2017)
Helvella verruculosa (Sacc.) Harmaja, 1978	–	–	–	–	Abbott and Currah 1997	In need of reassessment (Skrede et al. 2017)

Materials and methods

Fungal materials and morphological observations

Collections of the cupulate Helvella species with ribbed stipes, deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS), were re-examined, including those originally deposited in the Mycological Herbarium of Shanxi University (MHSU). Specimens recently collected from Beijing, Inner Mongolia, Hubei and Sichuan provinces were identified (Table 3). Morphological observations were conducted following Wang and Zhuang (2019). In measurements, Q refers to length/width ratio of ascospores for which the medians are given.

Table 3.

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Fungal species and sequences used in phylogenetic analyses.

Species	Voucher	Locality	HSP90	ITS	LSU	TEF1	Label	Reference
Balsamia aestivalis (R. Heim & L. Rémy) K. Hansen, Skrede & T. Schumach.	KH.10.133	Sweden	–	–	MK100250	MK113869	Balsamia aestivalis	Hansen et al. 2019
	O-253217	Norway	–	–	MK100251	MK113870	Balsamia aestivalis	Hansen et al. 2019
Balsamia platyspora Berk.	TUR206101	Finland	–	–	MK100252	MK113871	Balsamia platyspora	Hansen et al. 2019
Dissingia confusa (Harmaja) K. Hansen & X.H. Wang	H437^*	Norway	KY784529	–	KY773164	–	Helvella confusa	Skrede et al. 2017
	HMAS 27728^*	Qinghai, China	MK652180	MK592119	–	–	Helvella confusa	This study
	HMAS 38328^*	Xinjiang, China	MK652181	MK592120	–	–	Acetabula leucomelas	This study
Dissingia crassitunicata (N.S. Weber) T. Schumach. & Skrede	H222^*	Canada	KY784342	–	KY773053	–	Helvella crassitunicata	Skrede et al. 2017
Dissingia leucomelaena (Pers.) K. Hansen & X.H. Wang	H404, epitype	Sweden	KY784500	–	–	–	Helvella leucomelaena	Skrede et al. 2017
	H115^*	USA	KY784253	–	KY772970	–	Helvella leucomelaena	Skrede et al. 2017
	KH.06.01 = H115	USA	–	–	KC012682	KC109207	Helvella leucomelaena	Hansen et al. 2013
	He273	Australia	–	–	JX993075	–	Helvella leucomelaena	Landeros et al. 2015
	He286, isotype	Italy	–	–	JX993051	–	Acetabula calyx	Landeros et al. 2015
	HMAS 61351	Denmark	MK652201	–	–	–	Helvella leucomelaena	This study
	HMAS 61356^*	Sweden	MK652202	MK592137	–	–	Helvella leucomelaena	This study
Dissingia oblongispora (Harmaja) T. Schumach. & Skrede	H132^*	Norway	KY784265	–	KY772983	–	Helvella oblongispora	Skrede et al. 2017
	HMAS 38329^*	Xinjiang, China	MK652203	MK592138	–	–	Helvella acetabulum	This study
	HMAS 74657^*	Gansu, China	MK652204	MK592139	–	–	Helvella leucomelaena	This study
	HMAS 75147^*	Sichuan, China	MK652205	MK592140	–	MK652162	Helvella leucomelaena	This study
	HMAS 75151	Sichuan, China	MK652206	MK592141	–	–	Helvella leucomelaena	This study
	HMAS 75183	Sichuan, China	MK652207	MK592142	–	–	Helvella leucomelaena	This study
	HMAS 75960	Sichuan, China	MK652208	MK592143	–	–	Helvella cupuliformis	This study
	HMAS 86050	Xinjiang, China	–	MK592144	–	–	Helvella acetabulum	This study
	HMAS 86051	Xinjiang, China	–	MK592145	–	MK652163	Helvella acetabulum	This study
	HMAS 86160	Shanxi, China	–	MK592146	–	–	Helvella leucomelaena	This study
Helvella acetabuloides X.C. Wang & W.Y. Zhuang	HMAS 279703^*, CFSZ 2044, holotype	Inner Mongolia, China	MK652219	MK592155	–	MK652168	Helvella acetabulum	This study
Helvella acetabuloides X.C. Wang & W.Y. Zhuang	HMAS 23842^*	Shaanxi, China	MK652220	–	–	–	Acetabula vulgaris	This study
Helvella acetabulum (L.) Quél.	H410, epitype	Sweden	KY784506	–	KY773154	–	Helvella acetabulum	Skrede et al. 2017
	H133^*	Norway	KY784266	–	KY772984	KY772875	Helvella acetabulum	Skrede et al. 2017
	HMAS 7046^*	Czech	MK652177	MK592116	–	–	Acetabula vulgaris	This study
	HMAS 61353	Denmark	MK652176	–	–	–	Helvella acetabulum	This study
	HMAS 243823^*	UK	MK652174	MK592114	MK592099	–	Helvella acetabulum	This study
	HMAS 23839	Qinghai, China	MK652171	MK592112	–	–	Helvella acetabulum	This study
	HMAS 23841	Beijing, China	MK652172	MK592113	–	–	Helvella acetabulum	This study
	HMAS 23843	Qinghai, China	MK652173	–	–	–	Acetabula vulgaris	This study
	HMAS 38129	Xinjiang, China	MK652175	MK592115	–	–	Helvella acetabulum	This study
Helvella acetabulum (L.) Quél.	HMAS 75176^*	Sichuan, China	MK652178	MK592117	–	MK652156	Helvella acetabulum	This study
Helvella arctoalpina Harmaja	H293, holotype	Norway	KY784406	–	–	–	Helvella arctoalpina	Skrede et al. 2017
Helvella arctoalpina Harmaja	H033^*	Norway	KY784207	–	KY772924	KY772841	Helvella arctoalpina	Skrede et al. 2017
Helvella calycina Skrede, T.A. Carlsen & T. Schumach.	H022^*, epitype	Norway	KY784198	–	KY772915	KY772833	Helvella calycina	Skrede et al. 2017
Helvella calycina Skrede, T.A. Carlsen & T. Schumach.	HMAS 279704^*, CFSZ 2658	Inner Mongolia, China	MK652179	MK592118	MK592100	MK652157	Helvella acetabulum	This study
Helvella costata Schwein.	H100^*	USA	KY784244	–	KY772962	–	Helvella costata	Skrede et al. 2017
Helvella costifera Nannf.	H298, epitype	Sweden	KY784409	–	–	–	Helvella costifera	Skrede et al. 2017
	H131^*	Norway	KY784264	–	KY772982	KY772874	Helvella costifera	Skrede et al. 2017
	HMAS 61361	Shanxi, China	MK652185	–	–	–	Helvella acetabulum	This study
	HMAS 71778	Beijing, China	MK652186	MK592124	–	–	Helvella costifera	This study
	HMAS 83510	Xinjiang, China	MK652187	MK592125	–	–	Helvella costifera	This study
	HMAS 88497	Shanxi, China	MK652188	MK592126	–	–	Helvella acetabulum	This study
	HMAS 139024^*	Shaanxi, China	MK652182	MK592121	MK592101	–	Helvella sp.	This study
	HMAS 187120^*	Beijing, China	MK652183	MK592122	MK592102	MK652158	Helvella sp.	This study
	HMAS 280301^*	Yunnan, China	MK652184	MK592123	MK592103	MK652159	Helvella sp.	This study
Helvella dryadophila Harmaja	H302, holotype	Norway	KY784412	–	–	–	Helvella dryadophila	Skrede et al. 2017
Helvella dryadophila Harmaja	H180^*	Norway	KY784309	–	KY773024	KY772883	Helvella dryadophila	Skrede et al. 2017
Helvella floriforma Q. Zhao & K.D. Hyde	HKAS 90224, Holotype	Yunnan, China	–	–	KX239771	–	Helvella floriforma	Hyde et al. 2016
Helvella griseoalba N.S. Weber	He164, holotype	USA	–	–	JX993066	–	Helvella griseoalba	Landeros et al. 2015
Helvella griseoalba N.S. Weber	H306^*	USA	KY784416	–	–	–	Helvella griseoalba	Skrede et al. 2017
Helvella hyperborea Harmaja	H491^*	Finland	KY784569	–	–	–	Helvella hyperborea	Skrede et al. 2017
	HMAS 23840	Gansu, China	MK652189	–	–	–	Helvella acetabulum	This study
	HMAS 38331	Xinjiang, China	MK652190	–	–	–	Helvella costifera	This study
	HMAS 83506	Xinjiang, China	MK652191	MK592127	–	–	Helvella costifera	This study
	HMAS 83507	Xinjiang, China	MK652192	MK592128	–	–	Helvella costifera	This study
	HMAS 83508	Xinjiang, China	MK652193	MK592129	–	–	Helvella costifera	This study
	HMAS 83509	Xinjiang, China	MK652194	MK592130	–	–	Helvella costifera	This study
	HMAS 83511	Xinjiang, China	MK652195	MK592131	–	MK652160	Helvella costifera	This study
Helvella hyperborea Harmaja	HMAS 83512	Xinjiang, China	MK652196	MK592132	–	–	Helvella costifera	This study
	HMAS 85476	Xinjiang, China	MK652197	MK592133	–	–	Helvella acetabulum	This study
	HMAS 85591^*	Shanxi, China	MK652198	MK592134	–	–	Helvella leucomelaena	This study
	HMAS 85673^*	Shanxi, China	MK652199	MK592135	–	–	Helvella solitaria	This study
	HMAS 86043^*	Xinjiang, China	MK652200	MK592136	–	MK652161	Helvella costifera	This study
Helvella robusta S.P. Abbott	He163, holotype	Canada	–	–	JX993079	–	Helvella robusta	Landeros et al. 2015
Helvella sichuanensis X.C. Wang & W.Y. Zhuang	10706^*, HMAS 254610, holotype	Sichuan, China	MK652221	MK592156	MK592107	MK652169		This study
Helvella solitaria P. Karst.	H370, epitype	Sweden	KY784470	–	–	–	Helvella solitaria	Skrede et al. 2017
	H004^*	Norway	KY784184	–	KY772902	KY772819	Helvella solitaria	Skrede et al. 2017
	He248, holotype	Finland	–	–	JX993085	–	Helvella ulvinenii	Landeros et al. 2015
	HMAS 41140^*	Netherlands	MK652211	MK592148	–	–	Helvella queletii	This study
	HMAS 58371	Czech	MK652212	–	–	–	Helvella queletii	This study
	HMAS 27727^*	Qinghai, China	MK652209	MK592147	–	–	Helvella confusa	This study
	HMAS 27951	Jilin, China	MK652210	–	–	–	Helvella confusa	This study
	HMAS 73509	Sichuan, China	MK652213	MK592149	–	–	Helvella acetabulum	This study
	HMAS 75175^*	Sichuan, China	MK652214	MK592150	–	MK652164	Helvella leucomelaena	This study
Helvella taiyuanensis B. Liu, Du & J.Z. Cao	HMAS 85689^*, neotype	Shanxi, China	MK652217	MK592153	–	–	Helvella taiyuanensis	This study
	HMAS 277500^*	Yunnan, China	MK652216	MK592152	MK592105	MK652166	Helvella sp.	This study
	11925^*, HMAS 254611	Beijing, China	MK652215	MK592151	MK592104	MK652165		This study
	MCCNNU 6499^*, HMAS 279702	Hubei, China	MK652218	MK592154	MK592106	MK652167	Helvella solitaria	This study
Helvella tianshanensis X.C. Wang & W.Y. Zhuang	HMAS 86040^*, holotype	Xinjiang, China	MK652222	MK592157	MK592108	MK652170	Helvella costifera	This study
Helvella tianshanensis X.C. Wang & W.Y. Zhuang	HMAS 88611^*	Xinjiang, China	MK652223	MK592158	–	–	Helvella acetabulum	This study
Helvella tinta Q. Zhao, B. Feng & K.D. Hyde	HKAS 82560, holotype	Sichuan, China	–	KX239842	KX239772	–	Helvella tinta	Hyde et al. 2016
Helvella crispa (Scop.) Fr.	H408^*, epitype	Sweden	KY784504	–	–	–	Helvella crispa	Skrede et al. 2017
	H135	Norway	KY784268	–	KY772986	–	Helvella crispa	Skrede et al. 2017
	HKAS 75434	Germany	–	JX462572	KR493479	KT254487	Helvella crispa	Zhao et al. 2015
Helvella elastica Bull.	H066^*	Sweden	KY784230	–	KY772950	KY772858	Helvella elastica	Skrede et al. 2017
Helvella lacunosa Afzel.	H407, epitype	Sweden	KY784503	–	KY773152	–	Helvella lacunosa	Skrede et al. 2017
Helvella lacunosa Afzel.	H039^*	Norway	KY784213	–	KY772930	KY772845	Helvella lacunosa	Skrede et al. 2017
Helvella macropus (Pers.) P. Karst.	H412, epitype	Sweden	KY784507	–	–	–	Helvella macropus	Skrede et al. 2017
Helvella macropus (Pers.) P. Karst.	H073^*	Norway	KY784233	–	KY772954	KY772863	Helvella macropus	Skrede et al. 2017
Midotis lingua Fr.	H283^*	Switzerland	KY784397	–	KY773093	–	Wynnella silvicola	Skrede et al. 2017
	HMAS 67962^*	Germany	MK652224	MK592159	MK592109	–	Wynnella auricula	This study
	HMAS 71896^*	Shanxi, China	MK652225	MK592160	MK592110	–	Wynnella silvicola	This study
	HMAS 74656	Gansu, China	MK652226	MK592161	MK592111	–	Helvella silvicola	This study
	HMAS 83548	Xinjiang, China	MK652227	MK592162	–	–	Wynnella auricula	This study
Pindara terrestris Velen.	KH.12.67	Sweden	–	–	MK100279	MK113889	Pindara terrestris	Hansen et al. 2019
	S-F327988	Sweden	–	–	MK100280	MK113896	Pindara terrestris	Hansen et al. 2019
	T. Kekki 168	Finland	–	–	MK100281	MK113897	Pindara terrestris	Hansen et al. 2019
Underwoodia columnaris Peck	Kanouse 1951	USA	–	–	U42685	–	Underwoodia columnaris	O'Donnell et al. 1997

DNA extraction, PCR amplification and sequencing

Well-preserved specimens were selected for DNA extraction using a Plant Genomic DNA Kit (DP305, TIANGEN Biotech, Beijing, China). Partial Hsp90, ITS2, LSU and TEF1 were amplified by PCR using primers H_hspf and H_hspr (Skrede et al. 2017), ITS3 and ITS4 (White et al. 1990), LROR and LR5 (Vilgalys and Hester 1990) and EF1-983F and EF1-1567R (Rehner and Buckley 2005). Products were sequenced on an ABI 3730 DNA Sequencer (Applied Biosystems).

Phylogenetic analyses

Sequences obtained from this study and those retrieved from GenBank are listed in Table 3. Four single gene datasets and two combined datasets were compiled. Sequences were aligned using MAFFT 7.221 (Katoh and Standley 2013) and subsequently processed with BioEdit 7.1.10 (Hall 1999). A Maximum-Likelihood (ML) tree for each single gene data was generated using MEGA 6.0.6 (Tamura et al. 2013) with the most suitable nucleotide substitution model and 1,000 replicates of bootstrap (BP) tests. For the combined four-gene dataset, the ML tree was determined using RAxML-HPC2 on XSEDE 8.2.12 on CIPRES Science Gateway (Miller et al. 2010) with the default GTRCAT model. Bayesian Inference (BI) analysis was performed with MrBayes 3.2.6 (Ronquist et al. 2012) using a Markov Chain Monte Carlo (MCMC) algorithm. Appropriate nucleotide substitution models and parameters were determined via ModelTest 3.7 (Posada and Crandall 1998). The first 25% of the trees were excluded as the burn-in phase and posterior probability (PP) values were estimated with the remaining 75% of trees. Helvella crispa (Scop.) Fr., H. elastica Bull., H. lacunosa Afzel. and H. macropus (Pers.) P. Karst. are the representatives of the formerly recognised sections Crispae, Elasticae, Lacunosae and Macropodes, respectively. Midotis lingua Fr. served as the outgroup taxon of the four-gene phylogeny and Underwoodia columnaris Peck worked for the two-gene analysis.

Results

Fifty-one specimens of the rib-stiped cupulate species of Helvella s. l. deposited in HMAS and five recent collections were examined. A total of 125 sequences of the Helvella and Dissingia samples and 11 of the outgroup taxa were submitted to GenBank (Table 3).

The combined four-locus dataset included 48 taxa of Helvella s. s. and Dissingia in an alignment of 1788 bp, including 236 bp of Hsp90, 348 bp of ITS2, 690 bp of LSU and 514 bp of TEF1. Kimura 2-parameter (K2) with gamma distribution (+G) was determined as the most suitable model for ML analysis. Tamura-Nei with gamma distribution and invariant sites (TrN+I+G) was selected by Akaike Information Criterion as the best fit for the BI analysis. As shown in Figure 1, three clades and some independent lineages were recognised amongst the cupulate taxa of Helvella s. s. Clade 1 consisted of H. calycina, H. costifera and H. tianshanensis; Clade 2 included H. solitaria and H. taiyuanensis; and Clade 3 contained H. acetabuloides, H. acetabulum, H. arctoalpina, H. costata and H. sichuanensis. Helvella dryadophila, as an independent lineage, was sister to Clade 3, which was not supported by two of the single gene analyses (Suppl. material 1: Figures S1 and S4). Helvella griseoalba and H. hyperborea were situated outside the clades in all analyses.

Figure 1.

Bayesian phylogenetic tree of Helvella and Dissingia inferred from combined Hsp90, ITS2, LSU and TEF1 dataset. Posterior probability values ≥ 0.90 (left) and bootstrap values ≥ 70% (right) are indicated at nodes.

The combined LSU and TEF1 dataset was comprised of 38 taxa of Balsamia, Dissingia, Helvella, Midotis, Pindara and Underwoodia. The alignment is of 1239 bp, including 711 bp of LSU and 528 bp of TEF1. Tamura-Nei with gamma distribution (TN93+G) was determined as the most suitable model for ML analysis. Clades 1–3 were supported and H. dryadophila was outside Clade 3 (Figure 2), which are congruent with the four-gene analysis (Figure 1).

Figure 2.

Maximum likelihood phylogeny of Helvellaceae inferred from combined LSU and TEF1 dataset. Bootstrap values ≥ 50% are indicated at nodes.

The Hsp90 dataset consisted of 84 sequences of Helvella and Dissingia. K2+G was determined as the most suitable model for ML analysis. Clades 2 and 3 were monophyletic, but Clade 1 was poorly supported (Suppl. material 1: Figure S1). The positions of the three undescribed species were consistent with that of the four-locus phylogeny.

The ITS2 dataset possessed 53 taxa of Helvella and Dissingia. Tamura 3-parameter with gamma distribution (T92+G) was determined as the most suitable model for ML analysis. Clades 1–3 were strongly supported. Helvella tinta, excluded from these clades, appeared to be sister of H. hyperborea (Suppl. material 1: Figure S2).

The LSU dataset comprised 40 sequences of Helvella and Dissingia. TN93+G was determined as the most suitable model for ML analysis. Clades 1–3 of Helvella were monophyletic, in which H. floriforma and H. robusta, absent in other trees, were located. Dissingia seemed to be not monophyletic (Suppl. material 1: Figure S3).

The TEF1 dataset consisted of 26 taxa of Helvella and Dissingia. K2+G was determined as the most suitable model for ML analysis. Clades 1–3 of Helvella were strongly supported (Suppl. material 1: Figure S4) and the phylogenetic positions of the three undescribed species recalled that of the multigene phylogeny (Figure 1).

Taxonomy

New species

Helvella acetabuloides X.C. Wang & W.Y. Zhuang, sp. nov.

Fungal Names: FN 570634

Figure 3a–d

Holotype

CHINA. Inner Mongolia Autonomous Region, Chifeng City, Harqin Banner, Shijia Town, Toudaoyingzi Village, 41°53'20"N, 119°1'1"E, on the ground under Ostryopsis davidiana Decne., 8 Aug 2002, T.Z. Liu & T.H. Liu, HMAS 279703 (= CFSZ 2044).

Etymology

The species epithet refers to its similarity to H. acetabulum.

Description

Apothecia stipitate to subsessile, cupulate, margin undulate, involute or revolute, 2.2–4.8 cm high and 2.5–4 cm diam. when dry; hymenium dull brown to reddish-brown when dry, receptacle surface light brown to brown when dry, glabrous; stipe terete or flattened, buff, light yellowish-brown to brown, surface ribbed, 0.5–3 × 0.4–1.3 cm, typically fluted with sharp-edged or rarely blunt ribs, ribs branching at the upper half of receptacle surface, reaching to the edge or ending 1–2 mm from the edge. Ectal excipulum of textura angularis, 75–100 µm thick, cells hyaline, outer cells arranged in chains, 16–21.5 × 7–8 µm. Medullary excipulum of textura intricata, 180–220 µm thick, hyphae hyaline. Asci subcylindrical, tapering and with crozier at base, 8-spored, 235–280 × 15–20 µm. Paraphyses filiform with apical portion very slightly enlarged, septate, hyaline, 4.5–5.5 µm wide at apex and 4–4.5 µm below. Ascospores ellipsoidal, hyaline, smooth, uniguttulate, 14–20 × 10–14.5 µm, median 16.2 × 12.3 µm, Q = 1.2–1.55, median 1.375, n = 50.

Additional specimen examined

CHINA. Shaanxi Province, Baoji City, Taibai County, Mt. Taibai, 34°1'53"N, 107°25'33"E, alt. 2270 m, on the ground in broad-leaf forest, 26 Jun 1958, J.H. Yu 106, HMAS 23842.

Notes

Helvella acetabuloides is nested with H. acetabulum, H. arctoalpina, H. costata and H. sichuanensis in Clade 3 (Figure 1). Its hymenium is reddish-brown when dry and different from that of H. acetabulum (brown when dry) and those of H. arctoalpina and H. sichuanensis (black when dry, Harmaja 1977b). The two specimens cited are identical in sequences of Hsp90. Helvella acetabuloides differs from H. acetabulum in 6 bp for Hsp90 (H410, epitype), 14 bp for ITS2 (HMAS 243823) and 17 bp for TEF1 (H133). It is distinguished from H. arctoalpina in 2 bp of Hsp90 (H293, holotype) and 11 bp of TEF1 (H033), from H. costata in 3 bp of Hsp90. It differs from H. sichuanensis in 1 bp of Hsp90, 20 bp of ITS2 and 11 bp of TEF1. PCR amplification of LSU failed.

Figure 3.

a–d Helvella acetabuloides: a mature apothecia when dry (CFSZ 2044) b asci (HMAS 23842) c, d ascospores in ascus (c: CFSZ 2044, d: HMAS 23842) e–g Helvella sichuanensis (HMAS 254610): e mature apothecia when dry f mature apothecia when fresh g ascospores in asci h–k Helvella tianshanensis (HMAS 86040): h, i Mature apothecium when dry j asci k ascospores in ascus. Scale bars: 1 cm (a, e); 0.75 cm (h, i); 50 μm (b, g, j); 20 μm (c, d, k).

Helvella sichuanensis X.C. Wang & W.Y. Zhuang, sp. nov.

Fungal Names: FN 570635

Figure 3e–g

Holotype

CHINA. Sichuan Province, Garzê Tibetan Autonomous Prefecture, Daocheng County, Yading National Nature Reserve, 28°25'6"N, 100°21'26"E, alt. 3900 m, on the ground of mixed forest, 18 Aug 2016, J.P. Wang & X.C. Wang 10706, HMAS 254610.

Etymology

The species epithet refers to the type locality of the fungus.

Description

Apothecia stipitate, shallow-cupulate, margin entire and flattened when fresh, undulate, involute or revolute when dry, 5–6 cm diam. when fresh and 2.5–3.5 cm high when dry; hymenium yellowish-brown when fresh, nearly black when dry, receptacle surface buff to light brown when fresh, light brown to dark brown when dry, glabrous; stipe terete or flattened, buff to light brown, surface ribbed, 2.5–3 × 1.5–3 cm when fresh, 2–2.5 × 0.5–1.5 cm when dry, typically fluted with sharp-edged or rarely blunt ribs, ribs branching at the upper half of receptacle surface, reaching to the edge or ending 3–5 mm from the edge. Ectal excipulum of textura angularis, 100–180 µm thick, cells hyaline to light brown, outer cells 15–45 × 9–35 µm. Medullary excipulum of textura intricata, 300–500 µm thick, hyphae hyaline. Asci subcylindrical, tapering and with crozier at base, 8-spored, 225–325 × 13–18.5 µm. Paraphyses filiform with apical portion obviously swollen, septate, hyaline to light brown, 7–10.5 µm wide at apex and 3–4.5 µm below. Ascospores ellipsoidal, hyaline, smooth, uniguttulate, 15.5–18.5 × 10–12.5 µm, median 16.9 × 11.2 µm, Q = 1.3–1.7, median 1.48, n = 40.

Notes

Helvella sichuanensis belongs to Clade 3 (Figure 1). Its hymenium is nearly black when dry, which is similar to that of H. arctoalpina, but different from those in H. acetabulum (brown when dry) and H. acetabuloides (reddish-brown when dry). When fresh, the hymenium is yellowish-brown, while that of H. arctoalpina is brown. Molecularly, it differs from H. acetabulum in 7 bp of Hsp90 (H410, epitype), 14 bp of ITS2 (HMAS 243823), 17 bp of LSU (H133) and 15 bp of TEF1 (H133); from H. arctoalpina in 1 bp of Hsp90 (H293, holotype), 25 bp of LSU (H033) and 11 bp of TEF1 (H033); and from H. costata in 2 bp of Hsp90 and 13 bp of LSU. The sequence divergences between H. sichuanensis and H. acetabuloides are 1 bp of Hsp90, 20 bp of ITS2 and 12 bp of TEF1.

Helvella tianshanensis X.C. Wang & W.Y. Zhuang, sp. nov.

Fungal Names: FN 570636

Figure 3h–k

Holotype

CHINA. Xinjiang Uygur Autonomous Region, Changji Hui Autonomous Prefecture, Jimsar County, 43°59'44"N, 89°10'31"E, alt. 1700 m, on the ground, 31 Jul 2003, W.Y. Zhuang & Y. Nong 4661, HMAS 86040.

Etymology

The species epithet refers to the type locality of the fungus.

Description

Apothecia stipitate, cupulate, margin undulate, involute, 2.5–3.5 cm high and 2–3 cm diam. when dry; hymenium greyish-brown, brown to dark brown, receptacle surface yellowish-brown to brown; stipe terete or flattened, buff, yellowish-brown, orange brown to brown, surface ribbed, 2–2.5 × 0.5–1.3 cm, typically fluted with rarely blunt ribs, ribs branching at the upper half of receptacle surface, reaching to the edge or ending 3–12 mm from the edge. Ectal excipulum of textura angularis, 120–150 µm thick, hyphae hyaline, outer cells 35–40 × 20–40 µm. Medullary excipulum of textura intricata, 350–600 µm thick, hyphae hyaline. Asci subcylindrical, tapering and with crozier at base, 8-spored, 240–275 × 12–24 µm. Paraphyses filiform, slightly enlarged at apical portion, septate, hyaline to light brown, 6–7.5 µm wide at apex and 3–4.5 µm below. Ascospores ellipsoidal, hyaline, smooth, uniguttulate, 17–21 × 11.5–13.5 µm, median 18.8 × 12.3 µm, Q = 1.35–1.7, median 1.51, n = 30.

Additional specimen examined

CHINA. Xinjiang Uygur Autonomous Region, Urumqi City, Urumqi County, 43°28'47"N, 87°27'27"E, 12 Aug 1985, L. Fan & K. Tao 161, HMAS 88611.

Notes

Helvella tianshanensis nested with H. calycina and H. costifera in Clade 1 (Figure 1). These three species are hardly separated by gross morphology and anatomic structures. Helvella tianshanensis differs from H. calycina in 4 bp of Hsp90 (H022, epitype), 16 bp of ITS2 (HMAS 279704), 9 bp of LSU (H022) and 15 bp of TEF1 (H022); and it is different from H. costifera in 3 bp of Hsp90 (H298, epitype), 12 bp of ITS2 (HMAS 187120), 11 bp of LSU (H131) and 13 bp of TEF1 (H131). The two specimens of the new species are identical in Hsp90 and ITS2.

New Chinese record

Helvella calycina Skrede, T.A. Carlsen & T. Schumach., Persoonia 39: 221, 2017

Specimen examined

CHINA. Inner Mongolia Autonomous Region, Xilingol League, Zhenglan Banner, Yihehaierhan Sumu, 42°23'8"N, 116°10'17"E, 21 August 2005, on the ground, T.Z. Liu & X.L. Bai, HMAS 279704 (= CFSZ 2658).

Notes

Helvella calycina is a new record for China. It was known only from Norway and Denmark. The Chinese collection extends its distribution to Asia. The Chinese collection is identical with the epitype in TEF1 but with 2 bp differences for Hsp90 and 1 bp for LSU.

Neotypification

Helvella taiyuanensis B. Liu, Du & J.Z. Cao, Acta Mycol. Sin. 4(4): 211, 1985

Fungal Names: FN 570637

Figure 4

Neotype is designated here

CHINA. Shanxi Province, Lvliang City, Jiaocheng County, Guandishan National Forest Park, 37°54'25"N, 111°35'40"E, on the ground in mixed forest, 16 Jul 1987, Y.M. Li, HMAS 85689 (= MHSU 758).

Additional specimens examined

CHINA. Beijing City, Mentougou District, Xiaolongmen National Forest Park, 39°58'2"N, 115°26'43"E, alt. 1100 m, on the ground in mixed forest, 4 Aug 2018, X.C. Wang et al. 11925, HMAS 254611. Hubei Province, Yichang City, Xingshan County, Longmenhe National Forest Park, 31°21'12"N, 110°30'40"E, on the ground, 23 Jul 2017, R. Wang & X. Zhang 420526MF0679, MCCNNU 6499, HMAS 279702. Yunnan Province, Diqing Tibetan Autonomous Prefecture, Dêqên County, Yunling Town, Meili Snow Mountain, 28°23'23"N, 98°47'49"E, alt. 3150 m, on the ground, 12 Aug 2016, Y. Li 920, HMAS 277500.

Notes

This species was originally described, based on a single specimen collected by Y.M. Li from Taiyuan City, Shanxi Province in 1983 (Holotype: HBSU 2449, Liu et al. 1985). Unfortunately, the type specimen was destroyed by a fire in MHSU in 1984 (Cao 1988, Cao et al. 1990). To protect fungal collections after the fire, the remaining specimens, deposited in MHSU, were moved to HMAS. The neotype specimen HMAS 85689 was collected by the same collector as the type specimen of H. taiyuanensis and identified by one of the original authors J.Z. Cao (Cao 1988). Its detailed morphological characteristics are in accordance with the original description. We thus treat it as authentic material. As other specimens were neither cited in the protologue nor filed under this name, we thus designate HMAS 85689 as the neotype specimen of H. taiyuanensis.

Helvella taiyuanensis was once treated as a synonym of H. solitaria sensu Dissing (1966), based on morphological features (Cao 1988), but the molecular differences between them are clear in the multigene analysis (Figure 1). It should be a tenable species. The four specimens of the fungus examined are variable in colour of the hymenium and receptacle surface when dry or fresh, but stable in cupulate to saddle-shaped apothecia (Figure 4). Phylogenetic analyses indicate that they belong to the same species (Figures 1, 2 and Suppl. material 1: S1–S4) although minor sequence divergences exist amongst collections. The maximum sequence divergences amongst collections are 1 bp in Hsp90, 6 bp in ITS2, 3 bp in LSU and 7 bp in TEF1.

Figure 4.

Helvella taiyuanensis a specimen sheet (HMAS 85689) b mature apothecia when dry (HMAS 85689) c mature apothecia when fresh (HMAS 254611) d mature apothecia when fresh (HMAS 277500) e mature apothecium when fresh (HMAS 279702) f–h ascospores in ascus (f, g: HMAS 85689, h: HMAS 254611). Scale bars: 0.8 cm (b, d); 2 cm (c); 20 μm (f), applies to g, h.

Discussion

A total of about 28 rib-stiped cupulate species of Helvella and Dissingia have been reported in the world (Table 2) and 17 of them were investigated in this study. With the discovery of the three new species and one new record, 13 species were confirmed to be distributed in China. Amongst them, six are known only from China, five (D. oblongispora, H. acetabulum, H. calycina, H. costifera and H. hyperborea) are found in Europe and China and D. confusa and H. solitaria are widespread in Europe, Asia and North America. Amongst the Chinese helvellas, H. acetabulum, H. costifera and H. taiyuanensis show a relatively wide distribution range and occur in at least four provinces. However, H. calycina, H. floriforma, H. sichuanensis, H. tianshanensis and H. tinta were known only from a single locality. Eight species are in northwest China (Gansu, Qinghai, Shaanxi and Xinjiang), eight in the southwest (Sichuan and Yunnan) and seven in the north (Beijing, Inner Mongolia and Shanxi). However, the Chinese record of H. leucomelaena (≡ D. leucomelaena) (Teng 1963, Tai 1979, Zhuang 1998) is questionable since many specimens in HMAS, filed under that name, were based on misidentifications (Table 3).

As shown in the multigene phylogeny (Figure 1), three clades were formed amongst the investigated species. The cupulate Helvella taxa are clustered or mixed with the saddle-shaped ones. This gives the hint that the apothecial shape changed several times during the evolution. Clade 2, Clade 3 and H. dryadophila belong to the acetabulum-solitaria lineage (Skrede et al. 2017); however, this lineage was not herein supported due to joining of the non-cupulate species H. crispa. Clade 1 is in accordance with the costifera lineage (Skrede et al. 2017) with the addition of H. tianshanensis. Our results clearly support the separation of Dissingia from Helvella s. l. (Hansen et al. 2019).

Supplementary DNA barcodes are essential for delimitation of Helvella species. LSU is the most commonly used region for Helvella species identification (Nguyen et al. 2013, Landeros et al. 2015, Skrede et al. 2017). LSU is capable of distinguishing cupulate Helvella species (Suppl. material 1: Figure S3); whereas, its PCR amplification success rate is low (10/56), especially for specimens subject to long storage. A similar situation is witnessed in TEF1, which was suggested as a secondary barcode for fungi (Stielow et al. 2015). Although the primers for this region were reported working well on DNAs extracted from fresh materials, the amplifications from dried Helvella specimens were not easy (Skrede et al. 2017). The amplification success rate of TEF1 in our study was again low (15/56). Hsp90 was first applied to Helvella by Skrede et al. (2017) and is recommended due to its short sequence length, high amplification success rate, usefulness in species delimitation and its reasonable phylogenetic informative properties. It was successfully amplified from 53 of the 56 specimens studied and is able to distinguish all the involved species (Suppl. material 1: Figure S1). RPB2 was also applied in the recent studies (Skrede et al. 2017, Hansen et al. 2019), but did not work well since the amplicons of the newly designed primers, H_rpb2r2 and H_rpb2f, had a lower species resolution than that of Hsp90. The fragment is also too short to align with the existing sequences in GenBank.

ITS is recommended as the universal barcode for fungi (Schoch et al. 2012), which is applied widely to elucidate species diversity of the pezizalean ectomycorrhizae (Tedersoo et al. 2006, Healy et al. 2013, Hwang et al. 2015). However, very limited ITS sequences of cupulate Helvella species were available in GenBank. The trials of obtaining ITS amplicons, using the universal primers for many Helvella species, usually failed owing to primer mismatch (Skrede et al. 2017). The success rate of ITS amplification in our work was extremely low (2/56) upon using the primer pairs ITS5 and ITS4. Functional Helvella-specific ITS primers are expected to be developed. Our amplifications of the ITS2 region by the primers ITS3 and ITS4 reached a relative high success rate (47/56) with the tested species well separated (Suppl. material 1: Figure S2). We thus propose to use Hsp90 and ITS2 as supplementary DNA barcodes for rib-stiped cupulate species of Helvella.

Acknowledgements

We are very grateful to Prof. Jie-Ping Wang (Fujian Academy of Agricultural Sciences) and Mr. Xian Zhang (Nanjing Normal University) for sending the specimen and photograph of a Helvella species as gift.

This work was supported by the National Natural Science Foundation of China (nos. 31750001, 31760004) and Key Research Program of Frontier Science, Chinese Academy of Sciences (QYZDY-SSW-SMC029).

References

Abbott SP, Currah RS (1988) The genus Helvella in Alberta. Mycotaxon 33: 229–250.

Abbott SP, Currah RS (1997) The Hevellaceae: systematic revision and occurrence in northern and northwestern North America. Mycotaxon 62: 1–125.

Ariyawansa HA, Hyde KD, Jayasiri SC, Buyck B, Chethana KWT, Dai DQ, Dai YC, Daranagama DA, Jayawardena RS, Lücking R, Ghobad-Nejhad M, Niskanen T, Thambugala KM, Voigt K, Zhao RL, Li G-J, Doilom M, Boonmee S, Yang ZL, Cai Q, Cui Y-Y, Bahkali AH, Chen J, Cui BK, Chen JJ, Dayarathne MC, Dissanayake AJ, Ekanayaka AH, Hashimoto A, Hongsanan S, Jones EBG, Larsson E, Li WJ, Li Q-R, Liu JK, Luo ZL, Maharachchikumbura SSN, Mapook A, McKenzie EHC, Norphanphoun C, Konta S, Pang KL, Perera RH, Phookamsak R, Phukhamsakda C, Pinruan U, Randrianjohany E, Singtripop C, Tanaka K, Tian CM, Tibpromma S, Abdel-Wahab MA, Wanasinghe DN, Wijayawardene NN, Zhang J-F, Zhang H, Abdel-Aziz FA, Wedin M, Westberg M, Ammirati JF, Bulgakov TS, Lima DX, Callaghan TM, Callac P, Chang C-H, Coca LF, Dal-Forno M, Dollhofer V, Fliegerová K, Greiner K, Griffith GW, Ho H-M, Hofstetter V, Jeewon R, Kang JC, Wen T-C, Kirk PM, Kytövuori I, Lawrey JD, Xing J, Li H, Liu ZY, Liu XZ, Liimatainen K, Lumbsch HT, Matsumura M, Moncada B, Nuankaew S, Parnmen S, de Azevedo Santiago ALCM, Sommai S, Song Y, de Souza CAF, de Souza-Motta CM, Su HY, Suetrong S, Wang Y, Wei S-F, Wen TC, Yuan HS, Zhou LW, Réblová M, Fournier J, Camporesi E, Luangsa-ard JJ, Tasanathai K, Khonsanit A, Thanakitpipattana D, Somrithipol S, Diederich P, Millanes AM, Common RS, Stadler M, Yan JY, Li XH, Lee HW, Nguyen TTT, Lee HB, Battistin E, Marsico O, Vizzini A, Vila J, Ercole E, Eberhardt U, Simonini G, Wen H-A, Chen X-H, Miettinen O, Spirin V (2015) Fungal diversity notes 111–252–taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity 75: 27–274. https://doi.org/10.1007/s13225-015-0346-5

Bonito G, Smith ME, Nowak M, Healy RA, Guevara G, Cazares E, Kinoshita A, Nouhra ER, Dominguez LS, Tedersoo L, Murat C, Wang Y, Moreno BA, Pfister DH, Nara K, Zambonelli A, Trappe JM, Vilgalys R (2013) Historical biogeography and diversification of truffles in the Tuberaceae and their newly identified southern hemisphere sister lineage. PLoS One 8: e52765. https://doi.org/10.1371/journal.pone.0052765

Cao JZ (1988) The genus Helvella in China. MSc Thesis, Shanxi University, China.

Cao JZ, Fan L, Liu B (1990) Some new species and new records of the genus Helvella from China II. Acta Mycologica Sinica 9: 184–190.

Cao JZ, Liu B (1990) A new species of Helvella from China. Mycologia 82: 642–643. https://doi.org/10.1080/00275514.1990.12025938

Dissing H (1966) The genus Helvella in Europe, with special emphasis on the species found in Norden. Dansk Botanisk Arkiv 25: 1–172.

Häffner J (1987) Die Gattung Helvella: morphologie und taxonomie. Beihefte zur Zeitschrift für Mykologie 7: 1–165.

Hall TA (1999) BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.

Hansen K, Perry BA, Dranginis AW, Pfister DH (2013) A phylogeny of the highly diverse cup-fungus family Pyronemataceae (Pezizomycetes, Ascomycota) clarifies relationships and evolution of selected life history traits. Molecular Phylogenetics and Evolution 67: 311–335. https://doi.org/10.1016/j.ympev.2013.01.014

Hansen K, Pfister DH (2006) Systematics of the Pezizomycetes–the operculate discomycetes. Mycologia 98: 1029–1040. https://doi.org/10.1080/15572536.2006.11832631

Hansen K, Schumacher T, Skrede I, Huhtinen S, Wang XH (2019) Pindara revisited – evolution and generic limits in Helvellaceae. Persoonia 42: 186–204. https://doi.org/10.3767/persoonia.2019.42.07

Harmaja H (1976) New species and combinations in the genera Gyromitra, Helvella and Otidea. Karstenia 15: 29–32. https://doi.org/10.29203/ka.1976.111

Harmaja H (1977a) A note on Helvella solitaria (syn. H. queletii) and H. confusa n. sp. Karstenia 17: 40–44. https://doi.org/10.29203/ka.1977.123

Harmaja H (1977b) A revision of the Helvella acetabulum group (Pezizales) in Fennoscandia. Karstenia 17: 45–48. https://doi.org/10.29203/ka.1977.124

Harmaja H (1978) New species and combinations in Helvella and Gyromitra. Karstenia 18: 1–57. https://doi.org/10.29203/ka.1978.139

Harmaja H (1979) Studies on cupulate species of Helvella. Karstenia 19: 33–45. https://doi.org/10.29203/ka.1979.184

Healy RA, Smith ME, Bonito GM, Pfister DH, Ge ZW, Guevara GG, Williams G, Stafford K, Kumar L, Lee T, Hobart C, Trappe J, Vilgalys R, Mclaughlin DJ (2013) High diversity and widespread occurrence of mitotic spore mats in ectomycorrhizal Pezizales. Molecular Ecology 22: 1717–1732. https://doi.org/10.1111/mec.12135

Hwang J, Zhao Q, Yang ZL, Wang Z, Townsend JP (2015) Solving the ecological puzzle of mycorrhizal associations using data from annotated collections and environmental samples–an example of saddle fungi. Environmental Microbiology Reports 7: 658–667. https://doi.org/10.1111/1758-2229.12303

Hyde KD, Hongsanan S, Jeewon R, Bhat DJ, McKenzie EHC, Jones EBG, Phookamsak R, Ariyawansa HA, Boonmee S, Zhao Q, Abdel-Aziz FA, Abdel-Wahab MA, Banmai S, Chomnunti P, Cui B-K, Daranagama DA, Das K, Dayarathne MC, de Silva NI, Dissanayake AJ, Doilom M, Ekanayaka AH, Gibertoni TB, Góes-Neto A, Huang S-K, Jayasiri SC, Jayawardena RS, Konta S, Lee HB, Li W-J, Lin C-G, Liu J-K, Lu Y-Z, Luo Z-L, Manawasinghe IS, Manimohan P, Mapook A, Niskanen T, Norphanphoun C, Papizadeh M, Perera RH, Phukhamsakda C, Richter C, de A Santiago ALCM, Drechsler-Santos ER, Senanayake IC, Tanaka K, Tennakoon TMDS, Thambugala KM, Tian Q, Tibpromma S, Thongbai B, Vizzini A, Wanasinghe DN, Wijayawardene NN, Wu H-X, Yang J, Zeng X-Y, Zhang H, Zhang J-F, Bulgakov TS, Camporesi E, Bahkali AH, Amoozegar MA, Araujo-Neta LS, Ammirati JF, Baghela A, Bhatt RP, Bojantchev D, Buyck B, da Silva GA, de Lima CLF, de Oliveira RJV, de Souza CAF, Dai Y-C, Dima B, Duong TT, Ercole E, Mafalda-Freire F, Ghosh A, Hashimoto A, Kamolhan S, Kang J-C, Karunarathna SC, Kirk PM, Kytövuori I, Lantieri A, Liimatainen K, Liu Z-Y, Liu X-Z, Lücking R, Medardi G, Mortimer PE, Nguyen TTT, Promputtha I, Raj KNA, Reck MA, Lumyong S, Shahzadeh-Fazeli SA, Stadler M, Soudi MR, Su H-Y, Takahashi T, Tangthirasunun N, Uniyal P, Wang Y, Wen T-C, Xu J-C, Zhang Z-K, Zhao Y-C, Zhou J-L, Zhu L (2016) Fungal diversity notes 367–490: taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity 80: 1–270. https://doi.org/10.1007/s13225-016-0373-x

Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010

Laessoe T, Hansen K (2007) Truffle trouble: what happened to the Tuberales? Mycological Reseach 111: 1075–1099. https://doi.org/10.1016/j.mycres.2007.08.004

Landeros F, Iturriaga T, Guzmán-Dávalos L (2012) Type studies in Helvella (Pezizales) 1. Mycotaxon 119: 35–63. https://doi.org/10.5248/119.35

Landeros F, Iturriaga T, Rodríguez A, Vargas-Amado G, Guzmán-Dávalos L (2015) Advances in the phylogeny of Helvella (Fungi: Ascomycota), inferred from nuclear ribosomal LSU sequences and morphological data. Revista Mexicana de Biodiversidad 86: 856–871. https://doi.org/10.1016/j.rmb.2015.09.005

Landvik S, Kristiansen R, Schumacher T (1999) Pindara: a miniature Helvella. Mycologia 91: 278–285. https://doi.org/10.1080/00275514.1999.12061018

Liu B, Cao JZ (1988) Some new species and new records of the genus Helvella from China (I). Acta Mycologica Sinica 7: 198–204.

Liu B, Du F, Cao JZ (1985) New species and new combination of the genus Helvella. Acta Mycologica Sinica 4: 208–217.

Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, 1–8. https://doi.org/10.1109/GCE.2010.5676129

Nguyen NH, Landeros F, Garibay-Orijel R, Hansen K, Vellinga EC (2013) The Helvella lacunosa species complex in western North America: cryptic species, misapplied names and parasites. Mycologia 105: 1275–1286. https://doi.org/10.3852/12-391

O’Donnell K, Cigelnik E, Weber NS, Trappe JM (1997) Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89: 48–65. https://doi.org/10.1080/00275514.1997.12026754

Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817–818. https://doi.org/10.1093/bioinformatics/14.9.817

Rehner SA, Buckley E (2005) A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97: 84–98. https://doi.org/10.1080/15572536.2006.11832842

Ronquist F, Teslenko M, Van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029

Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Fungal Barcoding C (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America 109: 6241–6246. https://doi.org/10.1073/pnas.1117018109

Shameem N, Kamili AN, Ahmad M, Masoodi FA, Parray JA (2016) Antioxidant potential and DNA damage protection by the slate grey saddle mushroom, Helvella lacunosa (Ascomycetes), from Kashmir Himalaya (India). International Journal of Medicinal Mushrooms 18: 631–636. https://doi.org/10.1615/IntJMedMushrooms.v18.i7.80

Skrede I, Carlsen T, Schumacher T (2017) A synopsis of the saddle fungi (Helvella: Ascomycota) in Europe–species delimitation, taxonomy and typification. Persoonia 39: 201–253. https://doi.org/10.3767/persoonia.2017.39.09

Stielow JB, Levesque CA, Seifert KA, Meyer W, Iriny L, Smits D, Renfurm R, Verkley GJ, Groenewald M, Chaduli D, Lomascolo A, Welti S, Lesage-Meessen L, Favel A, Al-Hatmi AM, Damm U, Yilmaz N, Houbraken J, Lombard L, Quaedvlieg W, Binder M, Vaas LA, Vu D, Yurkov A, Begerow D, Roehl O, Guerreiro M, Fonseca A, Samerpitak K, van Diepeningen AD, Dolatabadi S, Moreno LF, Casaregola S, Mallet S, Jacques N, Roscini L, Egidi E, Bizet C, Garcia-Hermoso D, Martin MP, Deng S, Groenewald JZ, Boekhout T, de Beer ZW, Barnes I, Duong TA, Wingfield MJ, de Hoog GS, Crous PW, Lewis CT, Hambleton S, Moussa TA, Al-Zahrani HS, Almaghrabi OA, Louis-Seize G, Assabgui R, McCormick W, Omer G, Dukik K, Cardinali G, Eberhardt U, de Vries M, Robert V (2015) One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes. Persoonia 35: 242–263. https://doi.org/10.3767/003158515X689135

Tai FL (1979) Sylloge Fungorum Sinicorum. Science Press, 1527 pp.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725–2729. https://doi.org/10.1093/molbev/mst197

Tedersoo L, Hansen K, Perry BA, Kjoller R (2006) Molecular and morphological diversity of pezizalean ectomycorrhiza. New Phytologist 170: 581–596. https://doi.org/10.1111/j.1469-8137.2006.01678.x

Teng SC (1963) Fungi of China. Science Press, 808 pp. https://doi.org/10.1136/bmj.1.5333.808

Tibpromma S, Hyde KD, Jeewon R, Maharachchikumbura SSN, Liu J-K, Bhat DJ, Jones EBG, McKenzie EHC, Camporesi E, Bulgakov TS, Doilom M, de Azevedo Santiago ALCM, Das K, Manimohan P, Gibertoni TB, Lim YW, Ekanayaka AH, Thongbai B, Lee HB, Yang J-B, Kirk PM, Sysouphanthong P, Singh SK, Boonmee S, Dong W, Raj KNA, Latha KPD, Phookamsak R, Phukhamsakda C, Konta S, Jayasiri SC, Norphanphoun C, Tennakoon DS, Li J, Dayarathne MC, Perera RH, Xiao Y, Wanasinghe DN, Senanayake IC, Goonasekara ID, de Silva NI, Mapook A, Jayawardena RS, Dissanayake AJ, Manawasinghe IS, Chethana KWT, Luo Z-L, Hapuarachchi KK, Baghela A, Soares AM, Vizzini A, Meiras-Ottoni A, Mešić A, Dutta AK, de Souza CAF, Richter C, Lin C-G, Chakrabarty D, Daranagama DA, Lima DX, Chakraborty D, Ercole E, Wu F, Simonini G, Vasquez G, da Silva GA, Plautz HL, Ariyawansa HA, Lee H, Kušan I, Song J, Sun J, Karmakar J, Hu K, Semwal KC, Thambugala KM, Voigt K, Acharya K, Rajeshkumar KC, Ryvarden L, Jadan M, Hosen MI, Mikšík M, Samarakoon MC, Wijayawardene NN, Kim NK, Matočec N, Singh PN, Tian Q, Bhatt RP, de Oliveira RJV, Tulloss RE, Aamir S, Kaewchai S, Marathe SD, Khan S, Hongsanan S, Adhikari S, Mehmood T, Bandyopadhyay TK, Svetasheva TY, Nguyen TTT, Antonín V, Li W-J, Wang Y, Indoliya Y, Tkalčec Z, Elgorban AM, Bahkali AH, Tang AMC, Su H-Y, Zhang H, Promputtha I, Luangsa-ard J, Xu J, Yan J, Ji-Chuan K, Stadler M, Mortimer PE, Chomnunti P, Zhao Q, Phillips AJL, Nontachaiyapoom S, Wen T-C, Karunarathna SC (2017) Fungal diversity notes 491–602: taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity 83: 1–261. https://doi.org/10.1007/s13225-017-0378-0

Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4239–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990

Wang M, Zhao YC, Zhao Q, Zhou DQ (2016) Helvella sublactea sp. nov. (Helvellaceae) from southwestern China. Phytotaxa 253: 131–138. https://doi.org/10.11646/phytotaxa.253.2.2

Wang XC, Zhuang WY (2019) A three-locus phylogeny of Gyromitra (Discinaceae, Pezizales) and discovery of two cryptic species. Mycologia 111: 69–77. https://doi.org/10.1080/00275514.2018.1515456

Wang YZ, Chen CM (2002) The genus Helvella in Taiwan. Fungal Science 17: 11–17.

Weber NS (1972) The genus Helvella in Michigan. The Michigan Botanist 11: 147–201.

Weber NS (1975) Notes on western species of Helvella. I. Nova Hedwigia 51: 25–38.

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, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

Xu AS (2002) Notes on Helvella in Xizang. Mycosystema 21: 188–191.

Yu ZH, Zhuang WY, Chen SL, Decock C (2000) Preliminary survey of discomycetes from the Changbai Mountains, China. Mycotaxon 75: 395–408.

Zhao Q, Sulayman M, Zhu XT, Zhao YC, Yang ZL, Hyde KD (2016a) Species clarification of the culinary Bachu mushroom in western China. Mycologia 108: 828–836. https://doi.org/10.3852/16-002

Zhao Q, Tolgor B, Zhao Y, Yang ZL, Hyde KD (2015) Species diversity within the Helvella crispa group (Ascomycota: Helvellaceae) in China. Phytotaxa 239: 130–142. https://doi.org/10.11646/phytotaxa.239.2.2

Zhao Q, Zhang X, Li S, Chai H, Bahkali AH, Hyde KD (2016b) New species and records of saddle fungi (Helvella, Helvellaceae) from Jiuzhaigou Natural Reserve, China. Mycoscience 57: 422–430. https://doi.org/10.1016/j.myc.2016.07.005

Zhuang WY (1989) Some common discomycetes in Shennongjia, Hubei Province. In: Academia Sinica (Eds) Fungi and Lichens of Shennongjia. World Publishing Corp, Beijing, 98–106.

Zhuang WY (1995) Some new species and new records of discomycetes in China. V. Mycotaxon 56: 31–40.

Zhuang WY (1996) Some new species and new records of discomycetes in China. VI. Mycotaxon 59: 337–342.

Zhuang WY (1997) Fungal flora of the Daba Mountains: Discomycetes. Mycotaxon 61: 3–12.

Zhuang WY (1998) Notes on discomycetes from Qinghai Province, China. Mycotaxon 66: 439–444.

Zhuang WY (2004) Preliminary survey of the Helvellaceae from Xinjiang, China. Mycotaxon 90: 35–42.

Zhuang WY, Wang Z (1998a) Discomycetes of tropical China. I. Collections from Hainan Island. Mycotaxon 67: 21–31.

Zhuang WY, Wang Z (1998b) Some new species and new records of discomycetes in China. VIII. Mycotaxon 66: 429–438.

Zhuang WY, Yang ZL (2008) Some pezizalean fungi from alpine areas of southwestern China. Mycologia Montenegrina 10: 235–249.

Zhuang WY, Zheng HD, Zeng ZQ (2018) Species catalogue of China. Volume 3 Fungi. Cup-Fungi. Science Press, 1–142.

Supplementary material

Supplementary material 1

Figures S1–S4

Xin-Cun Wang, Tie-Zhi Liu, Shuang-Lin Chen, Yi Li, Wen-Ying Zhuang

Data type: phylogenetic data

Explanation note: Figure S1. Maximum-likelihood phylogenetic tree of Helvella and its allies inferred from Hsp90 dataset. Bootstrap values ≥ 50% are indicated at nodes. Figure S2. Maximum-likelihood phylogenetic tree of Helvella and its allies inferred from ITS2 dataset. Bootstrap values ≥ 50% are indicated at nodes. Figure S3. Maximum-likelihood phylogenetic tree of Helvella and its allies inferred from LSU dataset. Bootstrap values ≥ 50% are indicated at nodes. Figure S4. Maximum-likelihood phylogenetic tree of Helvella and its allies inferred from TEF1 dataset. Bootstrap values ≥ 50% are indicated at nodes.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

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