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

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

Introduction

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

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 , , , , , and . Amongst them, the sections and 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 species having ribbed stipes and their infrageneric positions are summarised in Table 2. Recently, sensu stricto was found to be associated with Vittad., K. Hansen, X.H. Wang & T. Schumach., Fr., Velen. and Peck in (Hansen and Pfister 2006; Hansen et al. 2019). Amongst them, was proposed to accommodate the species formerly placed in (Hansen et al. 2019).

Table 1.

Comparison of the taxonomic systems established in .

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 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
Section Elasticae Dissing	Section Elasticae Dissing	Section Elasticae Dissing	Subgenus Elasticae (Dissing) S.P. Abbott
Section Ephippium Dissing	Section Ephippium Dissing	Section Ephippium Dissing
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.

Table 2.

A checklist of cupulate 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)

Syn.: synonym; * indicates the species originally described from China.

With the development of molecular phylogenetics, the taxonomy of has been re-evaluated. Sequences of nuclear large and small subunit ribosomal DNA (LSU and SSU) were adopted for phylogenetic inference of 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 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 , , and 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 . In their updated study, Hansen et al. (2019) defined s. s., treated the cupulate (Pers.) Nannf. lacking crozier at the ascus base as a separate genus , retrieved the generic name , and transferred (R. Heim & L. Rémy) Dissing & Raitv. to . Brief comparisons amongst different taxonomic treatments are shown in Table 1.

In China, Teng (1963) recorded 11 species of 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 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, N.S. Weber and Harmaja. There are about 51 species currently known from the country.

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

Materials and methods

Fungal materials and morphological observations

Collections of the cupulate species with ribbed stipes, deposited in the Herbarium Mycologicum Academiae Sinicae (), were re-examined, including those originally deposited in the Mycological Herbarium of Shanxi University (). 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.

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
	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
	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
	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
	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
	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
	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
	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
	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

* Taxa included in the four-locus sequence analysis; Note: GenBank accession numbers in bold indicating the newly generated sequences.

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. (Scop.) Fr., Bull., Afzel. and (Pers.) P. Karst. are the representatives of the formerly recognised sections , , and , respectively. Fr. served as the outgroup taxon of the four-gene phylogeny and Peck worked for the two-gene analysis.

Results

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

The combined four-locus dataset included 48 taxa of s. s. and 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 s. s. Clade 1 consisted of , and ; Clade 2 included and ; and Clade 3 contained , , , and . , 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). and were situated outside the clades in all analyses.

Figure 1.

Bayesian phylogenetic tree of and 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 , , , , and . 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 was outside Clade 3 (Figure 2), which are congruent with the four-gene analysis (Figure 1).

Figure 2.

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

The Hsp90 dataset consisted of 84 sequences of and . 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 and . Tamura 3-parameter with gamma distribution (T92+G) was determined as the most suitable model for ML analysis. Clades 1–3 were strongly supported. , excluded from these clades, appeared to be sister of (Suppl. material 1: Figure S2).

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

The TEF1 dataset consisted of 26 taxa of and . K2+G was determined as the most suitable model for ML analysis. Clades 1–3 of 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

X.C. Wang & W.Y. Zhuang sp. nov.

325488B8-66BA-5A9C-9FFE-655038853D58

Figure 3a–d

Figure 3.

a–d: a mature apothecia when dry (CFSZ 2044) b asci (HMAS 23842) c, d ascospores in ascus (c: CFSZ 2044, d: HMAS 23842) e–g (HMAS 254610): e mature apothecia when dry f mature apothecia when fresh g ascospores in asci h–k (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).

Holotype.

CHINA. Inner Mongolia Autonomous Region, Chifeng City, Harqin Banner, Shijia Town, Toudaoyingzi Village, , on the ground under Decne., 8 Aug 2002, T.Z. Liu & T.H. Liu, HMAS 279703 (= CFSZ 2044).

Etymology.

The species epithet refers to its similarity to .

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, , alt. 2270 m, on the ground in broad-leaf forest, 26 Jun 1958, J.H. Yu 106, HMAS 23842.

Notes.

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

X.C. Wang & W.Y. Zhuang sp. nov.

B26640F5-749E-5827-9B98-508F5CAF8DE7

Figure 3e–g

CHINA. Sichuan Province, Garzê Tibetan Autonomous Prefecture, Daocheng County, Yading National Nature Reserve, , alt. 3900 m, on the ground of mixed forest, 18 Aug 2016, J.P. Wang & X.C. Wang 10706, HMAS 254610.

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

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.

belongs to Clade 3 (Figure 1). Its hymenium is nearly black when dry, which is similar to that of , but different from those in (brown when dry) and (reddish-brown when dry). When fresh, the hymenium is yellowish-brown, while that of is brown. Molecularly, it differs from 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 in 1 bp of Hsp90 (H293, holotype), 25 bp of LSU (H033) and 11 bp of TEF1 (H033); and from in 2 bp of Hsp90 and 13 bp of LSU. The sequence divergences between and are 1 bp of Hsp90, 20 bp of ITS2 and 12 bp of TEF1.

X.C. Wang & W.Y. Zhuang sp. nov.

09A48BF3-ECA7-5100-9576-E424981BD683

Figure 3h–k

CHINA. Xinjiang Uygur Autonomous Region, Changji Hui Autonomous Prefecture, Jimsar County, , alt. 1700 m, on the ground, 31 Jul 2003, W.Y. Zhuang & Y. Nong 4661, HMAS 86040.

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

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.

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.

nested with and in Clade 1 (Figure 1). These three species are hardly separated by gross morphology and anatomic structures. differs from 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 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

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

FA734B9A-7D79-59C4-A722-A3FFE6D51D54

Specimen examined.

CHINA. Inner Mongolia Autonomous Region, Xilingol League, Zhenglan Banner, Yihehaierhan Sumu, , 21 August 2005, on the ground, T.Z. Liu & X.L. Bai, HMAS 279704 (= CFSZ 2658).

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

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

72A5BD1B-AD94-5CBC-8462-2F9222C29E07

Figure 4

Figure 4.

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.

Neotype is designated here.

CHINA. Shanxi Province, Lvliang City, Jiaocheng County, Guandishan National Forest Park, , 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, , 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, , 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,, alt. 3150 m, on the ground, 12 Aug 2016, Y. Li 920, HMAS 277500.

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 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 .

was once treated as a synonym of 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.

Discussion

A total of about 28 rib-stiped cupulate species of and 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 (, , , and ) are found in Europe and China and and are widespread in Europe, Asia and North America. Amongst the Chinese helvellas, , and show a relatively wide distribution range and occur in at least four provinces. However, , , , and 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 (≡ ) (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 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 belong to the - lineage (Skrede et al. 2017); however, this lineage was not herein supported due to joining of the non-cupulate species . Clade 1 is in accordance with the lineage (Skrede et al. 2017) with the addition of . Our results clearly support the separation of from s. l. (Hansen et al. 2019).

Supplementary DNA barcodes are essential for delimitation of species. LSU is the most commonly used region for species identification (Nguyen et al. 2013, Landeros et al. 2015, Skrede et al. 2017). LSU is capable of distinguishing cupulate 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 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 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 species were available in GenBank. The trials of obtaining ITS amplicons, using the universal primers for many 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 -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 .