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

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 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.

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.

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.

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

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.
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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 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, 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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