11urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3MycoKeysMC1314-40571314-4049Pensoft Publishers10.3897/mycokeys.60.3818638186Research ArticleAscomycotaHelvellaceaePezizalesPezizomycetesBiodiversity & ConservationDNA barcodingMolecular systematicsPhylogenyTaxonomyAsiaCentral AsiaChinaA four-locus phylogeny of rib-stiped cupulate species of Helvella (Helvellaceae, Pezizales) with discovery of three new speciesWangXin-Cunhttps://orcid.org/0000-0003-1780-77351LiuTie-Zhi2ChenShuang-Lin3LiYi4ZhuangWen-Yingzhuangwy@im.ac.cn1State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaInstitute of Microbiology, Chinese Academy of SciencesBeijingChinaCollege of Life Sciences, Chifeng University, Chifeng, Inner Mongolia 024000, ChinaChifeng UniversityChifengChinaCollege of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, ChinaNanjing Normal UniversityNanjingChinaCollege of Food Science and Engineering, Yangzhou University, Yangzhou, Jiangsu 225127, ChinaYangzhou UniversityYangzhouChina
2019311020196045678003A613-9B10-5088-8874-E5F93ABFF32235323871107201918092019Xin-Cun Wang, Tie-Zhi Liu, Shuang-Lin Chen, Yi Li, Wen-Ying ZhuangThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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. Helvellacalycina is a new record for China, while Dissingialeucomelaena should be excluded from Chinese mycota. Hsp90 and ITS2 are recommended as useful supplementary barcodes for species identifications of the genus.
AscomycotaDNA barcodephylogenytaxonomytypificationChinese Academy of Sciences501100002367http://doi.org/10.13039/501100002367National Natural Science Foundation of China501100001809http://doi.org/10.13039/501100001809Citation
Wang X-C, Liu T-Z, Chen S-L, Li Y, Zhuang W-Y (2019) A four-locus phylogeny of rib-stiped cupulate species of Helvella (Helvellaceae, Pezizales) with discovery of three new species. MycoKeys 60: 45–67. https://doi.org/10.3897/mycokeys.60.38186
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 HelvellasectionLeucomelaenae (Hansen et al. 2019).
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.
A checklist of cupulate Helvella species sensu lato with ribbed stipes.
Species
Section Acetabulum
Section Leucomelaenae
Section Solitariae
Section Macropodes
Subgenus Leucomelaenae
Remark
Acetabulacalyx 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)
Balsamiaaestivalis (R. Heim & L. Rémy) K. Hansen, Skrede & T. Schumach, 2019
–
Häffner 1987
–
–
Abbott and Currah 1997
as Helvellaaestivalis
Dissingiacrassitunicata (N.S. Weber) T. Schumach & Skrede, 2019
–
Weber 1975, Häffner 1987
–
–
Abbott and Currah 1997
as Helvellacrassitunicata
Dissingiaconfusa (Harmaja) K. Hansen & X.H. Wang, 2019
–
Harmaja 1977a, Häffner 1987
–
–
Syn. of H.leucomelaena (Abbott and Currah 1997)
as Helvellaconfusa
Dissingialeucomelaena (Pers.) K. Hansen & X.H. Wang, 2019
–
Dissing 1966, Weber 1975, Häffner 1987
–
–
Abbott and Currah 1997
as Helvellaleucomelaena
Dissingiaoblongispora (Harmaja) T. Schumachand Skrede, 2019
–
Harmaja 1978, Häffner 1987
–
–
Abbott and Currah 1997
as Helvellaoblongispora
Helvellaacetabulum (L.) Quél, 1874
Dissing 1966, Weber 1972, Häffner 1987
–
–
–
Abbott and Currah 1997
Valid species
Helvellaarctoalpina Harmaja, 1977
Harmaja 1977b, Häffner 1987
–
–
–
Syn. of H.verruculosa (Abbott and Currah 1997)
Valid species
Helvellacalycina Skrede, T.A. Carlsen & T. Schumach, 2017
–
–
–
–
–
Valid species
Helvellacostata Schwein, 1822
–
–
–
–
Syn. of H.acetabulum (Abbott and Currah 1997)
Valid species
Helvellacostifera Nannf, 1953
Dissing 1966, Häffner 1987
–
–
–
Abbott and Currah 1997
Valid species
Helvelladryadophila Harmaja, 1977
Harmaja 1977b, Häffner 1987
–
–
–
Syn. of H.verruculosa (Abbott and Currah 1997)
Valid species
Helvellafloriforma Q. Zhao & K.D. Hyde, 2016*
–
–
–
–
–
Valid species
Helvellagriseoalba N.S. Weber, 1972
Weber 1972, Häffner 1987
–
–
–
Syn. of H.costifera (Abbott and Currah 1997)
Valid species
Helvellahelvellula (Durieu) Dissing, 1966
–
Dissing 1966
–
–
–
Member of lasunosa clade (Skrede et al. 2017)
Helvellahyperborea Harmaja, 1978
Harmaja 1978, Häffner 1987
–
–
–
Abbott and Currah 1997
Valid species
Helvellajiaohensis J.Z. Cao, L. Fan & B. Liu, 1990*
–
–
–
–
–
Holotype lost
Helvellajilinensis J.Z. Cao, L. Fan & B. Liu, 1990*
–
–
–
–
–
Holotype lost
Helvellapedunculata Harmaja, 1978
–
Harmaja 1978, Häffner 1987
–
–
Syn. of H.leucomelaena (Abbott and Currah 1997)
?Syn. of H.costifera (Skrede et al. 2017)
Helvellapocillum Harmaja, 1976
Häffner 1987
Harmaja 1976
–
–
–
Syn. of B.aestivalis (Hansen et al. 2019)
Helvellaqueletii 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)
Helvellarobusta S.P. Abbott, 1988
Abbott and Currah 1988
–
–
–
Abbott and Currah 1997
Valid species
Helvellasolitaria P. Karst, 1871
–
Dissing 1966
Häffner 1987
–
Abbott and Currah 1997
Valid species
Helvellataiyuanensis B. Liu, Du & J.Z. Cao, 1985*
–
–
–
–
–
Neotypification here
Helvellatinta Q. Zhao, B. Feng & K.D. Hyde, 2016*
–
–
–
–
–
Valid species
Helvellaulvinenii Harmaja, 1979
Harmaja 1979
–
Häffner 1987
–
Abbott and Currah 1997
Syn. of H.solitaria (Landeros et al. 2015)
Helvellaunicolor (Boud.) Dissing, 1966
Dissing 1966, Häffner 1987
–
–
–
Abbott and Currah 1997
In need of reassessment (Skrede et al. 2017)
Helvellaverruculosa (Sacc.) Harmaja, 1978
–
–
–
–
Abbott and Currah 1997
In need of reassessment (Skrede et al. 2017)
Syn.: synonym; * indicates the species originally described from China.
Materials and methodsFungal 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.
Fungal species and sequences used in phylogenetic analyses.
Species
Voucher
Locality
HSP90
ITS
LSU
TEF1
Label
Reference
Balsamiaaestivalis (R. Heim & L. Rémy) K. Hansen, Skrede & T. Schumach.
KH.10.133
Sweden
–
–
MK100250
MK113869
Balsamiaaestivalis
Hansen et al. 2019
O-253217
Norway
–
–
MK100251
MK113870
Balsamiaaestivalis
Hansen et al. 2019
Balsamiaplatyspora Berk.
TUR206101
Finland
–
–
MK100252
MK113871
Balsamiaplatyspora
Hansen et al. 2019
Dissingiaconfusa (Harmaja) K. Hansen & X.H. Wang
H437*
Norway
KY784529
–
KY773164
–
Helvellaconfusa
Skrede et al. 2017
HMAS 27728*
Qinghai, China
MK652180
MK592119
–
–
Helvellaconfusa
This study
HMAS 38328*
Xinjiang, China
MK652181
MK592120
–
–
Acetabulaleucomelas
This study
Dissingiacrassitunicata (N.S. Weber) T. Schumach. & Skrede
H222*
Canada
KY784342
–
KY773053
–
Helvellacrassitunicata
Skrede et al. 2017
Dissingialeucomelaena (Pers.) K. Hansen & X.H. Wang
H404, epitype
Sweden
KY784500
–
–
–
Helvellaleucomelaena
Skrede et al. 2017
H115*
USA
KY784253
–
KY772970
–
Helvellaleucomelaena
Skrede et al. 2017
KH.06.01 = H115
USA
–
–
KC012682
KC109207
Helvellaleucomelaena
Hansen et al. 2013
He273
Australia
–
–
JX993075
–
Helvellaleucomelaena
Landeros et al. 2015
He286, isotype
Italy
–
–
JX993051
–
Acetabulacalyx
Landeros et al. 2015
HMAS 61351
Denmark
MK652201
–
–
–
Helvellaleucomelaena
This study
HMAS 61356*
Sweden
MK652202
MK592137
–
–
Helvellaleucomelaena
This study
Dissingiaoblongispora (Harmaja) T. Schumach. & Skrede
H132*
Norway
KY784265
–
KY772983
–
Helvellaoblongispora
Skrede et al. 2017
HMAS 38329*
Xinjiang, China
MK652203
MK592138
–
–
Helvellaacetabulum
This study
HMAS 74657*
Gansu, China
MK652204
MK592139
–
–
Helvellaleucomelaena
This study
HMAS 75147*
Sichuan, China
MK652205
MK592140
–
MK652162
Helvellaleucomelaena
This study
HMAS 75151
Sichuan, China
MK652206
MK592141
–
–
Helvellaleucomelaena
This study
HMAS 75183
Sichuan, China
MK652207
MK592142
–
–
Helvellaleucomelaena
This study
HMAS 75960
Sichuan, China
MK652208
MK592143
–
–
Helvellacupuliformis
This study
HMAS 86050
Xinjiang, China
–
MK592144
–
–
Helvellaacetabulum
This study
HMAS 86051
Xinjiang, China
–
MK592145
–
MK652163
Helvellaacetabulum
This study
HMAS 86160
Shanxi, China
–
MK592146
–
–
Helvellaleucomelaena
This study
Helvellaacetabuloides X.C. Wang & W.Y. Zhuang
HMAS 279703*, CFSZ 2044, holotype
Inner Mongolia, China
MK652219
MK592155
–
MK652168
Helvellaacetabulum
This study
HMAS 23842*
Shaanxi, China
MK652220
–
–
–
Acetabulavulgaris
This study
Helvellaacetabulum (L.) Quél.
H410, epitype
Sweden
KY784506
–
KY773154
–
Helvellaacetabulum
Skrede et al. 2017
H133*
Norway
KY784266
–
KY772984
KY772875
Helvellaacetabulum
Skrede et al. 2017
HMAS 7046*
Czech
MK652177
MK592116
–
–
Acetabulavulgaris
This study
HMAS 61353
Denmark
MK652176
–
–
–
Helvellaacetabulum
This study
HMAS 243823*
UK
MK652174
MK592114
MK592099
–
Helvellaacetabulum
This study
HMAS 23839
Qinghai, China
MK652171
MK592112
–
–
Helvellaacetabulum
This study
HMAS 23841
Beijing, China
MK652172
MK592113
–
–
Helvellaacetabulum
This study
HMAS 23843
Qinghai, China
MK652173
–
–
–
Acetabulavulgaris
This study
HMAS 38129
Xinjiang, China
MK652175
MK592115
–
–
Helvellaacetabulum
This study
Helvellaacetabulum (L.) Quél.
HMAS 75176*
Sichuan, China
MK652178
MK592117
–
MK652156
Helvellaacetabulum
This study
Helvellaarctoalpina Harmaja
H293, holotype
Norway
KY784406
–
–
–
Helvellaarctoalpina
Skrede et al. 2017
H033*
Norway
KY784207
–
KY772924
KY772841
Helvellaarctoalpina
Skrede et al. 2017
Helvellacalycina Skrede, T.A. Carlsen & T. Schumach.
H022*, epitype
Norway
KY784198
–
KY772915
KY772833
Helvellacalycina
Skrede et al. 2017
HMAS 279704*, CFSZ 2658
Inner Mongolia, China
MK652179
MK592118
MK592100
MK652157
Helvellaacetabulum
This study
Helvellacostata Schwein.
H100*
USA
KY784244
–
KY772962
–
Helvellacostata
Skrede et al. 2017
Helvellacostifera Nannf.
H298, epitype
Sweden
KY784409
–
–
–
Helvellacostifera
Skrede et al. 2017
H131*
Norway
KY784264
–
KY772982
KY772874
Helvellacostifera
Skrede et al. 2017
HMAS 61361
Shanxi, China
MK652185
–
–
–
Helvellaacetabulum
This study
HMAS 71778
Beijing, China
MK652186
MK592124
–
–
Helvellacostifera
This study
HMAS 83510
Xinjiang, China
MK652187
MK592125
–
–
Helvellacostifera
This study
HMAS 88497
Shanxi, China
MK652188
MK592126
–
–
Helvellaacetabulum
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
Helvelladryadophila Harmaja
H302, holotype
Norway
KY784412
–
–
–
Helvelladryadophila
Skrede et al. 2017
H180*
Norway
KY784309
–
KY773024
KY772883
Helvelladryadophila
Skrede et al. 2017
Helvellafloriforma Q. Zhao & K.D. Hyde
HKAS 90224, Holotype
Yunnan, China
–
–
KX239771
–
Helvellafloriforma
Hyde et al. 2016
Helvellagriseoalba N.S. Weber
He164, holotype
USA
–
–
JX993066
–
Helvellagriseoalba
Landeros et al. 2015
H306*
USA
KY784416
–
–
–
Helvellagriseoalba
Skrede et al. 2017
Helvellahyperborea Harmaja
H491*
Finland
KY784569
–
–
–
Helvellahyperborea
Skrede et al. 2017
HMAS 23840
Gansu, China
MK652189
–
–
–
Helvellaacetabulum
This study
HMAS 38331
Xinjiang, China
MK652190
–
–
–
Helvellacostifera
This study
HMAS 83506
Xinjiang, China
MK652191
MK592127
–
–
Helvellacostifera
This study
HMAS 83507
Xinjiang, China
MK652192
MK592128
–
–
Helvellacostifera
This study
HMAS 83508
Xinjiang, China
MK652193
MK592129
–
–
Helvellacostifera
This study
HMAS 83509
Xinjiang, China
MK652194
MK592130
–
–
Helvellacostifera
This study
HMAS 83511
Xinjiang, China
MK652195
MK592131
–
MK652160
Helvellacostifera
This study
Helvellahyperborea Harmaja
HMAS 83512
Xinjiang, China
MK652196
MK592132
–
–
Helvellacostifera
This study
HMAS 85476
Xinjiang, China
MK652197
MK592133
–
–
Helvellaacetabulum
This study
HMAS 85591*
Shanxi, China
MK652198
MK592134
–
–
Helvellaleucomelaena
This study
HMAS 85673*
Shanxi, China
MK652199
MK592135
–
–
Helvellasolitaria
This study
HMAS 86043*
Xinjiang, China
MK652200
MK592136
–
MK652161
Helvellacostifera
This study
Helvellarobusta S.P. Abbott
He163, holotype
Canada
–
–
JX993079
–
Helvellarobusta
Landeros et al. 2015
Helvellasichuanensis X.C. Wang & W.Y. Zhuang
10706*, HMAS 254610, holotype
Sichuan, China
MK652221
MK592156
MK592107
MK652169
This study
Helvellasolitaria P. Karst.
H370, epitype
Sweden
KY784470
–
–
–
Helvellasolitaria
Skrede et al. 2017
H004*
Norway
KY784184
–
KY772902
KY772819
Helvellasolitaria
Skrede et al. 2017
He248, holotype
Finland
–
–
JX993085
–
Helvellaulvinenii
Landeros et al. 2015
HMAS 41140*
Netherlands
MK652211
MK592148
–
–
Helvellaqueletii
This study
HMAS 58371
Czech
MK652212
–
–
–
Helvellaqueletii
This study
HMAS 27727*
Qinghai, China
MK652209
MK592147
–
–
Helvellaconfusa
This study
HMAS 27951
Jilin, China
MK652210
–
–
–
Helvellaconfusa
This study
HMAS 73509
Sichuan, China
MK652213
MK592149
–
–
Helvellaacetabulum
This study
HMAS 75175*
Sichuan, China
MK652214
MK592150
–
MK652164
Helvellaleucomelaena
This study
Helvellataiyuanensis B. Liu, Du & J.Z. Cao
HMAS 85689*, neotype
Shanxi, China
MK652217
MK592153
–
–
Helvellataiyuanensis
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
Helvellasolitaria
This study
Helvellatianshanensis X.C. Wang & W.Y. Zhuang
HMAS 86040*, holotype
Xinjiang, China
MK652222
MK592157
MK592108
MK652170
Helvellacostifera
This study
HMAS 88611*
Xinjiang, China
MK652223
MK592158
–
–
Helvellaacetabulum
This study
Helvellatinta Q. Zhao, B. Feng & K.D. Hyde
HKAS 82560, holotype
Sichuan, China
–
KX239842
KX239772
–
Helvellatinta
Hyde et al. 2016
Helvellacrispa (Scop.) Fr.
H408*, epitype
Sweden
KY784504
–
–
–
Helvellacrispa
Skrede et al. 2017
H135
Norway
KY784268
–
KY772986
–
Helvellacrispa
Skrede et al. 2017
HKAS 75434
Germany
–
JX462572
KR493479
KT254487
Helvellacrispa
Zhao et al. 2015
Helvellaelastica Bull.
H066*
Sweden
KY784230
–
KY772950
KY772858
Helvellaelastica
Skrede et al. 2017
Helvellalacunosa Afzel.
H407, epitype
Sweden
KY784503
–
KY773152
–
Helvellalacunosa
Skrede et al. 2017
H039*
Norway
KY784213
–
KY772930
KY772845
Helvellalacunosa
Skrede et al. 2017
Helvellamacropus (Pers.) P. Karst.
H412, epitype
Sweden
KY784507
–
–
–
Helvellamacropus
Skrede et al. 2017
H073*
Norway
KY784233
–
KY772954
KY772863
Helvellamacropus
Skrede et al. 2017
Midotislingua Fr.
H283*
Switzerland
KY784397
–
KY773093
–
Wynnellasilvicola
Skrede et al. 2017
HMAS 67962*
Germany
MK652224
MK592159
MK592109
–
Wynnellaauricula
This study
HMAS 71896*
Shanxi, China
MK652225
MK592160
MK592110
–
Wynnellasilvicola
This study
HMAS 74656
Gansu, China
MK652226
MK592161
MK592111
–
Helvellasilvicola
This study
HMAS 83548
Xinjiang, China
MK652227
MK592162
–
–
Wynnellaauricula
This study
Pindaraterrestris Velen.
KH.12.67
Sweden
–
–
MK100279
MK113889
Pindaraterrestris
Hansen et al. 2019
S-F327988
Sweden
–
–
MK100280
MK113896
Pindaraterrestris
Hansen et al. 2019
T. Kekki 168
Finland
–
–
MK100281
MK113897
Pindaraterrestris
Hansen et al. 2019
Underwoodiacolumnaris Peck
Kanouse 1951
USA
–
–
U42685
–
Underwoodiacolumnaris
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. Helvellacrispa (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. Midotislingua Fr. served as the outgroup taxon of the four-gene phylogeny and Underwoodiacolumnaris 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. Helvelladryadophila, 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). Helvellagriseoalba and H.hyperborea were situated outside the clades in all analyses.
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.
https://binary.pensoft.net/fig/352437
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).
Maximum likelihood phylogeny of Helvellaceae inferred from combined LSU and TEF1 dataset. Bootstrap values ≥ 50% are indicated at nodes.
https://binary.pensoft.net/fig/352438
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. Helvellatinta, 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).
TaxonomyNew speciesFungiPezizalesHelvellaceae325488B8-66BA-5A9C-9FFE-655038853D58HelvellaacetabuloidesX.C. Wang & W.Y. Zhuangsp. nov.Figure 3a–dHolotype.
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 Ostryopsisdavidiana 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.
Helvellaacetabuloides 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. Helvellaacetabuloides 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.
a–dHelvellaacetabuloides: a mature apothecia when dry (CFSZ 2044) b asci (HMAS 23842) c, d ascospores in ascus (c: CFSZ 2044, d: HMAS 23842) e–gHelvellasichuanensis (HMAS 254610): e mature apothecia when dry f mature apothecia when fresh g ascospores in asci h–kHelvellatianshanensis (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).
https://binary.pensoft.net/fig/352439FungiPezizalesHelvellaceaeB26640F5-749E-5827-9B98-508F5CAF8DE7HelvellasichuanensisX.C. Wang & W.Y. Zhuangsp. nov.Figure 3e–gHolotype.
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.
Helvellasichuanensis 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.
FungiPezizalesHelvellaceae09A48BF3-ECA7-5100-9576-E424981BD683HelvellatianshanensisX.C. Wang & W.Y. Zhuangsp. nov.Figure 3h–kHolotype.
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.
Helvellatianshanensis nested with H.calycina and H.costifera in Clade 1 (Figure 1). These three species are hardly separated by gross morphology and anatomic structures. Helvellatianshanensis 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 recordFungiPezizalesHelvellaceaeFA734B9A-7D79-59C4-A722-A3FFE6D51D54HelvellacalycinaSkrede, T.A. Carlsen & T. Schumach., Persoonia 39: 221, 2017Specimen 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.
Helvellacalycina 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.
NeotypificationFungiPezizalesHelvellaceae72A5BD1B-AD94-5CBC-8462-2F9222C29E07HelvellataiyuanensisB. Liu, Du & J.Z. Cao, Acta Mycol. Sin. 4(4): 211, 1985Figure 4Neotype 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.
Helvellataiyuanensis 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.
Helvellataiyuanensisa 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.
https://binary.pensoft.net/fig/352440Discussion
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).
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Figures S1–S4
phylogenetic data
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.
https://binary.pensoft.net/file/352441This 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.Xin-Cun Wang, Tie-Zhi Liu, Shuang-Lin Chen, Yi Li, Wen-Ying Zhuang