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Article

Species Diversity of Amanita Section Vaginatae in Eastern China, with a Description of Four New Species

by
Yang-Yang Cui
1,2,
Yan-Jia Hao
3,
Ting Guo
4,5,6,
Zhu L. Yang
1,2,* and
Qing Cai
1,2,*
1
Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
2
Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming 650201, China
3
College of Horticulture, Anhui Agricultural University, Hefei 230036, China
4
Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
5
Key Laboratory of Edible Fungal Resources and Utilization (South), Ministry of Agriculture and Rural Affairs, Shanghai 201403, China
6
National Engineering Research Center of Edible Fungi, Shanghai 201403, China
*
Authors to whom correspondence should be addressed.
J. Fungi 2023, 9(8), 862; https://doi.org/10.3390/jof9080862
Submission received: 29 May 2023 / Revised: 9 August 2023 / Accepted: 16 August 2023 / Published: 19 August 2023
(This article belongs to the Section Fungal Evolution, Biodiversity and Systematics)
Browse Figures
Versions Notes

Abstract

:
Species of Amanita sect. Vaginatae (Fr.) Quél. are challenging to delimitate due to the morphological similarity or morphostasis among different taxa. In this study, a multi-locus (nuc rDNA region encompassing the internal transcribed spacers 1 and 2 with the 5.8S rDNA, the D1–D3 domains of nuc 28S rDNA, partial sequences of translation elongation factor 1-a, and the second largest subunit of RNA polymerase II) phylogeny was employed to investigate the species diversity of the section in eastern China. Sixteen species were recognized, including four new species; namely, A. circulata, A. multicingulata, A. orientalis, and A. sinofulva. They were documented with illustrated descriptions, ecological evidence, and comparisons with similar species. A key to the species of the section from eastern China is provided.

1. Introduction

Amanita Pers. is a cosmopolitan genus with about 700 accepted species [1,2,3,4,5,6,7,8,9,10,11,12,13]. According to the most recent comprehensive phylogenetic studies, Amanita is divided into three subgenera and 11 sections [2]. In these sections, A. sect. Vaginatae (Fr.) Quél. is the most species-rich (http://www.amanitaceae.org/; accessed on 1 January 2023). The section is characterized by a striate and non-appendiculate pileus, a bulbless stipe base with saccate volva or warts arranged in incomplete belts, mostly absent annulus, inamyloid basidiospores, and an absence of clamps.
Species of A. sect. Vaginatae are distributed worldwide and can form ectomycorrhizal associations with plants of more than ten families, such as Fagaceae, Pinaceae, and Dipterocarpaceae [1,14,15,16,17,18]. It is challenging to delimitate and recognize the species of the section due to the morphological similarity or morphostasis among different taxa. As currently circumscribed, ca. 145 species have been described and accepted [2,4,5,14,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34] (http://www.amanitaceae.org/; accessed on 1 January 2023). Asia and Europe are the most species-rich areas of the section, in which 48 and 47 taxa have been reported, respectively. Thirty and five taxa have been described from North and South America, respectively. In Africa and Oceania, seventeen species have been described (http://www.amanitaceae.org/; accessed on 1 January 2023).
In China, 33 taxa of A. sect. Vaginatae have been reported [1,2,21,35]. Twenty-seven of them can be found in southwestern China. Southern and eastern China are the second and third most species-rich areas, with six and five species. Three species have been reported in central and northeastern China, respectively. The majority of them are distributed in more than one area. There are nine endemic species in southwestern China. Only one species is restricted in the southern, central, and northeastern parts, respectively. The five taxa in eastern China—namely A. cingulata J.W. Liu and Zhu L. Yang, A. hamadae Nagas. and Hongo, A. olivaceofusca Y.Y. Cui et al., A. ovalispora Boedijn, and A. pallidozonata Y.Y. Cui et al.—also occur in central, southern, and southwestern China [2].
During our investigations of macrofungi in eastern China, numerous specimens of A. sect. Vaginatae were collected. In this study, we applied integrative taxonomy, including morphological characters, multi-locus phylogenetic evidence, and ecological data, to elucidate the species diversity of A. sect. Vaginatae in eastern China. A key to all of the species found in the area is provided.

2. Materials and Methods

2.1. Taxon Sampling

Sixty-one specimens of A. sect. Vaginatae were examined. Among them, 50 were collected from eastern China, and the rest were from southwestern China. For each collection, a part of the basidioma was dried with silica gel for DNA extraction. The remaining materials were then dried at 45–50 °C with an electronic food dehydrator. At the same time, the specimen information, host trees, altitudes, locations, collectors, and dates were recorded, and photos of the fruiting bodies were taken. The location information and ecological habits of the specimens mentioned above are stated in the results section. All specimens examined in this work were deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS), and the Edible-medicinal Fungal Herbarium of Anhui Agriculture University (EFHAAU).

2.2. Morphological Observation

The macroscopic descriptions are based on detailed field notes and photographs of fresh basidiomata. Color codes indicated in the descriptions are from Kornerup and Wanscher [36]. Microscopic features were studied with light microscopy using dried material rehydrated in 5% KOH and, when necessary, dyed with Congo Red. Melzer’s reagent was used to check the amyloidity of basidiospores. In the description of the basidiospores, the abbreviation (n/m/p) represents n basidiospores measured from m basidiomata of p collections. Dimensions for the basidiospores are given using a range notation of the form (a–) b–c (–d). The range b–c contains a minimum of 90% of the measured values. Extreme values, a or d, are given in parentheses. Q represents the ‘length/width ratio’ of a basidiospore in the side view. Qm means the average Q of all basidiospores measured ± sample standard deviation.

2.3. DNA Extraction, PCR Amplification, and Sequencing

Genomic DNA was extracted from silica gel-dried material or herbarium specimens using the modified CTAB method [37]. The primer pairs LR0R/LR5 [38], ITS1F/ITS4 [39], 983F/1567R [40], and Am-6F/Am-7R [41] were used to amplify the large subunit D1–D3 domains of nuc 28S rDNA (nrLSU), nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS), partial sequences of translation elongation factor 1-a (tef1-α), and the second largest subunit of RNA polymerase II (rpb2), respectively. Protocols for the polymerase chain reactions (PCR) and sequencing followed those in Cai et al. [41] and the reference therein. All sequences analyzed in this study were deposited at GenBank and are listed in Table 1.

2.4. Sequence Alignments and Phylogenetic Analyses

According to the most recent comprehensive multi-locus phylogenetic analyses of A. sect. Vaginatae, the ITS sequences were not included [33]. Therefore, two datasets, the multi-locus dataset (nrLSU, tef1-α and rpb2) and the ITS sequences matrix, were compiled to infer the phylogeny of the section, respectively. For the ITS dataset, sequences of the new taxa were initially blasted in GenBank. The most closely related sequences (nucleotide identities >90%) were retrieved to complement the ITS matrix with one to two representatives per species. For the combined dataset, all known species of the section with sequences of the gene fragments employed in this study were included [2,4,5,21,22,33]. According to recent phylogenetic analyses, A. muscaria (L.) Lam., A. parvipantherina Zhu L. Yang et al., A. caesarea (Scop.) Pers., and A. yuaniana Zhu L. Yang of A. sect. Amanita and sect. Caesareae Singer ex Singer were selected as outgroups [2]. Sequences of all gene fragments were separately aligned with MAFFT 7 [42] and manually optimized in BioEdit 7 [43]. For the two datasets, the introns of tef1-α and rpb2 were excluded because of the difficulty in alignment. The ambiguously aligned regions of nrLSU and ITS were eliminated using Gblocks 0.91b [44] with the “less stringent selection” parameter set. The final alignments of both datasets were deposited in TreeBASE (30635).
Single-gene analyses were carried out for the concatenated matrix to detect possible incongruence among individual genes based on the Maximum Likelihood (ML) method. Because no well-supported bootstrap value (BS > 70%) was detected (Figures S1–S3), the resulting alignments of nrLSU, tef1-α, and rpb2 were concatenated using Geneious v9.1.3 [45]. The best partition schemes and evolutionary models of the two datasets were selected using PartitionFinder V2.1.1 [46]. ITS was divided into three blocks: ITS1, ITS2, and 5.8S. The concatenated matrix was partitioned according to gene fragments and codon positions, including nrLSU, tef1-α_codon1, tef1-α_codon2, tef1-α_codon3, rpb2_codon1, rpb2_codon2, and rpb2_codon3. Thus, three and seven blocks were predefined for the ITS and combined datasets, respectively. The two datasets were then analyzed using RAxML v8.2.4 [47] and MrBayes v3.2.6 [48] for ML and Bayesian Inference (BI) analyses, respectively. In the ML analyses, the statistical supports were obtained using rapid bootstrapping with 1000 replicates, and the other parameters used the default settings. Some of the selected models could not be implemented in RAxML; thus, the GTR + I + G model, which included all of the parameters of the selected model, was used for all partitioned data. For BI analyses, four Markov Chain Monte Carlo (MCMC) chains were run simultaneously for 5 million generations under the best partition schemes and evolutionary models selected using PartitionFinder, and trees were sampled every 1000 generations. Runs were automatically terminated when the average standard deviation of split frequencies fell below 0.01 and the ESS values exceeded 200 [48]. Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer/, accessed on 20 January 2023) was used to confirm the chain convergence. Subsequently, the sampled trees were summarized, and posterior probabilities were obtained by discarding the first 25% of generations as burn-in.
Table 1. Information on specimens used in multi-locus phylogenetic analyses and their GenBank accession numbers. Sequences newly generated in this study are indicated in bold.
Table 1. Information on specimens used in multi-locus phylogenetic analyses and their GenBank accession numbers. Sequences newly generated in this study are indicated in bold.
SpeciesVoucherLocalityGenBank No.Reference
nrLSUtef1-αrpb2
A. albidostipesHKAS57358ChinaMH486756MH508983–[2]
A. albidostipesHKAS95189ChinaMH486757––[2]
A. angustilamellataHKAS24158ChinaAF024440––[2]
A. cf. angustilamellataHKAS83453ChinaMH486430––[2]
A. cf. angustilamellataHKAS89451ChinaMH486431MH508716MH485910[2]
A. annulataMHKMU L. P. Tang 1671ChinaMZ005570––[21]
A. basianaRET 308-4ItalyKP258987––Direct sub.
A. battarraeHKAS92090ChinaMH486388MH508689MH485880[2]
A. battarraeMB-000643GermanyMH486389MH508690MH485881[2]
A. brunneofuligineaHKAS29508ChinaAF024442––[2]
A. brunneofuligineaHKAS89226ChinaMH486391MH508691MH485883[2]
A. brunneoproceraBZ2015-24ThailandMF461553–MF440412[4]
A. brunneoproceraEFHAAU3796ChinaOR042681OR046329OR051723This study
A. brunneoproceraEFHAAU4162ChinaOR042682OR046330OR051724This study
A. brunneoproceraHKAS97514ChinaMH486390–MH485882[2]
A. brunneoproceraHKAS98435ChinaMH486391MH508691MH485883[2]
A. brunneosquamataBZ2015-73ThailandMF461563–MF440422[4]
A. brunneoumbonataBZ2015-67ThailandMF461561–MF440420[4]
A. brunneoumbonataEFHAAU131ChinaOR042683OR046370OR051725This study
‘A. ceciliae’ASIS26247South KoreaKU139437––Direct sub.
‘A. ceciliae’ASIS26935South KoreaKU139439––Direct sub.
A. ceciliaeC. Bas9341The NetherlandsAF024444––[49]
‘A. ceciliae’KA12-0758South KoreaKF021668––[32]
‘A. ceciliae’KA12-0916South KoreaKF021669––[32]
A. changtuiaHKAS92100ChinaMH486442MH508724MH485919[2]
A. chiuiHKAS76328ChinaMH486447MH508727MH485930[2]
A. cinctipesHKAS101388ChinaMH486448––[2]
A. cinctipesHKAS78465ChinaMH486449––[2]
A. cingulataHKAS100640ChinaMH486454MH508731MH485935[2]
A. cingulataHKAS75600ChinaKY949583––[50]
A. cinnamomeaBZ2015-45ThailandMF461555–MF440414[4]
A. cinnamomeaBZ2015-48ThailandMF461557–MF440416[4]
A. circulataHKAS127629ChinaOR042717OR046356OR051749This study
A. circulataHKAS127639ChinaOR042715OR046355OR051747This study
A. circulataHKAS101238ChinaOR042718OR046357OR051750This study
A. circulataHKAS56815ChinaOR042719––This study
A. circulataHKAS57535ChinaOR042720––This study
A. circulataHKAS57543ChinaOR042721OR046358–This study
A. circulataHKAS67955ChinaOR042722OR046359–This study
A. circulataHKAS76411ChinaOR042723––This study
A. circulataHKAS97054ChinaOR042724OR046360OR051751This study
A. circulataHKAS97543ChinaOR042725OR046361OR051752This study
A. circulataHKAS97784ChinaOR042726OR046362OR051753This study
A. circulataHKAS128052ChinaOR042716OR046354OR051748This study
A. cf. circulataEFHAAU4143ChinaOR042684––This study
A.cf. circulataHKAS128051ChinaOR042704OR046331–This study
A. cistetorumRET 293-5ItalyMK536604––Direct sub.
A. colombianaANDES_F910_NVE410ColombiaKT008041KT008012–[23]
A. constrictaBW_Mycoblitz IV_2USAHQ539684––Direct sub.
A. corneliiCAL 1337IndiaKX528072––Direct sub.
A. craseodermaINPA No. 265158BrazilON392651ON492138ON492104[33]
A. crebresulcataINPA No. 265178BrazilON392663ON540337ON492115[33]
A. cf. daimonioctantesTRTC155757CanadaON392639ON492125–[33]
A. emodotrygonMFM-219PakistanMF491881––[5]
A. emodotrygonSUA902PakistanMF491880––[5]
A. flammeolaJD960DR Congo––MF440424[4]
A. flavidocereaBZ2015-59ThailandMF461559–MF440418[4]
A. flavidocereaBZ2015-60ThailandMF461560–MF440419[4]
A. flavidogriseaBZ2015-44ThailandMF461554–MF440413[4]
A. friabilisAF2529Belgium––MF440404[4]
A. fuligineodiscaANDES_F823_NVE324ColombiaKT008039KT008011–[23]
‘A. fulva’ASIS26398 South KoreaKU139446––Direct sub.
A. fulvaHKAS96168AustriaMH486555MH508826MH486022[2]
A. fulvaN. Arnold2The NetherlandsAF024455––[49]
A. aff. fulvaHKAS29518ChinaAF024456––[49]
A. glareaLAH35044PakistanKY781175––[19]
A. griseofoliaEFHAAU555ChinaOR042689OR046334OR051728This study
A. griseofoliaHKAS38159ChinaAY436488––[51]
A. griseofoliaHKAS54443ChinaMH486564MH508835MH486029[2]
A. griseofuscaLAH35366PakistanMH241056MH282854–[20]
A. griseofuscaSWAT000137PakistanMH241058––[20]
A. griseoumbonataHKAS92103ChinaMH486578MH508847MH486040[2]
A. hamadaeEFHAAU3013ChinaOR042690OR046335OR051729This study
A. hamadaeHKAS79081ChinaMH486585–MH486047[2]
A. hamadaeHKAS83451ChinaMH486586MH508853MH486048[2]
A. lignitinctaHKAS29512ChinaAF024461––[49]
A. lignitinctaHKAS69411ChinaMH486625–MH508883[2]
A. lippiaeRET 418-2BrazilNG_057062––Direct sub.
A. liquiiHKAS36611ChinaAY436493––[51]
A. liquiiHKAS54568ChinaJF710794KU714525KU714584[52]
A. liquiiHKAS93915ChinaMH486628MH508886MH486078[2]
A. luteoparvaBZ2015-46ThailandMF461556–MF440415[4]
A. madagascariensisJEIC0515GuineaON843338ON894331ON854980[33]
A. magnivolvataAF2528BelgiumMF461551–MF440403[4]
A. malleataAM91-255Belgium––MF440406[4]
A. mansehraensisLAH31005PakistanMG195982MH495970–[22]
A. mansehraensisLAH31006PakistanMG195983––[22]
A. multicingulataHKAS128054ChinaOR042711OR046350OR051743This study
A. multicingulataHKAS128049ChinaOR042712OR046351OR051744This study
A. multicingulataHKAS127630ChinaOR042713OR046352OR051745This study
A. multicingulataHKAS127631ChinaOR042714OR046353OR051746This study
A. multicingulataHKAS127632ChinaOR042705OR046346OR051739This study
A. multicingulataHKAS127633ChinaOR042706OR046347OR051740This study
A. multicingulataHKAS127634ChinaOR042707OR046348OR051741This study
A. multicingulataHKAS127635ChinaOR042708OR046349OR051742This study
A. multicingulataHKAS127636ChinaOR042709––This study
A. multicingulataHKAS127637ChinaOR042710––This study
A. neocinctipesHKAS79627ChinaMH486653MH508910MH486103[2]
A. neocinctipesHKAS78463China––MH486102[2]
A. nivalisR. Watling 17489The NetherlandsAF024466––[49]
A. olivaceofuscaHKAS97581ChinaMH486691–MH486127[2]
A. olivaceofuscaHKAS80243ChinaMH486689MH508934MH486125[2]
A. olivaceogriseaAF2427The Netherlands––MF440402[4]
A. olivovaginataSUA138PakistanMF491875––Direct sub.
A. olivovaginataSUA939PakistanMF491873––Direct sub.
A. orientalisEFHAAU1367ChinaOR042698–OR051735This study
A. orientalisHKAS127638ChinaOR042697OR046340OR051734This study
A. orienticroceaEFHAAU2919ChinaOR042691OR046336OR051730This study
A. orienticroceaEFHAAU4837ChinaOR042692OR046337OR051731This study
A. orienticroceaHKAS80029ChinaMH486700––[2]
A. orienticroceaHKAS90455ChinaMH486701MH508942MH486133[2]
A. orientifulvaHKAS32522ChinaAY436496––[51]
A. orientifulvaHKAS87937ChinaMH486704MH509154MH486136[2]
‘A. orientifulva’KA12-0642South KoreaKF021679––[32]
‘A. orientifulva’STDS-2-10JapanLC098763––Direct sub.
A. ovalisporaHKAS101406ChinaMH486720MH508955MH486148[2]
A. pachycoleaHKAS101422USAMH486724–MH486152[2]
A. pallidocarneaHKAS97678ChinaMH486728–MH486156[2]
A. pallidozonataEFHAAU114ChinaOR042693–OR051722This study
A. pallidozonataEFHAAU1594ChinaOR042694–OR051732This study
A. pallidozonataEFHAAU373ChinaOR042695OR046338OR051733This study
A. pallidozonataEFHAAU542ChinaOR042696OR046339–This study
A. pallidozonataHKAS57718ChinaMH486740MH508973–[2]
A. pallidozonataHKAS100608ChinaMH486739–MH486164[2]
A. pekeoidesJAC13244New ZealandMT862269MT977108MT993777Direct sub.
A. populiphilaRET 068-7USAKP221315––Direct sub.
A. prudensMP220407SpainOP279613––[34]
A. pseudovaginataHKAS70138ChinaMH486791–MH486205[2]
A. retentaHKAS70020ChinaMH486802MH509028MH486215[2]
A. shennongjianaHKAS75553ChinaMH486862MH509085MH486270[2]
A. shennongjianaHKAS75554ChinaMH486863––[2]
A. simulansJM0303Belgium––MF440425[4]
A. sinofulvaEFHAAU207ChinaOR042700OR046342–This study
A. sinofulvaEFHAAU313ChinaOR042701OR046343–This study
A. sinofulvaEFHAAU118ChinaOR042699OR046341OR051736This study
A. sinofulvaHKAS75058ChinaOR042702OR046344OR051737This study
A. sinofulvaHKAS92355ChinaOR042703OR046345OR051738This study
A. aff. sinicoflavaTRTC156849 CanadaON392637ON492153–[33]
A. aff. sinicoflavaTRTC156851CanadaON392638ON492154–[33]
A. sororculaANDES_F2088_NVE587ColombiaKT008030KT008013–[23]
A. strobilaceovolvataJEIC0609Ivory CoastON843372ON894364ON855006[33]
A. submembranaceaMB -001174GermanyMH486916MH509135–[2]
A. suborientifulvaBZ2013-55ThailandMF461564––[4]
A. suborientifulvaEFHAAU3559ChinaOR042687––This study
A. suborientifulvaOR1276ThailandMF461567–MF440426[4]
A. cf. suborientifulvaEFHAAU4437ChinaOR042685OR046332OR051726This study
A. cf. suborientifulvaEFHAAU5291ChinaOR042688OR046333OR051727This study
A. subovalisporaBZ2014-06ThailandMF461565–MF440409[4]
A. subovalisporaBZ2015-70ThailandMF461562–MF440421[4]
A. subovalisporaHKAS128053ChinaOR042727OR046363OR051754This study
A. subovalisporaEFHAAU2621ChinaOR042728––This study
A. subovalisporaEFHAAU3558ChinaOR042729––This study
A. subovalisporaEFHAAU4075ChinaOR042730––This study
A. subovalisporaEFHAAU4480ChinaOR042731OR046364OR051755This study
A. subovalisporaHKAS128050ChinaOR042732OR046365OR051756This study
A. subtropicanaTM 15-995IndiaMG923799––Direct sub.
A. sulcatissimaTRTC176558BrazilON392674ON540324ON492094[33]
A. cf. sulcatissimaTRTC176754BrazilON470140ON540342ON492103[33]
A. tenuifulvaHKAS87120ChinaMH486929MH509146MH486322[2]
A. tenuifulvaHKAS58877ChinaMH486928MH509145–[2]
A. tomentosivolvaHKAS108152ChinaOR042733OR046366OR051758This study
A. umbrinoluteaHKAS89201ChinaMH486933MH509150MH486326[2]
A. umbrinoluteaMB-000658GermanyMH486937–MH486330[2]
A. vaginata var. vaginataHAvdAasn_HolandThe NetherlandsAF024482––[49]
A. cf. velosaTRTC157486CanadaON392642ON492127–[33]
A. verrucosivolvaHKAS28253ChinaAF024483––[49]
A. verrucosivolvaHKAS75608ChinaMH486939MH509156MH486332[2]
A. vladimiriiBRNM825829 Czech RepublicMW208921MW208626–[31]
A. zonataEFHAAU607ChinaOR042734OR046367 This study
A. zonataEFHAAU709ChinaOR042735OR046368 This study
A. zonataEFHAAU755ChinaOR042736OR046369OR051757This study
A. zonataHKAS97240ChinaMH486959MH509179MH486352[2]
A. zonataHKAS97244ChinaMH486960MH509180MH486353[2]
A.cf. zonataEFHAAU2254ChinaOR042686– This study
Amanita sp. RET 732-8USAMT013999––Direct sub.
Amanita sp.RET 374-3USAMN614413––Direct sub.
Amanita sp.TRTC156902CanadaON392647ON492128–[33]
Amanita sp.TRTC176759BrazilON392666ON540336ON492124[33]
Amanita sp.JEIC0513GuineaON843345ON894338ON854987[33]
Amanita sp.JEIC0674BeninON843350ON894344ON854993[33]
Amanita sp.JEIC0737BeninON843357ON894351ON854997[33]
Amanita sp.JEIC0592TogoON843359ON894353–[33]
Amanita sp.JEIC0510GuineaON843360ON894355ON854998[33]
Amanita sp.JEIC0583BeninON843362ON894357–[33]
Amanita sp.JEIC0625BeninON843366ON894358ON855001[33]
Amanita sp.JEIC0599TogoON843367ON894359ON855002[33]
Amanita sp.JEIC0691Benin–ON931616ON855003[33]
Amanita sp.INPA No. 265223BrazilON392669ON492134ON492118[33]
Amanita sp.JEIC0723BeninON843369ON894361ON855005[33]
Amanita sp.JEIC0602Ivory CoastON843370ON894362–[33]
Amanita sp.JEIC0598TogoON843371ON894363–[33]
Amanita spTRTC157487CanadaON392648ON520571–[33]
Amanita sp.INPA No. 265290BrazilON392671ON492136ON492120[33]
Amanita sp.TRTC176599BrazilON392673ON540335ON492121[33]
Amanita sp.JEIC0652BeninON843339–ON854981[33]
Amanita sp.JEIC0724BeninON843343ON894336ON854985[33]
Outgroup
A. caesareaHKAS96166ItalyMH486418MH508705MH485898[2]
A. muscariaMB-001171GermnayMH486652MH508909MH486101[2]
A. parvipantherinaHKAS54723ChinaKR824780KR824807 KR824802[53]
A. yuanianaHKAS58807ChinaMH486954MH509174MH486347[2]
Quotation marks are added to indicate the uncertain taxonomic positions, – represents missing corresponding sequences.

3. Results

3.1. Phylogenetic Analyses

Overall, 163 sequences—including 56 for nrLSU, 42 for tef1-α, 37 for rpb2, and 28 for ITS—were newly generated in this study, and they were aligned with the sequences downloaded from GenBank. The sequences retrieved from GenBank and obtained in this study are listed in Table 1 and Table S1. The concatenated dataset (nrLSU, tef1-α, and rpb2) included 429 sequences from 201 samples representing 112 taxa (Table 1). The raw concatenated dataset comprised 2146 positions, and the final matrix retained 1846 positions, with 646 parsimony-informative sites, after excluding introns and poorly aligned regions. In the ITS dataset, 128 sequences from 64 taxa were included (Table S1). The dataset comprised 903 positions, with 384 parsimony-informative sites, and 570 positions of the ambiguously aligned regions were excluded. Six and two subsets were selected for the combined and ITS datasets, respectively. The best partition schemes and corresponding best-fits models are summarized in Table 2.
The phylogenetic trees inferred from the ML and BI analyses were similar in topology. Therefore, only the trees obtained from the ML analyses were presented (Figure 1 and Figures S1–S4). In the phylogenetic tree based on the combined matrix, the collections from eastern China were clustered into 19 lineages, including 12 known species and seven undescribed taxa (Figure 1). Four of them were described as new species, namely A. circulata, A. multicingulata, A. orientalis, and A. sinofulva (Figure 1 and Figures S1–S4). Amanita circulata formed a monophyletic clade with A. flavidocerea Thongbai et al. from Thailand, A. pekeoides G.S. Ridl. from New Zealand, A. verrucosivolva Zhu L. Yang from China, and three undescribed taxa (A. aff. fulva HKAS29518 China, Amanita sp. RET 732-8, and 374-3 USA, A. cf. circulata China) (Figure 1 and Figure S4). Amanita multicingulata was sister to A. liquii Zhu L. Yang et al. from China. Amanita orientalis formed a monophyletic group with A. griseofolia Zhu L. Yang and another two species from South Korea, erroneously identified as A. ceciliae (Berk. & Broome) Bas. In the phylogenetic tree inferred from the ITS dataset, the taxon was more closely related to the sample (JL2) from China, which was labeled as A. griseofolia, and two collections (SUA441 and SUA510) from Pakistan, with moderate support (Figure S4). Amanita sinofulva was clustered in the clade formed by A. orientifulva Zhu L. Yang et al., A. suborientifulva Raspé et al., and another five collections from China, Japan, and South Korea, which were labeled as A. cf. suborientifulva (EFHAAU4437 and EFHAAU5291), or erroneously identified as A. orientifulva (STDS-2-10 and KA12-0642) and A. fulva Fr. (ASIS26398), respectively (Figure 1 and Figure S4).
The remaining lineages represented three putatively new taxa. As only one or two collections were included in every species, they will be described in the future with adequate samples.

3.2. Taxonomy

Amanita circulata Y.Y. Cui, Q. Cai and Zhu L. Yang, sp. nov., Figure 1, Figure 2 and Figure 3.
Fungal Names: FN 571585.
Etymology: circulata from circular, referring to its circular zone on the pileus.
Diagnosis: Similar to A. pallidozonata, but differs in its more filamentous hyphae in the volval remnants on the stipe base.
Type: CHINA. YUNNAN PROVINCE: Puer, Lancang Lahu Autonomous County, in a broad-leaved forest with trees of Fagaceae, altitude 1780 m, 20 August 2016, LC-LJW 39 (Holotype, HKAS 97543, GenBank Acc. Nos.: nrLSU = OR042725, ITS = OR042765, rpb2 = OR051752, tef1-α = OR046361).
Description: Basidioma small to medium-sized. Pileus 3–7.5 cm diam., convex, plano-convex to applanate, umbonate; surface gray-brown (4E2–4) to dark brown (3F6–8) at center and margin, forming a distinctly pale colored [brown (3D2–4) to brownish (2C2–4)] ring-like zone at proximal end of marginal striations; volval remnants on pileus absent; margin striate (0.2–0.5 R), non-appendiculate; trama white (1A1), unchanging. Lamellae free, crowded, white (1A1); lamellar edges white (1A1); lamellulae truncate, plentiful. Stipe 9–18 cm long × 0.5–1.5 cm diam., slender, subcylindric, slightly tapering upwards, with apex slightly expanded, white (1A1), gray (1B1), brownish (2B2–4) to gray-brown (2C2–4); context white (1A1), hollow in center; basal bulb absent; volva saccate, membranous, both surfaces white (1A1). Annulus absent. Odor indistinct.
Lamellar trama bilateral. Mediostratum 20–40 μm wide, composed of abundant, ellipsoid inflated cells (25–60 × 10–30 μm); filamentous hyphae abundant, 2–8 μm wide; vascular hyphae scarce. Lateral stratum composed of abundant, ellipsoid to fusiform inflated cells (20–40 × 10–25 μm), diverging at an angle of ca. 30° to 60° to mediostratum; filamentous hyphae abundant and 2–7 μm wide. Subhymenium 30–40 μm thick, with 2–3 layers of ellipsoid to fusiform or irregularly arranged cells, 5–10 × 5–10 μm. Basidia 45–60 × 15–20 μm, clavate, 4-spored; sterigmata 5–8 μm long; basal septa lacking clamps. Basidiospores [60/3/3] (10.5–) 11–13 (–13.5) × (9.5–) 10–12.5 (–13) μm, Q = 1–1.15 (–1.21), Qm = 1.08 ± 0.05, globose to subglobose, occasionally broadly ellipsoid, inamyloid, colorless, thin-walled, smooth; apiculus small. Lamellar edge appearing as a sterile strip, composed of subglobose to ellipsoid or sphaeropedunculate inflated cells (15–50 × 10–45 μm), single and terminal or in chains of 2–3, thin-walled, colorless; filamentous hyphae abundant, 2–6 μm wide, irregularly arranged or ± running parallel to lamellar edge. Pileipellis 50–90 μm thick; upper layer (15–40 μm thick) gelatinized, composed of radially arranged to interwoven, thin-walled, colorless, filamentous hyphae 2–5 μm wide; lower layer (35–50 μm thick) composed of radially arranged, filamentous hyphae 3–6 μm wide, colorless to brownish; vascular hyphae scarce. Interior of volval remnants on stipe base composed of longitudinally arranged elements: filamentous hyphae dominant and very abundant, 3–10 μm wide, colorless, thin-walled, branching, anastomosing; inflated cells rare, globose, subglobose, ellipsoid to fusiform, 50–80 × 40–50 μm, colorless, thin-walled, mostly terminal or sometimes in chains of 2–3. Outer and inner surface of volval remnants on stipe base similar to structure of interior part, but with inner surface gelatinized. Stipe trama composed of longitudinally arranged, clavate terminal cells, 80–250 × 15–40 μm; filamentous hyphae scattered to abundant, 2–10 μm wide; vascular hyphae scarce. Clamps absent in all parts of basidioma.
Habitat: Solitary to scattered on soil in subtropical mixed forests with Fagaceae and Pinaceae.
Distribution: known from eastern and southwestern China.
Additional specimens examined: CHINA. ANHUI PROVINCE: Huangshan, in a broad-leaved forest with trees of Fagaceae, altitude 620 m, 13 July 2018, Hong-Yu Chen 32 (HKAS 127629); same location, in a broad-leaved forest with trees of Fagaceae, altitude 610 m, 12 July 2018, Ting Guo 979 (HKAS 127639). YUNNAN PROVINCE: Baoshan, Tengchong, in a mixed forest with trees of Pinus, Quercus and Keteleeria, altitude 1900 m, 20 July 2009, Li-Ping Tang 858 (HKAS 56815); same county, in a forest with trees of Pinus armandii Franch. and Keteleeria fortune (A. Murray bis) Carrière, altitude 2010 m, 14 August 2010, Qing Cai 391 (HKAS 67955); same city, Changning County, forest type unknown, altitude 2000 m, 25 July 2009, Gang Wu 4 (HKAS 57535); Kunming, Panlong District, in a mixed forest with trees of Fagaceae and Pinaceae, altitude 1990 m, 21 August 2016, Xiao-Xia Ding 111 (HKAS 97054); same city, Wuhua District, in a mixed forest with trees of Fagaceae and Pinaceae, altitude 1990 m, 6 September 2012, Yan-Jia Hao 753 (HKAS 76411); Lincang, Fengqing County, in a mixed forest with trees of Fagaceae and Pinaceae, altitude 1800 m, 26 July 2009, Gang Wu 12 (HKAS 57543); Puer, Lancang Lahu Autonomous County, in a mixed forest with trees of Fagaceae and Pinaceae, altitude 1780 m, 29 September 2016, LC-LJW 280 (HKAS 97784); same county, in a forest dominated with trees of Fagaceae, altitude 1350 m, 31 August 2017, Zhu L. Yang 6049 (HKAS 101238).
Notes: Amanita circulata is somewhat related to A. flavidocerea (Figure 1). However, the latter can be easily distinguished from the former species by its non-umbonate pileus, which is yellow and lacks a ring-like zone at the proximal end of marginal striations [4]. Amanita pallidozonata and A. zonata Y.Y. Cui et al. might be confused with A. circulata due to the pronounced ring-like zones at the proximal end of the marginal striations. However, A. pallidozonata differs from A. circulata by its more inflated cells in the inner part of volval remnants on the stipe base [2]. Amanita zonata has relatively smaller basidiospores (9–10.5 × 8.5–10 μm) [2].
Amanita multicingulata Y.Y. Cui, Q. Cai and Zhu L. Yang, sp. nov., Figure 1, Figure 2 and Figure 4.
Fungal Names: FN 571586.
Etymology: multicingulata named after its tomentose volval remnants often arranged in incomplete rings on the stipe base.
Diagnosis: Close to A. liquii, but differs in its longer striations on pileal margin, white to dirty white lamellae without obvious color change when dried, smaller basidiospores and distributions in subtropical forests dominated with Fagaceae, sometimes mixed with Pinus.
Type: CHINA. ANHUI PROVINCE: Huangshan, in a forest dominated with Fagaceae, altitude 1390 m, 13 July 2018, Ting Guo 1017 (Holotype, HKAS 127630, GenBank Acc. Nos.: nrLSU = OR042713, ITS = OR042750, rpb2 = OR051745, tef1-α = OR046352).
Description: Basidioma small, medium-sized to large. Pileus 3–11 cm diam., plano-convex to applanate, surface gray-brown (2C2–4), brown (3E3–5) to yellow-brown (4C4–6), often darker at center; volval remnants on pileus verrucose to felted, dark gray (1E1–3) to gray (1C1–3), often dirty white (1B1) at apical part; margin striate (0.3–0.6 R), non-appendiculate; trama white (1A1) to dirty white (1B1), unchanging. Lamellae free, crowded, white (1A1) to dirty white (1B1), sometimes with brownish (3B2–3) tinge; lamellar edges gray (1B1), gray-brown (1B2) to brown (3B2–4); lamellulae truncate, plentiful. Stipe 10–17 cm long × 0.5–1.5 cm diam., slender, subcylindric, slightly tapering upwards, with apex slightly expanded, grayish (1B1), brownish (1B2) to gray-brown (2D2–4), covered with concolor squamules; context white (1A1) to dirty white (1B1), hollow in center; basal bulb absent; volval remnants on stipe base tomentose, arranged in incomplete rings, gray (1C1–3) to gray-brown (1B2). Annulus absent. Odor indistinct.
Lamellar trama bilateral. Mediostratum 20–30 μm wide, composed of abundant, clavate inflated cells (50–80 × 10–20 μm); filamentous hyphae abundant, 2–8 μm wide; vascular hyphae scarce. Lateral stratum composed of abundant, ellipsoid to fusiform inflated cells (20–40 × 10–20 μm), diverging at an angle of ca. 30° to 60° to mediostratum; filamentous hyphae abundant and 2–8 μm wide. Subhymenium 30–50 μm thick, with 2–3 layers of ellipsoid to fusiform or irregularly arranged cells, 10–30 × 8–20 μm. Basidia 40–60 × 13–18 μm, clavate, 4-spored; sterigmata 5–8 μm long; basal septa lacking clamps. Basidiospores [40/2/2] (9.5–) 10–12 (–12.5) × (9–) 9.5–11 (–11.5) μm, Q = 1–1.13 (–1.15), Qm = 1.06 ± 0.03, globose to subglobose, inamyloid, colorless, thin-walled, smooth; apiculus small. Lamellar edge appearing as a sterile strip, composed of subglobose, ellipsoid to clavate inflated cells (10–45 × 10–30 μm), single and terminal or in chains of 2–3, thin-walled, colorless; filamentous hyphae abundant, 2–8 μm wide, irregularly arranged or ± running parallel to lamellar edge. Pileipellis 50–100 μm thick; upper layer (30–50 μm thick) gelatinized, composed of radially arranged to interwoven, thin-walled, colorless filamentous hyphae 2–5 μm wide; lower layer (40–50 μm thick) composed of radially arranged filamentous hyphae 4–7 μm wide, colorless; vascular hyphae scarce. Volval remnants on pileus composed of more or less vertically arranged elements: inflated cells very abundant to dominant, globose, subglobose, ellipsoid to fusiform, 10–60 × 10–50 μm, brown to brownish or colorless, thin-walled, mostly terminal or sometimes in chains of 2–3; filamentous hyphae rare, 3–7 μm wide, brown to brownish or colorless, thin-walled, branching, anastomosing. Volval remnants on stipe base composed of longitudinally arranged elements, becoming horizontally arranged towards upper parts: inflated cells very abundant to nearly dominant, globose, subglobose, ellipsoid, fusiform to clavate, 20–80 × 10–50 μm, brown to brownish or colorless, thin-walled, mostly terminal or sometimes in chains of 2–3; filamentous hyphae rare to fairly abundant, 2–8 μm wide, brown to brownish or colorless, thin-walled, branching, anastomosing. Stipe trama composed of longitudinally arranged, clavate terminal cells, 100–400 × 15–40 μm; filamentous hyphae scattered to abundant, 2–10 μm wide; vascular hyphae scarce. Clamps absent in all parts of basidioma.
Habitat: Solitary to scattered on soil in subtropical broad-leaved forests dominated with Fagaceae, sometimes in mixed forests with fagaceous and Pinus plants.
Distribution: Known from eastern China.
Additional specimens examined: CHINA. ANHUI PROVINCE: Huangshan, in a forest dominated with Fagaceae, altitude 1300 m, 13 July 2018, Ting Guo 1018 (HKAS 127631); same location, in a forest dominated with Fagaceae, altitude 670 m, 13 July 2018, Rui-Heng Yang 73 (HKAS 127632); same location, in a forest with Fagaceae and Pinaceae, altitude 940 m, 15 July 2018, Rui-Heng Yang 123 (HKAS 127633); same location, in a forest dominated with Fagaceae, altitude 760 m, 15 July 2018, Rui-Heng Yang 127 (HKAS 127634); same location, in a forest dominated with Fagaceae, altitude 1220 m, 17 July 2018, Rui-Heng Yang 184 (HKAS 127635); same location, in a forest dominated with Fagaceae, altitude 1200 m, 17 July 2018, Rui-Heng Yang 186 (HKAS 127636); same location, in a forest dominated with Fagaceae, altitude 940 m, 15 July 2018, Hong-Yu Chen 77 (HKAS 127637).
Notes: Based on molecular phylogenetic analysis, A. multicingulata is closely related to A. liquii (Figure 1 and Figure S4), but the latter species has shorter striations on pileal margin (0.1–0.3 R), larger basidiospores (11.5–15 × 11–14.5 μm) and occurs in subalpine forests dominated by trees of Picea and Abies [2,35,54]. In addition, A. liquii has white to grayish lamellae that turn dark gray to dark brown when dried [2,35,54]. Amanita cinctipes Corner and Bas, A. griseofolia and A. neocinctipes Zhu L. Yang et al. of A. sect. Vaginatae with a nonsaccate volva were also reported from China. Smaller basidiospores (9–10.5 × 8–9.5 μm) and shorter striations on the pileal margin (0.3–0.4 R) distinguish A. cinctipes from A. multicingulata [2,29]. Amanita griseofolia differs from A. multicingulata by its more grayish pileus and slightly larger basidiospores (10–13.5 × 9.5–13 μm) [1,2,55]. White lamellae and subglobose to broadly ellipsoid basidiospores (8.0–10.5 × 7.0–9.0 μm, Q = 1.09–1.29, Qm = 1.19 ± 0.07) in A. neocinctipes set it apart from A. multicingulata [2].
Amanita orientalis Q. Cai, Y.Y. Cui and Zhu L. Yang, sp. nov., Figure 1, Figure 2 and Figure 5.
Fungal Names: FN 571587
Etymology: orientalis means eastern, namely after its type locality from East Asia.
Diagnosis: Close to A. griseofolia but differs in its more brownish pileus and mostly subglobose to broadly ellipsoid basidiospores.
Type: CHINA. ANHUI PROVINCE: Huangshan, in a mixed forest with Fagaceae and Pinaceae, altitude 860 m, 15 September 2018, Guo-Qi Chu 153 (Holotype, EFHAAU 1367, GenBank Acc. Nos.: nrLSU = OR042698, ITS = OR042759, rpb2 = OR051735).
Description: Basidioma small to medium-sized. Pileus 5–7 cm diam., plano-convex to applanate, surface gray-brown (3E3–4) to brown (3D2–4), often darker at center; volval remnants on pileus verrucose to felted, gray (3E1–3), often dirty white (1B1) at apical part; margin striate (0.3–0.5 R), non-appendiculate; trama white (1A1), unchanging. Lamellae free, crowded, white (1A1); lamellar edges white (1A1) to slightly grayish (1B1); lamellulae truncate, plentiful. Stipe 8.5–14.5 cm long × 0.5–1 cm diam., slender, subcylindric, slightly tapering upwards, with apex slightly expanded, dirty white, gray (1B1) to brownish (1B2), covered with gray (1B1) to gray-brown (3C1–3) squamules; context white (1A1), hollow in center; basal bulb absent; volval remnants on stipe tomentose, arranged in incomplete rings, gray (1B1) to gray-brown (3D2–4). Annulus absent. Odor indistinct.
Lamellar trama bilateral. Mediostratum 15–30 μm wide, composed of abundant, clavate inflated cells (30–60 × 10–20 μm); filamentous hyphae abundant, 2–7 μm wide; vascular hyphae scarce. Lateral stratum composed of abundant, ellipsoid to fusiform inflated cells (5–15 × 5–15 μm), diverging at an angle of ca. 30° to 45° to mediostratum; filamentous hyphae abundant and 2–8 μm wide. Subhymenium 20–45 μm thick, with 2–3 layers of ellipsoid to fusiform or irregularly arranged cells, 10–25 × 8–20 μm. Basidia 40–65 × 15–18 μm, clavate, 4-spored; sterigmata 4–6 μm long; basal septa lacking clamps. Basidiospores [40/2/2] (10–) 10.5–13 × 9–12 (–13) μm, Q = 1–1.26 (–1.31), Qm = 1.13 ± 0.07, subglobose to broadly ellipsoid, sometimes globose, inamyloid, colorless, thin-walled, smooth; apiculus small. Lamellar edge appearing as a sterile strip, composed of subglobose, ellipsoid to clavate inflated cells (10–50 × 10–30 μm), single and terminal or in chains of 2–3, thin-walled, colorless; filamentous hyphae abundant, 3–7 μm wide, irregularly arranged or ± running parallel to lamellar edge. Pileipellis 50–100 μm thick; upper layer (30–50 μm thick) gelatinized, composed of radially arranged to interwoven, thin-walled, colorless to brownish filamentous hyphae 2–6 μm wide; lower layer (30–40 μm thick) composed of radially arranged filamentous hyphae 3–8 μm wide, brownish to brown; vascular hyphae scarce. Volval remnants on stipe base composed of longitudinally arranged elements: inflated cells abundant, globose, subglobose, ellipsoid, fusiform to clavate, 20–40 × 10–40 μm, yellow-brown to gray-brown, thin-walled, mostly terminal or sometimes in chains of 2–3; filamentous hyphae abundant, 2–7 μm wide, yellow-brown to gray-brown, thin-walled, branching, anastomosing. Stipe trama composed of longitudinally arranged, clavate terminal cells, 80–300 × 15–40 μm; filamentous hyphae scattered to abundant, 2–10 μm wide; vascular hyphae scarce. Clamps absent in all parts of basidioma.
Habitat: Solitary to scattered on soil in subtropical forests with Fagaceae and Pinaceae.
Distribution: Known from eastern China.
Additional specimens examined: CHINA. ANHUI PROVINCE: Huangshan, in a mixed forest with Fagaceae and Pinaceae, altitude 760 m, 14 July 2018, Rui-Heng Yang 117 (HKAS 127638).
Notes: By having a more grayish pileus and globose to subglobose basidiospores (10–13.5 × 9.5–13 μm, Q = 1.0–1.1, Qm = 1.04 ± 0.03), A. griseofolia can be distinguished from A. orientalis [2,35,55]. Due to the similarity of their volval remnants on the base of the stipe, the Chinese records of A. multicingulata, A. cinctipes Corner and Bas, A. neocinctipes, and A. liquii can be confused with A. orientalis. Nevertheless, A. multicingulata differs from A. orientalis by having basidiospores that are rounder (globose to subglobose, 10–12 × 9.5–11 μm, Q = 1–1.13, Qm = 1.06 ± 0.03). Amanita cinctipes differs from A. orientalis by its more grayish pileus and smaller and rounder basidiospores (9–10.5 × 8–9.5 μm, Q = 1.0–1.16, Qm = 1.08 ± 0.04) [2,29,35]. The more grayish pileus and smaller basidiospores (8–10.5 × 7–9 μm) of A. neocinctipes distinguish it from A. orientalis [2]. Amanita liquii can be distinguished from A. orientalis by its larger basidioma, dark brown to black pileus with shorter striations on its margin (0.1–0.3 R), white to grayish lamellae that turn dark gray when dried, larger and rounded basidiospores (11.5–15 × 11–14.5 μm, Q = 1.0–1.09, Qm = 1.05 ± 0.04), and distribution in alpine to subalpine forests [2,35,54].
Multi-locus phylogenetic research reveals that A. griseofolia and the South Korean ‘A. ceciliae’ are relatives of A. orientalis (Figure 1). The European A. ceciliae can be distinguished from A. orientalis by its robust basidioma with a brown pileus with a yellow tint and globose to subglobose basidiospores [1,35,56,57,58,59]. Phylogenetically, the aforementioned three taxa seem to be close but clearly different (Figure 1).
Amanita sinofulva Q. Cai, Y.Y. Cui and Zhu L. Yang, sp. nov., Figure 1, Figure 2 and Figure 6.
Fungal Names: FN 571588.
Etymology: sinofulva refers to the fact that this species is found in China and resembles A. fulva.
Diagnosis: Close to A. orientifulva and A. suborientifulva, but A. orientifulva has slightly narrower basidiospores and grows in subalpine forests. Amanita suborientifulva has a non-umbonate pileus and globose to subglobose or broadly ellipsoid basidiospores.
Type: CHINA. YUNNAN PROVINCE: Dali, Nanjian Yizu Autonomous County, in a broad-leaved forest with trees of Fagaceae, altitude 2515 m, 27 June 2015, Kuan Zhao 727 (Holotype, HKAS 92355, GenBank Acc. Nos.: nrLSU = OR042703, ITS = OR042741, rpb2 = OR051738, tef1-α = OR046345).
Description: Basidioma small to medium-sized. Pileus 3–9 cm diam., plano-convex to applanate, umbonate at center, surface brown (4E6–8) to yellow-brown (5D6–8), often darker at center; volval remnants on pileus absent; margin striate (0.2–0.5 R), non-appendiculate; trama white (1A1), unchanging. Lamellae free, crowded, white (1A1); lamellar edges white (1A1), brownish (4B3–5) to brown (5B3–4); lamellulae truncate, plentiful. Stipe 6–18 cm long × 0.5–1.5 cm diam., slender, subcylindric, slightly tapering upwards, with apex slightly expanded, brown (5B3–4) to brownish (4B3–5); context white (1A1), hollow in center; basal bulb absent; volva saccate, membranous, outer surface white (1A1) with yellow-brown (4B2–4) stains, often yellow-brown (4B2–4) at upper margin, inner surface brownish (5B2–4). Annulus absent. Odor indistinct.
Lamellar trama bilateral. Mediostratum 20–40 μm wide, composed of abundant, fusiform, ellipsoid to clavate inflated cells (25–100 × 10–30 μm); filamentous hyphae abundant, 2–10 μm wide; vascular hyphae scarce. Lateral stratum composed of abundant, ellipsoid to fusiform inflated cells (5–15 × 5–15 μm), diverging at an angle of ca. 30° to 60° to mediostratum; filamentous hyphae abundant and 3–8 μm wide. Subhymenium 30–50 μm thick, with 2–3 layers of ellipsoid to fusiform or irregularly arranged cells, 5–20 × 5–20 μm. Basidia 50–70 × 15–20 μm, clavate, 4-spored; sterigmata 6–8 μm long; basal septa lacking clamps. Basidiospores [40/2/2] (10.5–) 11–13.5 (–18) × (9–) 9.5–12 (–13) μm, Q = (1.01–) 1.05–1.23 (–1.34), Qm = 1.14 ± 0.08, subglobose to broadly ellipsoid, occasionally globose or ellipsoid, inamyloid, colorless, thin-walled, smooth; apiculus small. Lamellar edge appearing as a sterile strip, composed of subglobose, ellipsoid to clavate inflated cells (10–30 × 10–25 μm), single and terminal or in chains of 2–3, thin-walled, colorless; filamentous hyphae abundant, 2–8 μm wide, irregularly arranged or ± running parallel to lamellar edge. Pileipellis 50–90 μm thick; upper layer (30–50 μm thick) gelatinized, composed of radially arranged to interwoven, thin-walled, colorless to brownish filamentous hyphae 2–5 μm wide; lower layer (35–50 μm thick) composed of radially arranged filamentous hyphae 3–7 μm wide, colorless to brownish; vascular hyphae scarce. Interior of volval remnants on stipe base composed of longitudinally arranged elements: filamentous hyphae dominant and very abundant, 3–7 μm wide, colorless, thin-walled, branching, anastomosing; inflated cells rare to fairly abundant, subglobose, ellipsoid to fusiform, 40–65 × 15–50 μm, colorless, thin-walled, mostly terminal or sometimes in chains of 2–3. Stipe trama composed of longitudinally arranged, clavate terminal cells, 80–300 × 15–50 μm; filamentous hyphae scattered to abundant, 3–10 μm wide; vascular hyphae scarce. Clamps absent in all parts of basidioma.
Habitat: Solitary to scattered on soil in subtropical forests dominated with Fagaceae, sometimes mixed with Pinus.
Distribution: Known from eastern, central, and southwestern China. Based on the phylogenetic tree inferred from the ITS dataset, it also occurs in Tibet autonomous region and Hunan province (Figure S4).
Additional specimens examined: CHINA. ANHUI PROVINCE: Liuan, Jinzhai County, in a forest dominated with Fagaceae, altitude 1110 m, 21 July 2017, Yan-Jia Hao 1520 (HKAS 100610); same county, in a mixed forest with Castanea seguinii Dode and Pinus taiwanensis Hayata, altitude 840 m, Yan-Jia Hao 1609 (EFHAAU 207); same county, in a mixed forest with Fagaceae and Pinaceae, altitude 1000 m, Yan-Jia Hao 1715 (EFHAAU 313). YUNNAN PROVINCE: Nujiang Lisu Autonomous Prefecture, Lanping Bai and Pumi Autonomous County, in a subtropical forest dominated with Quercus, mixed with Pinus yunnanensis, altitude 2150 m, Gang Wu 743 (HKAS 75058).
Notes: Amanita orientifulva and A. suborientifulva can be confused with A. sinofulva. According to our multi-gene phylogenetic analysis (Figure 1), the first two species also share close relationships with A. sinofulva. However, A. orientifulva has slightly narrower basidiospores (10.0–14.0 × 9.5–13.0 μm, Q = 1.0–1.12, Qm = 1.06 ± 0.04) and is found in subalpine forests dominated by trees of Abies and Picea [2,54]. The non-umbonate pileus and globose to subglobose or broadly ellipsoid basidiospores of A. suborientifulva set it apart from A. sinofulva [4]. The European A. fulva is also similar to A. sinofulva, but differs in the globose to subglobose basidiospores and in the saccate volva, with inflated cells dominant in its outer part [1,2].

4. Discussion

4.1. Species Delimitation and Recognition within Amanita sect. Vaginatae

Our data revealed that several macro- and microscopic characteristics could be useful for the delimitation of species in A. sect. Vaginatae. Five of them are most informative, viz. the color of the basidiomata, the striations on the pileal margin, the presence or absence of the annulus, the volval remnants on the stipe base, and the size of the basidiospores. In this study, A. cingulata is the only species with a white basidioma, while the other species from eastern China have basidiomata ranging from yellow, to gray, to brown. The striations on the pileal margin of A. zonata, A. pallidozonata, and A. circulata form a ring-like zone at the proximal end, while the remaining taxa in eastern China are without this zone [2]. Most species in the section are ringless, with only seven taxa with an annulus [12,21,50]. The volval remnants on the stipe base of several species are saccate, while some of them are tomentose, arranged in incomplete rings, viz. A. griseofolia, A. multicingulata, and A. orientalis. Species with an annulus, a ring-like zone at the proximal end, or with incomplete rings of volval remnants on the stipe base are clustered in non-monophyletic groups.
Given that it is difficult to delimitate these species based solely on morphological studies, integrative taxonomy is indispensable in recognizing species of the section. This method, which delimits and describes taxa by integrating information from different types of data and methodologies (e.g., phylogeny, comparative morphology, habitat and preference of hosts, and behavior), is proven to be useful for species recognition in plants, animals, and fungi [2,60,61,62,63,64]. In this study, species with similar morphological characteristics are successfully recognized using this method.
For example, A. zonata, A. pallidozonata, and A. circulata are morphologically similar due to the pronounced ring-like zones at the proximal end of the marginal striations. However, they occupy different positions in the phylogenetic tree and are distantly related (Figure 1). Following detailed morphological studies, A. pallidozonata can be distinguished from A. circulata by its more inflated cells in the inner part of the volval remnants on the stipe base [2]. Amanita zonata differs from A. circulata by its smaller basidiospores (9–10.5 × 8.5–10 μm) [2].
Amanita sinofulva is phylogenetically close and morphologically similar to A. orientifulva. However, they are clustered into two independent lineages (Figure 1 and Figure S4), and differ in their geographic distributions and host plants. The former species is restricted to the subtropical forests dominated by the trees of Fagaceae and Pinus, while the latter is found in the subalpine forests under Picea spp., Abies spp., and Quercus spp. [2,54]. Furthermore, the latter can be distinguished from the former by its narrower basidiospores (10–14 × 9.5–13 μm).
Overall, the combination of morphological characteristics, multi-locus phylogeny, and ecological data can make the result of species delimitation more reliable and objective.

4.2. Phylogenetic Relationships of Amanita sect. Vaginatae Species in Southeast Asia and Southern Parts of China

In this study, 40 taxa of A. sect. Vaginatae were delimitated in China, including 33 known taxa [1,2,21], four species new to sciences (A. circulata, A. multicingulata, A. orientalis and A. sinofulva), and three species new to China (A. brunneoumbonata Thongbai et al., A. suborientifulva and A. subovalispora Thongbai et al.). Thirty-nine of them are reported from the southern parts of China—namely, southwestern, central, eastern, and southern China.
According to our phylogenetic analyses, species of the section from the southern parts of China are closely related to those reported from Southeast Asia. For example, of the 13 species reported from Southeast Asia [4,29,65,66], 7 of them also occur in the southern parts of China (Figure 1). Among them, A. angustilamellata (Höhn.) Boedijn, A. brunneoprocera Thongbai et al., A. cinctipes, and A. pallidocarnea (Höhn.) Boedijn are typical tropical elements restricted in the tropical areas of China. The other three species, viz. A. brunneosquamata Thongbai et al., A. suborientifulva, and A. subovalispora, extend their distribution from Southeast Asia to subtropical China. In addition, several taxa found in subtropical or subalpine temperate areas in southern parts of China are phylogenetically close to species from Southeast Asia. For example, A. circulata and A. zonata, reported from the subtropical regions of China, are sister to A. flavidocerea and A. flavidogrisea Thongbai et al. from Southeast Asia, respectively (Figure 1). Amanita pallidozonata from the subtropical areas and A. orientifulva from the subalpine forests of the southern parts of China are closely related to A. pallidocarnea from Southeast Asia and tropical China (Figure 1). Therefore, the species in the southern parts of China may have historical affinities in common with those of tropical Asia [67]. This was also consistent with the results of Codjia [33], in which part of the taxa in East Asia were indicated to have migrated from Southeast Asia.
Previously, only five species of A. sect. Vaginate were reported from eastern China [2]. In this study, 16 species were delimitated, with four new species and 12 newly recorded species. For the convenience of recognition, a key to them is provided.
Key to the Species of Amanita sect. Vaginatae from Eastern China
1.
Basidioma white; volval remnants on pileus present as patches; annulus present; basidiospores mostly ellipsoid to elongate…………….……………….…….…..………A. cingulata
1’.
Basidioma yellow, gray to brown; volval remnants on pileus usually absent, sometimes present as verrucae to felts; basidiospores mostly globose to subglobose, sometimes broadly ellipsoid………………………………………….….……………………….…………..2
2.
Volval remnants on pileus mostly present as verrucae to felts; volval remnants on stipe base tomentose, arranged in incomplete rings………………………….……….…………….3
2’.
Volval remnants on pileus often absent; volval remnants on stipe base saccate………………………………………………………………………………………………....5
3.
Basidiospores subglobose to broadly ellipsoid, 10.5–13 × 9–12 μm, Q = 1–1.26 (–1.31), Qm = 1.13 ± 0.07……………………………….………………………………………A. orientalis
3’.
Basidiospores more rounded, globose to subglobose……………………………………..4
4.
Basidioma more grayish; basidiospores slightly larger, 10–13.5 × 9.5–13 μm………………………………………………………………..……….…….……A. griseofolia
4’.
Basidioma more brownish; basidiospores slightly smaller, 10–12 × 9.5–11 μm………………………………………………………………….……………A. multicingulata
5.
Pileal surface forming a distinctive ring-like zone at proximal end of marginal striations………………………………….…………………………………..………………………...6
5’.v
Pileal surface without a distinctive ring-like zone at proximal end of marginal striations………………………………….……………………………………………..……………...8
6.
Pileal margin with relatively shorter striations, 0.15–0.3 R; basidiospores slightly smaller and rounder, globose to subglobose, 9–10.5 × 8.5–10 μm, Q = 1.00–1.11, Qm = 1.05 ± 0.04……………………………………...……………………………………..………….A. zonata
6’.
Pileal margin with relatively longer striations; basidiospores slightly larger, globose, subglobose to broadly ellipsoid……………………….………………………………………...7
7.
Basidiospores slightly smaller, 10–12 × 9–11 μm; volval remnants on stipe base with abundant inflated cells in inner part……………………………………………A. pallidozonata
7’.
Basidiospores slightly larger, 11–13 × 10–12.5 μm; volval remnants on stipe base mainly with abundant filamentous hyphae………………………….……………………...A. circulata
8.
Basidioma with distinctive yellow color…………………...…………………………..…….9
8’.
Basidioma gray to brown, without yellow color…………………………………………..10
9.
Striations on pileal margin relatively longer, 0.2–0.5 R; basidiospores broadly ellipsoid to ellipsoid, 10–12 × 8–9 μm, Q = 1.22–1.37, Qm = 1.3 ± 0.07……………….………A. hamadae
9’.
Striations on pileal margin relatively shorter, 0.2–0.3 R; basidiospores globose to subglobose, 9–11 × 9–11 μm, Q = 1–1.11, Qm = 1.05 ± 0.04……………………………….A. crocea
10.
Basidioma orange-brown to yellow-brown…………………….…………….…………..11
10’.
Basidioma gray to gray-brown………………………………………………….………...12
11.
Pileus without an umbo……………………………...……….……………A. suborientifulva
11’.
Pileus with a distinctive umbo at center……………...…………………...…...A. sinofulva
12.
Pileus gray-brown to yellow-brown, with distinct olivaceous tinge; basidiospores broadly ellipsoid to ellipsoid, relatively larger, 10.5–13 × 8.5–10 μm, Q = 1.05–1.45, Qm = 1.25 ± 0.09………………………….………………………………………………A. olivaceofusca
12’.
Pileus gray to brown, without olivaceous tinge; basidiospores globose to subglobose, or broadly ellipsoid to ellipsoid, relatively smaller…………………………………………13
13.
Pileus gray, without umbo at the center; basidiospores broadly ellipsoid to ellipsoid……………………………………………………………………………….………………14
13’.
Pileus gray-brown to brown, umbonate at center; basidiospores globose to subglobose………………………………………………………………………………………….……15
14.
Pileal margin with shorter striations, 0.36–0.4 R………………………….A. subovalispora
14’.
Pileal margin with longer striations, 0.4–0.6 R………...………………….......A. ovalispora
15.
Pileus dark brownish, with darker colored central disk; pileal margin with longer striations, 0.33–0.42 R…………………………………………………………..A. brunneoumbonata
15’.
Pileus grayish brown; pileal margin with shorter striations, 0.18–0.21 R………………………………………………………………………………..A. brunneoprocera

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof9080862/s1, Figure S1: Phylogenetic tree of Amanita sect. Vaginatae inferred from maximum likelihood analyses based on the nrLSU sequences. Bootstrap values over 50% are shown along the branches. Sequences from type collections are indicated with (T), and new species are in boldface; Figure S2: Phylogenetic tree of Amanita sect. Vaginatae inferred from maximum likelihood analyses based on the tef1-α sequences. Bootstrap values over 50% are shown along the branches. Sequences from type collections are indicated with (T), and new species are in boldface; Figure S3: Phylogenetic tree of Amanita sect. Vaginatae inferred from maximum likelihood analyses based on the rpb2 sequences. Bootstrap values over 50% are shown along the branches. Sequences from type collections are indicated with (T), and new species are in boldface; Figure S4: Phylogenetic tree of Amanita sect. Vaginatae inferred from maximum likelihood analyses based on the ITS sequences. Bootstrap values over 50% and Bayesian posterior probabilities over 0.90 are shown along the branches. Sequences from type collections are indicated with (T), and new species are in boldface; Table S1: Voucher information and GenBank accession numbers of the samples used in the phylogenetic analyses of ITS sequences. Sequences newly generated in this study are indicated in bold.

Author Contributions

Conceptualization: Q.C., Z.L.Y. and Y.-Y.C.; filed sampling: Q.C., Y.-J.H. and T.G.; molecular experiments and data analysis: Q.C. and Y.-Y.C.; original draft—writing: Y.-Y.C. and Q.C.; initial draft—review and editing: Z.L.Y. and Q.C. All authors have read and agreed to the published version of the manuscript.

Funding

The research is funded by the National Natural Science Foundation of China (Nos. 31970023, 32100015 and 31600031), the Natural Science Foundation of Yunnan Province (2018FB029 and 202101AT070165), the Yunnan Provincial High-Level Talents Support Program (YNWR-QNBJ-2020-295), and Ten-Thousand-Talents Plan–Yunling Scholar Project.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Publicly available datasets were analyzed in this study (https://www.ncbi.nlm.nih.gov/, accessed on 1 May 2023; https://nmdc.cn/fungalnames/, accessed on 25 May 2023).

Acknowledgments

We express our gratitude to X.X. Ding, J.W. Liu, G. Wu (Kunming Institute of Botany, Chinese Academy of Sciences), L.P. Tang (Kunming Medical University), and K. Zhao (Jiangxi Science and Technology Normal University) for providing samples. The reviewers’ comments and suggestions for improving the manuscript are highly appreciated.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree of Amanita sect. Vaginatae inferred from maximum likelihood analyses based on the combined dataset (nrLSU, tef1-α and rpb2). Bootstrap values over 50% and Bayesian posterior probabilities over 0.90 are shown along the branches. Sequences from type collections are indicated with (T), and new species are in boldface.
Figure 1. Phylogenetic tree of Amanita sect. Vaginatae inferred from maximum likelihood analyses based on the combined dataset (nrLSU, tef1-α and rpb2). Bootstrap values over 50% and Bayesian posterior probabilities over 0.90 are shown along the branches. Sequences from type collections are indicated with (T), and new species are in boldface.
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Figure 2. Fresh basidiomata of novel species in Amanita sect. Vaginatae from eastern China. (a,b) A. circulata ((a) HKAS 97784, (b) Holotype, HKAS 97543); (c,d) A. multicingulata (Holotype, HKAS 127630); (e,f) A. orientalis ((e) HKAS 127638, (f) EFHAAU 1367); (g,h) A. sinofulva (Holotype, HKAS92355). Bar = 2 cm.
Figure 2. Fresh basidiomata of novel species in Amanita sect. Vaginatae from eastern China. (a,b) A. circulata ((a) HKAS 97784, (b) Holotype, HKAS 97543); (c,d) A. multicingulata (Holotype, HKAS 127630); (e,f) A. orientalis ((e) HKAS 127638, (f) EFHAAU 1367); (g,h) A. sinofulva (Holotype, HKAS92355). Bar = 2 cm.
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Figure 3. Microscopic features of A. circulata (Holotype, HKAS 97543). (a). Hymenium and subhymenium; (b). Basidiospores; (c). Interior of volval remnants on the stipe base. Bars: (a,b) = 10 µm, (c) = 20 µm.
Figure 3. Microscopic features of A. circulata (Holotype, HKAS 97543). (a). Hymenium and subhymenium; (b). Basidiospores; (c). Interior of volval remnants on the stipe base. Bars: (a,b) = 10 µm, (c) = 20 µm.
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Figure 4. Microscopic features of A. multicingulata (Holotype, HKAS 127630). (a). Hymenium and subhymenium; (b). Basidiospores; (c). Volval remnants on the pileus; (d) Volval remnants on the stipe base (right side indicates outer part). Bars: (a,b) = 10 µm, (c,d) = 20 µm.
Figure 4. Microscopic features of A. multicingulata (Holotype, HKAS 127630). (a). Hymenium and subhymenium; (b). Basidiospores; (c). Volval remnants on the pileus; (d) Volval remnants on the stipe base (right side indicates outer part). Bars: (a,b) = 10 µm, (c,d) = 20 µm.
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Figure 5. Microscopic features of A. orientalis (Holotype, EFHAAU 1367). (a). Hymenium and subhymenium; (b). Basidiospores; (c). Interior of volval remnants on the stipe base (right side indicates outer part). Bars: (a,b) = 10 µm, (c) = 20 µm.
Figure 5. Microscopic features of A. orientalis (Holotype, EFHAAU 1367). (a). Hymenium and subhymenium; (b). Basidiospores; (c). Interior of volval remnants on the stipe base (right side indicates outer part). Bars: (a,b) = 10 µm, (c) = 20 µm.
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Figure 6. Microscopic features of A. sinofulva (Holotype, HKAS 92355). (a). Hymenium and subhymenium; (b). Basidiospores; (c). Interior of volval remnants on the stipe base. Bars: (a,b) = 10 µm, (c) = 20 µm.
Figure 6. Microscopic features of A. sinofulva (Holotype, HKAS 92355). (a). Hymenium and subhymenium; (b). Basidiospores; (c). Interior of volval remnants on the stipe base. Bars: (a,b) = 10 µm, (c) = 20 µm.
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Table 2. The best partition schemes and models selected by PartitionFinder.
Table 2. The best partition schemes and models selected by PartitionFinder.
Subsets in the Best-Fit Partition Scheme The Base Positions of Each Subset Best-Fit Model
nrLSU1–775TIM+I+G
tef1-α_condon1776–1184\3TVMef+I+G
tef1-α_condon2, rpb2_condon3777–1184\3, 1187–1846\3SYM+I+G
tef1-α_condon3778–1184\3TIM+I+G
rpb2_condon11185–1846\3GTR+I+G
rpb2_condon21186–1846\3HKY+I+G
ITS1, ITS21–92, 250–333HKY+I+G
5.8S94–249TIMef+I+G
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MDPI and ACS Style

Cui, Y.-Y.; Hao, Y.-J.; Guo, T.; Yang, Z.L.; Cai, Q. Species Diversity of Amanita Section Vaginatae in Eastern China, with a Description of Four New Species. J. Fungi 2023, 9, 862. https://doi.org/10.3390/jof9080862

AMA Style

Cui Y-Y, Hao Y-J, Guo T, Yang ZL, Cai Q. Species Diversity of Amanita Section Vaginatae in Eastern China, with a Description of Four New Species. Journal of Fungi. 2023; 9(8):862. https://doi.org/10.3390/jof9080862

Chicago/Turabian Style

Cui, Yang-Yang, Yan-Jia Hao, Ting Guo, Zhu L. Yang, and Qing Cai. 2023. "Species Diversity of Amanita Section Vaginatae in Eastern China, with a Description of Four New Species" Journal of Fungi 9, no. 8: 862. https://doi.org/10.3390/jof9080862

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