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Research Article
Two new Inosperma (Inocybaceae) species with unexpected muscarine contents from tropical China
expand article infoLun-Sha Deng‡, Rui Kang§, Nian-Kai Zeng‡, Wen-Jie Yu‡, Cheng Chang|, Fei Xu, Wang-Qiu Deng#, Liang-Liang Qi¤, Yu-Ling Zhou§, Yu-Guang Fan‡
‡ Hainan Medical University, Haikou, China
§ Hainan Institute for Food Control, Haikou, China
| Jilin Provincial Joint Key Laboratory of Changbai Mountain Biocoenosis and Biodiversity, Changbai Mountain Academy of Sciences, Yanbian, China
¶ Physical and Chemical Department, Ningxia Hui Autonomous Region Center for Disease Control and Prevention, Yinchuan, China
# Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China
¤ Microbiology Research Institute, Guangxi Academy of Agriculture Sciences, Haikou, China

Corresponding author: Yu-Guang Fan ( mycena@qq.com )

Academic editor: Thorsten Lumbsch
© 2021 Lun-Sha Deng, Rui Kang, Nian-Kai Zeng, Wen-Jie Yu, Cheng Chang, Fei Xu, Wang-Qiu Deng, Liang-Liang Qi, Yu-Ling Zhou, Yu-Guang Fan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Deng L-S, Kang R, Zeng N-K, Yu W-J, Chang C, Xu F, Deng W-Q, Qi L-L, Zhou Y-L, Fan Y-G (2021) Two new Inosperma (Inocybaceae) species with unexpected muscarine contents from tropical China. MycoKeys 85: 87-108. https://doi.org/10.3897/mycokeys.85.71957
Open Access

Abstract

An accurate identification of poisonous mushrooms and the confirmation of the toxins involved are both of great importance in the treatment of mushroom poisoning incidents. In recent years, cases of mushroom poisoning by Inosperma spp. have been repeatedly reported from tropical Asia. It is urgent to know the real species diversity of Inosperma in this region. In the present study, we proposed two new Inosperma species from tropical Asia, namely I. muscarium and I. hainanense. They were described based on morphology and multilocus phylogeny. Detailed descriptions, color photographs and the discussion with other closely related species of the two new taxa were provided. In addition, a comprehensive muscarine determination of these two new species using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) approach has been performed. Results showed that these two species were muscarine positive, with a content of 16.03 ± 1.23 g/kg in I. muscarium and a content of 11.87 ± 3.02 g/kg in I. hainanense, much higher than the known species I. virosum. Recovery of muscarine ranged from 93.45% to 97.25%, and the average recovery is 95.56%.

Keywords

Agaricales, muscarine, new species, phylogeny, taxonomy

Introduction

Muscarine C9H20NO2+, CAS number: 300–54–9, is a toxic alkaloid found in Inocybaceae, Clitocybe and several other mushroom genera (Patocka et al. 2021). The ingestion of muscarine-containing mushrooms would cause diaphoresis, salivation, urination, nausea, vomiting, gastrointestinal effects and muscular cramp, and fatal muscarinic syndromes like miosis, bronchoconstriction, and bradycardias in humans (Wilson 1947; Lurie et al. 2009; Chandrasekharan et al. 2020; Latha et al. 2020; Patocka et al. 2021), or even death (Pauli et al. 2005; Işıloğlu et al. 2009; Zosel et al. 2015). Many species of Inocybaceae are known to contain muscarine (Malone et al. 1962), especially in Inocybe sensu stricto, and Pseudosperma (Kosentka et al. 2013; Matheny et al. 2020). Inosperma, a genus in Inocybaceae, is supposed to contain only a small number of muscarine positive species (Kosentka et al. 2013). However, mushroom poisoning events caused by Inosperma species were repeatedly reported from tropical Asia in recent years (Chandrasekharan et al. 2020; Li et al. 2021; Parnmen et al. 2021). Accordingly, it is urgent to enrich the knowledge of species diversity of the genus and to detect their muscarine toxin contents in tropical Asia.

Inosperma was erected as a subgenus of Inocybe with Inocybe calamistrata (Fr.) Gillet as type (Kühner 1980), and is now treated as genus rank (Matheny et al. 2020). Members in this genus are characterized by small to medium-sized basidiomata, rimose to scaly pileus, often rubescent context, phaseoliform to subglobose basidiospores, thin-walled cheilocystidia, lack of pleurocystidia, and often with distinctive odors. Inosperma species are widespread and there are seventy-one taxa documented globally (http://www.indexfungorum.org, retrieved 7 Oct. 2021). The tropical elements of Inosperma comprise several recently described, and still a few undescribed taxa, which were divided into two separate Old World tropical clades (Kropp et al. 2013; Matheny et al. 2020; Aïgnon et al. 2021; Deng et al. 2021). Interestingly, most of the taxa from Old World tropical clade 1 were mainly distributed in western Africa (Matheny et al. 2020; Aïgnon et al. 2021), and species in Old World tropical clade 2 were mainly from tropical Asia (Deng et al. 2021).

During our field works around the tropical China, two new Inosperma species were discovered. The present study aims to describe these two new tropical species using a combined data of morphology and phylogeny, and to determine their muscarine contents, in order to provide an accurate data for the prevention and clinical treatment of potential Inosperma poisoning accidents.

Materials and methods

Research area and specimens sampling

Our collections were made from Castanopsis dominated forests in Hainan, Guangdong Provinces, and Guangxi Zhuang Autonomous Region of China, with a tropical or subtropical climate. Specimens were photographed in the field using a digital camera and then described soon after collection. The specimens were dried through an electronic drier at 45 °C overnight, and were then preserved in plastic bags and sealed. After study, dried specimens were deposited in the Fungal Herbarium of Hainan Medical University (FHMU), Haikou City, Hainan Province of China, or in the Fungarium of Guangdong Institute of Microbiology (GDGM), Guangzhou, China.

Morphological study

Marcoscopic features were made from field notes and photographs. Color notations follow Kornerup and Wanscher (1978). Microscopic characters from dried materials mounted in KOH (5%) or mixed with Congo Red (1%) solution were observed with a microscope and photographed using a digital camera. Randomly selected twenty basidiospores and ten basidia for each specimen, the length and width of each basidiospore and basidium were measured, excluding the apiculus and sterigmata respectively (Kobayashi 2009). Numbers in square brackets [n/m/p] represent “n” basidiospores measured from “m” basidiomata of “p” specimens (Zhang et al. 2019). The dimensions of basidiospores and Q values are expressed as (a) b–c (d), “a” and “d” denote extreme values (“a” < 5th percentile; “d” > 95th percentile), while the ranges “b–c” means 5th to 95th percentile values. The quotient Q = length/width ratio for individual basidiospore, and Qm means the average of Q values (Dramani et al. 2020).

DNA extraction, PCR and sequencing

Genomic DNA was extracted from dried specimens using the NuClean Plant Genomic DNA kit (ComWin Biotech, Beijing). The following primers were used: ITS1F/ITS4 for ITS (Gardes and Bruns 1993), LR0R/LR7 for LSU (Vilgalys and Herster 1990), bRPB2-6F/bRPB2-7.1R for rpb2 (Matheny 2005). The volume of polymerase chain reaction (PCR) mixture solution was 25 μL, containing 9.5 μL dd H2O, 12.5 μL 2×Taq Plus MasterMix (Dye), 1 μL of each primer, and 1 μL of template DNA. PCR conditions for ITS, LSU and rpb2 followed Wang et al. (2021), that the conditions of PCR for three different gene regions are all the same as denaturation at 95 °C for 1 min at first, then followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 8 min. Afterwards, the products of amplifications were sent to the Beijing Genomics Institute for purification and sequenced as soon as possible.

Analysis of sequence data

Sequences in this study were prepared and compared with closely related Inosperma sequences that were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/) through BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) or literature survey (Larsson et al. 2009; Kropp et al. 2013; Horak et al. 2015; Nasser et al. 2017; Bau and Fan 2018; Matheny and Kudzma 2019; Matheny et al. 2020; Deng et al. 2021; Aïgnon et al. 2021; Cervini et al. 2021; Bandini et al. 2021). Then sequences from three genes were aligned respectively using MAFFT online service (https://mafft.cbrc.jp/alignment/server/) (Katoh et al. 2019) and were edited by BioEdit version 7.0.9.0 (Hall 1999). Two taxa in Auritella (A. hispida and A. spiculosa) were served as outgroups (Matheny et al. 2020). MrModeltest v2.3 was used to select the best-fit model for each gene partition for Bayes analysis (Nylander 2004). The datasets of each locus were combined in MEGA 5.02 (Tamura 2011). Maximum likelihood (ML) was inferred under partitioned models using W-IQ-TREE Web Service (http://iqtree.cibiv.univie.ac.at/), and the ultrafast bootstrapping was done with 1000 replicates (Trifinopoulos et al. 2016). Bayesian analysis was performed in MrBayes v.3.2.7a (Ronquist et al. 2012).

Muscarine toxin detection

Methods for sample preparation and analysis through UPLC-MS/MS were followed by Xu et al. (2020) with some modifications. Dried samples were ground to a fine power respectively, to 20 mg of each homogenised portion, 2 mL methanol-water solution (5:95 v/v) was added. The extraction was vortexed in a vortex mixer for 30 min, the mixture was further extracted by using an ultrasonic bath for another 30 min, and centrifuged for 5 min with 10000 rpm speed. Total supernatant was collected, using 0.22 μm organic filter membrane to filtrate for UPLC-MS/MS analysis and diluted with methanol-water (5:95, v/v) when necessary. The blank sample used here was Lentinula edodes. The optimal MS parameters and product ion confirmation settings followed Xu et al. (2020), while the chromatographic column we used was ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 µm). The muscarine content was estimated in the mushroom extract by using standard muscarine (Sigma-Aldrich, Chemical purity ≥ 98%). The analytical results are reported as Mean ± SD g/kg, where Mean is the average content of muscarine in the mushroom from each experimental species, and SD represents its standard deviation.

Results

Phylogenetic inference

The final multilocus dataset (Table 1) includes 94 taxa and 3130 characters, and 37 new sequences (14 ITS, 12 LSU and 11 rpb2) were generated in this study and then submitted to GenBank. The alignment was deposited in TreeBase (28515). The best-fit models for each gene selected by MrModelGUI are GTR+I+G equally. The Maximum likelihood (ML) and Bayesian analyses for the combined dataset provide a best scoring tree is shown in Fig. 1. Three ectomycorrhizal samples (KIC27, KI54, and KIB1) and an environmental sample grouped together with eight specimens of I. muscarium with significant support (BP = 100%, PP = 1). In addition, two specimens (TJB10045 and NW972) from Thailand and an environmental sample (CROP denovo 1461) from China grouped together with six specimens of I. hainanense with high support (BP = 99%, PP = 0.99). The two new Inosperma species formed separate lineages and were sister with significant support (BP = 88%, PP = 0.96) to each other. These two new species formed a subclade in the Old World tropical clade 2. The subclade was sister to I. virosum (K.B. Vrinda, C.K. Pradeep, A.V. Joseph & T.K. Abraham ex C.K. Pradeep, K.B. Vrinda & Matheny) Matheny & Esteve-Rav., I. gregarium (K.P.D. Latha & Manimohan) Matheny & Esteve-Rav., and an undescribed specimen I. sp. (TR220-06) from Papua New Guinea with full support (BP = 100%, PP = 1).

Table 1.
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Taxon sampling information and DNA sequences used for phylogenetic analyses

Taxa Collection number/Herbaium Locality GenBank accession number Reference
ITS LSU rpb2
Auritella hispida TH10009 Cameroon KT378203 KT378207 KT378215 Matheny et al. (2020)
Auritella spiculosa TH9866 Cameroon KT378204 KT378206 KT378214 Matheny et al. (2020)
Inosperma adaequatum JV16501F Finland – AY380364 AY333771 Matheny et al. (2020)
Inosperma aff. lanatodiscum PBM3051 USA JQ801401 JN975026 JQ846485 Pradeep et al. (2016)
Inosperma aff. calamistratum DED8134 Thailand GQ892983 GQ892937 – Pradeep et al. (2016)
Inosperma aff. calamistratum REH8420 Costa Rica JQ801390 JN975018 JQ846471 Pradeep et al. (2016)
Inosperma aff. fastigiellum PBM3325 USA JQ801399 JQ815419 JQ846477 Pradeep et al. (2016)
Inosperma aff. latericium TR109-02 Papua New Guinea JQ801405 JN975023 JQ846487 Pradeep et al. (2016)
Inosperma aff. maculatum PBM2446 USA DQ241778 AY745700 EU569863 Pradeep et al. (2016)
Inosperma africanum MR00387 Togo MN096189 MN097881 MT770739 Aïgnon et al. (2021)
Inosperma africanum HLA0383 (Type) Benin MT534298 MT560733 – Aïgnon et al. (2021)
Inosperma africanum HLA0353 Benin MT534299 – – Aïgnon et al. (2021)
Inosperma akirnum CAL1358 India KY440085 KY549115 KY553236 Matheny et al. (2020)
Inosperma apiosmotum PBM3020 USA JQ801385 JN975021 JQ846463 Matheny et al. (2020)
Inosperma bicoloratum ZT12187 Malaysia GQ892984 GQ892938 JQ846464 Pradeep et al. (2016)
Inosperma bongardii JV7450F Finland – EU555448 – Pradeep et al. (2016)
Inosperma bulbomarginatum MR00357 (Type) Benin MN096190 MN097882 MN200775 Aïgnon et al. (2021)
Inosperma bulbomarginatum HLA0417 Benin MT534300 MT560734 – Aïgnon et al. (2021)
Inosperma bulbomarginatum HLA0373 Benin MT534301 – – Aïgnon et al. (2021)
Inosperma bulbomarginatum HLA0389 Benin MT534302 – – Aïgnon et al. (2021)
Inosperma bulbomarginatum PC96082 Benin JQ801412 JN975027 – Aïgnon et al. (2021)
Inosperma calamistratoides PBM3384 Australia JQ801393 JQ815415 KJ729949 Pradeep et al. (2016)
Inosperma calamistratum PBM1105 USA JQ801386 JQ815409 JQ846466 Pradeep et al. (2016)
Inosperma calamistratum EL1904 Sweden AM882938 AM882938 – Pradeep et al. (2016)
Inosperma calamistratum PBM2351 USA – AY380368 AY333764 Pradeep et al. (2016)
Inosperma calamistratum TR74-06 Papua New Guinea JQ801391 JN975020 JQ846472 Pradeep et al. (2016)
Inosperma carnosibulbosum TBGT12047 India KT329448 KT329454 KT329443 Pradeep et al. (2016)
Inosperma cervicolor TURA4761 Finland JQ801395 JQ815417 JQ846474 Pradeep et al. (2016)
Inosperma cf. lanatodiscum TURA1812 Finland JQ408763 JQ319694 JQ846484 Pradeep et al. (2016)
Inosperma cf. reisneri MCA646 Japan – EU555463 – Pradeep et al. (2016)
Inosperma changbaiense FYG2010156 (Type) China MH047251 MG844976 MT086755 Bau and Fan (2018)
Inosperma cyanotrichium I37 Australia JQ801396 JN975033 JQ846476 Pradeep et al. (2016)
Inosperma dodonae SMNS-STU-F-0901253 Netherlands MW647615 – – Bandini et al. (2021)
Inosperma erubescens JV9070F Finland EU569846 – Pradeep et al. (2016)
Inosperma flavobrunneum HLA0372 Benin MT534290 MT536756 – Aïgnon et al. (2021)
Inosperma flavobrunneum HLA0367 (Type) Benin MN096199 MT536754 – Aïgnon et al. (2021)
Inosperma geraniodorum EL10606 Sweden FN550945 FN550945 – Pradeep et al. (2016)
Inosperma gregarium ZT8944 India – EU600903 EU600902 Pradeep et al. (2016)
Inosperma gregarium CAL1309 India KX852305 KX852306 KX852307 Latha and Manimohan. (2016)
Inosperma hainanense Zeng4936 China MZ374069 MZ374760 MZ388103 The present study
Inosperma hainanense Zeng4937 (Type) China MZ374070 MZ374761 MZ388104 The present study
Inosperma hainanense Zeng4935 China MZ374071 MZ374762 MZ388105 The present study
Inosperma hainanense FYG4386 China MZ374072 – – The present study
Inosperma hainanense FYG4390 China MZ374073 MZ374763 – The present study
Inosperma hainanense FYG4394 China MZ374068 – – The present study
Inosperma ismeneanum STU:SMNS-STU-F-0901561 Germany MW647625 – – Bandini et al. (2021)
Inosperma lanatodiscum PBM2451 USA JQ408759 JQ319690 JQ846483 Pradeep et al. (2016)
Inosperma latericium PDD92382 New Zealand GU233367 GU233413 – Pradeep et al. (2016)
Inosperma maculatum EL12604 Sweden AM882964 AM882964 – Pradeep et al. (2016)
Inosperma maximum PBM2222 USA EU569854 – Pradeep et al. (2016)
Inosperma misakaense PC96234 Zambia JQ801409 EU569875 AY333767 Pradeep et al. (2016)
Inosperma monastichum STU:SMNS-STU-F-0901533 Germany MW647631 – – Bandini et al. (2021)
Inosperma mucidiolens DG1824 (Type) Canada HQ201339 HQ201340 – Pradeep et al. (2016)
Inosperma muscarium Zeng4720 China MZ373978 MZ373988 MZ388089 The present study
Inosperma muscarium Zeng4736 China MZ373979 MZ373989 MZ388090 The present study
Inosperma muscarium Zeng4737 China MZ373980 – MZ388091 The present study
Inosperma muscarium Zeng4719 China MZ373981 MZ373990 MZ388092 The present study
Inosperma muscarium FYG6091 (Type) China MZ373982 MZ373991 MZ388093 The present study
Inosperma muscarium FYG6092 China MZ373983 MZ373992 MZ388094 The present study
Inosperma muscarium FYG6093 China MZ373984 MZ373993 MZ388095 The present study
Inosperma muscarium GDGM76077 China MZ520549 MZ520550 MZ542730 The present study
Inosperma neobrunnescens PBM2452 USA – EU569868 EU569867 Pradeep et al. (2016)
Inosperma neobrunnescens var. leucothelotum SAT0427406 USA JQ801411 JN975025 JQ846489 Pradeep et al. (2016)
Inosperma proximum ZT13015 Thailand EU600839 EU600840 Matheny et al. (2020)
Inosperma quietiodor EL11504 Sweden AM882960 AM882960 Pradeep et al. (2016)
Inosperma rhodiolum EL223-06 France FJ904175 FJ904175 Pradeep et al. (2016)
Inosperma rimosoides PBM2459 USA DQ404391 AY702014 DQ385884 Pradeep et al. (2016)
Inosperma rubricosum PBM3784 Australia KP308817 KP170990 KM406230 Pradeep et al. (2016)
Inosperma saragum CAL1360 India KY440103 KY549133 KY553249 Latha and Manimohan (2017)
Inosperma shawarense ASSE79 Pakistan KY616964 KY616966 Naseer et al. (2018)
Inosperma sp. PBM2871 USA HQ201348 HQ201348 JQ846475 Pradeep et al. (2016)
Inosperma sp. BB3233 Zambia JQ801415 EU600885 Pradeep et al. (2016)
Inosperma sp. L-GN3a Papua New Guinea JX316732 JX316732 Pradeep et al. (2016)
Inosperma sp. TJB10045 Thailand KT600658 KT600659 KT600660 Pradeep et al. (2016)
Inosperma sp. TR22006 Papua New Guinea JQ801416 JN975017 JQ846496 Pradeep et al. (2016)
Inosperma sp. China LS983441 Unpublished
Inosperma sp. CROP China MF532817 Unpublished
Inosperma sp. China LS975930 Unpublished
Inosperma sp. NW972 Thailand MN492637 Unpublished
Inosperma sp. KIB1 China JX456867 Unpublished
Inosperma sp. KIC27 China JX456949 Unpublished
Inosperma sp. KI54 China JX456860 Unpublished
Inosperma sp. PC96013 Zambia JQ801383 EU600883 EU600882 Pradeep et al. (2016)
Inosperma sp. PC96073 Zambia JQ801417 EU600870 EU600869 Pradeep et al. (2016)
Inosperma subhirsutum JV11950 Latvia EU555452 AY333763 Pradeep et al. (2016)
Inosperma subsphaerosproum FYG5848 (Type) China MW403825 MW397171 MW404237 Deng et al. (2021)
Inosperma subsphaerosproum FYG5847 China MW403826 MW397172 MW404238 Deng et al. (2021)
Inosperma subsphaerosproum FYG5846 China MW403827 MW397173 MW404239 Deng et al. (2021)
Inosperma vinaceobrunneum PBM2951 USA HQ201353 JQ846478 Pradeep et al. (2016)
Inosperma vinaceum AMB18747 Italy MW561108 MW561120 Cervini et al. (2021)
Inosperma viridipes I153 Australia KP641646 KP171095 KM656139 Pradeep et al. (2016)
Inosperma virosum TBGT753 India KT329452 KT329458 KT329446 Pradeep et al. (2016)
Inosperma virosum CAL1383 India KY440108 KY549138 KY553253 Latha and Manimohan (2017)
Figure 1. 

Phylogram generated by Bayesian Inference (BI) analyses based on sequences of a combined data set from nuclear genes (rDNA-ITS, nrLSU, and rpb2), rooted with Auritella hispida and A. spiculosa. Bayesian Inference posterior probabilities (BI-PP) ≥0.95 and ML bootstrap proportions (ML-BP) ≥70 are represented as BI-PP/ML-BP. I. muscarium sp. nov. and I. hainanense sp. nov. are two newly described taxa.

Taxonomy

Inosperma muscarium Y.G. Fan, L.S. Deng, W.J. Yu & N.K. Zeng, sp. nov.

MycoBank No: MB840527
Figures 2, 3

Etymology

“muscarium” refers to its high content of muscarine.

Holotype. China, Hainan Province, Ledong Li Autonomous County, Yinggeling substation of Hainan Tropical Rainforest National Park, under Castanopsis forest, at 19°1'20"N, 109°23'33"E, alt. 550 m, 26 April 2021, FYG6091 (FHMU3162), GenBank accession number: ITS (MZ373982); LSU (MZ373991) and rpb2 (MZ388093).

Diagnosis

Basidiomata small to medium-sized. Pileus rimulose to rimose with an indistinct umbo, lamellae rather crowded. Basidiospores smooth, enlongate ellipsoid to ellipsoid. Cheilocystidia clavate. Under Castanopsis forest. Differs from I. hainanense by its more robust habit, elongate basidiospores, and narrower cheilocystidia.

Figure 2. 

Basidiomata of Inosperma muscarium a–e basidiomata f–h rimose to rimulose pileus i lamellae j–k lamellae edge l–m stipe surface. a–b, d, f–g, i–m FHMU3162 (holotype) c, e FYG6092 (FHMU3163) h FYG6093 (FHMU3164). Scale bars: 10 mm (a–m). Photos by Y.-G. Fan.

Figure 3. 

Microscopic features of Inosperma muscarium (FHMU3162, holotype) a–b basidiospores c–d basidia e–h cheilocystidia in clusters i oleiferous hyphae j pileipellis and pileal trama k terminal hyphae at the stipe apex l hymenophoral trama m stipitipellis and stipe trama. Scale bars: 10 μm (a–m). Photos by L.-S. Deng

Basidiomata

small to medium-sized. Pileus 25–60 mm diam., conical convex to convex when young, becoming broadly convex to plano-convex with a small indistinct umbo when mature, margin slightly incurved when young, becoming somewhat reflexed with age. Surface dry, smooth with distinct ivory white (5A1) veil layer around the disc when young, then appressed with indistinct veil remnants, fibrillose-rimulose elsewhere, margin usually strongly rimose with age; yellowish brown (5D8) to chocolate brown (5E8) around the center and on the fibrils, yellowish brown (5C6) elsewhere, yellowish brown (6C6) to slightly dark brown (6E7) all over the basidiomata when overmatured. Lamellae rather crowded, adnexed, initially pure white to pale off-white (4B1), becoming grayish white (5B1) to yellowish white (4A2), dirty yellow (4A3) to yellowish brown (5B4) when overmatured, 1.5–3 mm wide, edge fimbriate, faint serrate to somewhat wavy. Stipe 35–72 × 3–8 mm, central, solid, terete, equal with a slightly swollen apex and base; with sparse fibrils at apex, longitudinally fibrillose downwards the stipe, with white tomentose hyphae at the base; initially white (5A1) to cream white(3A2), yellowish (4A3) or brownish (5A3) with age, brown (5B6) to dark brown (5C5) when old. Context solid, fleshy in pileus, 0.5–1 mm thick at mid-radius, 1.5–4.5 mm under the umbo, white to ivory white (5A1) at first, becoming brownish white (5B2); fibrillose and striate in the stipe, white to yellowish (4A2) or flesh color (4B3). Odor fungoid, slightly grassy or mild.

Basidiospores

[180/9/9] 8–10(11) × 5–6 (6.5) μm, Q = (1.15)1.42–1.86(2.00), Qm=1.63, mostly ellipsoid to enlongate ellipsoid, occasionally sub-phaseoliform, smooth, thick-walled, yellowish, apiculus small, indistinct, with a spherical to ellipsoid yellowish brown oil-droplet inside. Basidia 17–24 × 7–9 μm, clavate to broadly clavate, obtuse at apex, slightly tapering towards the base, 4-spored, sterigmata 2–4 μm in length, thin-walled, hyaline or pale yellow, with oily drops in various sizes with age. Pleurocystidia none. Lamella edge sterile. Cheilocystidia 36–50 × 9–14 μm, abundant and crowded, mostly clavate, broadly clavate to enlongate-clavate, rarely balloon-shaped, apices rounded to obtuse, or occasionally subcapitate, thin- to slightly thick-walled, septate, often constricted at septa, colorless to yellowish, sometimes with golden yellow inclusions. Hymenophoral trama 75–108 μm thick, sub-regular, colorless to yellowish, composed of thin-walled, smooth, cylindric to mostly inflated, hyphae 12–25 μm wide, somewhat constricted at the both ends of per hyphae. Pileipellis a cutis, sub-regular, composed of thin-walled, brown to yellowish brown, cylindrical, slightly encrusted hyphae 4–10 μm wide. Pileal trama colorless, regular to subregular, hyphae 12–25 μm wide. Stipitipellis a cutis, regularly arranged, occasionally with small clusters of terminal cheilocystidoid cells at the stipe apex, cheilocystidoid cells 31–47 × 9–10 μm, rare, clavate to enlongate clavate, hyaline or pale yellow, thin- to slightly thick-walled, some with golden yellow inclusions. Caulocystidia not observed. Oleiferous hyphae 4–13 μm wide, scattered in pileus and stipe tramal tissue, yellow or bright golden yellow, smooth, often bent, sometimes diverticulate. Clamp connections present, common in all tissues.

Habitat

Gregarious in clusters, usually scattered with numerous clusters under Castanopsis forest, late March to August in tropical China.

Known distribution

China (Hainan, Guangdong, Guangxi), Thailand.

Additional materials examined

China. Hainan Province, Ledong Li Autonomous County, Yinggeling substation of Hainan Tropical Rainforest National Forest Park, under Castanopsis forest, 13 August 2020, N.K. Zeng, Zeng4720 (FHMU3158); Same location, under Castanopsis forest, 14 August 2020, N.K. Zeng Zeng4736 (FHMU3159); Zeng4737 (FHMU3160), Same location, 26 April 2021, Y.G. Fan, L.S. Deng & Q.Q. Chen, FYG6092 (FHMU3163); FYG6093 (FHMU3164); FYG6094 (FHMU3173); Guangdong Province, Yangchun City, Gangmei Town, Lunshui Village, under Castanopsis forest, 29 March 2019, W.Y. Huang, GDGM76077; Guangxi Zhuang Autonomous Region: Wuzhou City, Cangwu Country, Wangfu Town, 23°40'28"N, 111°29'6"E, alt. 30 m, Under Castanopsis dominated forest, 29 May 2021, L.L. Qi, WSW10286, (FHMU3174).

Inosperma hainanense Y.G. Fan, L.S. Deng, W.J. Yu & N.K. Zeng, sp. nov.

MycoBank No: MB840528
Figures 4, 5

Etymology

“hainanense” refers to the its type locality.

Holotype. China, Hainan Province, Changjiang Li Autonomous County, Bawangling substation of Hainan Tropical Rainforest National Park, under Castanopsis dominated forest, at 19°7'12.43"N, 109°7'6.29"E, alt. 630 m, 2 September, 2020, N.K. Zeng, Zeng4937 (FHMU3166), GenBank accession number: ITS (MZ374070); LSU (MZ374761) and rpb2 (MZ388104).

Diagnosis

Distinguishes from I. muscarium by its slender basidiomata, ellipsoid to ovoid basidiospores, and mostly vesiculose cheilocystidia.

Figure 4. 

Basidiomata of Inosperma hainanense a–e basidiomata f–g rimose to rimulose pileus h lamellae i lamellae edge j–k stipe surface. c FHMU3166 (holotype) a–b, d–g, i–k FHMU6511 h FHMU3168. Scale bars: 10 mm (a–k). a–b, d–k: photos by L.-S. Deng; c: photos by N.-K. Zeng

Figure 5. 

Microscopic features of Inosperma hainanense (FHMU3166, holotype) a–b basidiospores c–d basidia e–k cheilocystidia in clusters l pileipellis and pileal trama n hymenophoral trama m, o oleiferous hyphae p stipitipellis and stipe trama. Scale bars: 10 μm (a–k). Photos by L.-S. Deng

Basidiomata

small to medium-sized. Pileus 25–53 mm diam., conical to convex at young age, becoming applanate to uplifted with age, with a broad to subacute umbo, margin initially decurved, straight to somewhat wavy when mature; surface dry, smooth when young, fibrillose-rimulose elsewhere, strongly rimose towards the margin with age; chocolate brown (5D8) to somewhat dark brown (5F7) around the disc, straw yellow (4A6) to yellowish brown (4B5) elsewhere, background pallid to cream white (4B1), becoming brown (5B4) to dark brown (5C6) with age; Lamellae rather crowded, adnexed, initially ivory white (5A1) to grayish white (5B2), becoming dirty yellowish (5B5) to brownish (5C7) when matured, completely brown (5D6) after drying, 2–3 mm in width, edge fimbriate, slightly serrate. Stipe 40–72 × 3–5 mm, central, nearly terete, equal with a slightly swollen apex, base somewhat swollen; nearly smooth and longitudinally striate all over the stipe; initially ivory (5A1) to yellowish white (5A2) at the upper half, yellowish to brownish (4B5) downwards, becoming uniformly yellowish brown (4B7) to brown (4C7) with age. Context solid, fleshy in pileus, white to grayish white (4B1), pale brown under the umbo (4B2), 1–2 mm thick at mid-radius, 4–5 mm thick under the umbo, fibrillose in stipe, pallid to yellowish (4A2) or brownish (4B2), striate, shiny. Odor indistinct or slightly acid.

Basidiospores

[180/9/9] 8–9(10.5) × 5–7 μm, Q = (1.18)1.28–1.64 (1.78), Qm = 1.43, mostly ellipsoid to ovoid, occasionally subphaseoliform, smooth, slightly thick-walled, brown to yellowish brown, apiculus small, indistinct, with a spherical to ellipsoid yellowish brown oil-droplet. Basidia 21–28 × 6–9 μm, clavate, often obtuse at apex, slightly tapered towards the base, thin-walled, 4-spored, sometimes 2-spored, sterigmata 4–6 μm in length, with spherical yellowish brown to golden yellow brown oily inclusions. Pleurocystidia absent. Lamella edge sterile. Cheilocystidia 34–55 × 15–25 μm, abundant and crowded, mostly obovoid to balloon-shaped, occasionally broadly clavate, rarely enlongate-clavate, thin- to slightly thick-walled (up to 1 μm thick); often rounded or slightly obtuse at apex, colorless to pale yellow, sometimes with golden yellow pigments. Hymenophoral trama 75–138 μm thick, sub-regular, hyaline to slightly yellow, composed of cylindric to inflated hyphae 20–33 μm wide, slightly constricted at septa. Pileipellis a cutis, hyphae 2.5–10 μm wide, thin-walled, pale yellow to yellowish brown, cylindrical, sometimes slightly encrusted. Pileal trama regular to subregular, hyphae 12–30 μm wide, thin-walled, colorless. Stipitipellis a cutis, regularly arranged, walls yellowish to bright yellow. Oleiferous hyphae 2.5–10 μm wide, commonly scattered in pileus and stipe tramal tissues, straw yellow or bright golden yellow, smooth, often bent or diverticulate. Clamp connections observed in all tissues.

Habitat

Scattered or gregarious in small clusters under Castanopsis dominated forest, June to September in tropical China.

Known distribution

China (Hainan, Guangdong).

Additional materials examined

China. Hainan Province, Wuzhishan City, Maoyang Town, Maoyang Village, 11 August 2021, Y.G. Fan & L.S. Deng, FYG6440 (FHMU6513); Ganshiling Provincial Nature Reserve, L.S. Deng & Y.G. Fan, DLS0043 (FHMU6512); Changjiang Li Autonomous County, Bawangling substation of Hainan Tropical Rainforest National Park, under Castanopsis dominated forest, 2 September 2020, N.K. Zeng, Zeng4936 (FHMU3165); Zeng4935 (FHMU3167); Guangdong Province, Guangzhou City, Tianluhu Forest Park, 2 June 2019, Y.G. Fan & W.J. Yu, FYG4386 (FHMU3168); Shaoguan City, Danxiashan Nature Reserve, 4 June 2019, Y.G. Fan & W.J. Yu, FYG4388 (FHMU3175); 4390 (FHMU3169); FYG4394 (FHMU3170).

Muscarine detection

Representative chromatograms of muscarine were shown in Fig. 8. The muscarine toxin content was confirmed by linear equation according to the analysis of UPLC-MS/MS, it was found that both of the two new species contained muscarine toxin, and the content of Inosperma muscarium was 16.03 ± 1.23 g/kg while I. hainanense was 11.87 ± 3.02 g/kg. Muscarine was identified by comparing retention time (1.22 min) and relative deviation (0.82%) in the allowable relative range of 25 % base on the qualitative analysis. The calibration curve for muscarine generated during the validation was y = 2083.17 x–209.297 (r = 0.9988) for muscarine concentration in the range of 2–200 ng/mL (y represents the peak area, and x is muscarine concentration, r is correlation coefficient). Recovery of muscarine ranged from 93.45% to 97.25%, and the average recovery was 95.56%.

Discussion

New species delimitation

The phylogenetic results place both the two new species in the Old World tropical clade 2 in genus Inosperma (Kropp et al. 2013; Pradeep et al. 2016; Deng et al. 2021), and they are sister to each other with significant support (BP = 88%, PP = 0.96). Morphologically, they share yellowish brown pileus, longitudinally striate stipe, crowded lamellae, and elliptic basidiospores. It is really difficult to distinguish the two new species by their macromorphology, in spite of the fact that I. hainanense has a relatively more slender habit, more finely rimulose in pileus, and a smoother stipe surface. However, they could be easily distinguished by their outlines of basidiospores and cheilocystidia. As is shown in Figs 6–7, I. muscarium has more elongated basidiospores in outline, as well as narrower cheilocystidia (I. muscarium: 36–50 × 9–14 μm; I. hainanense: 34–55 × 15–25 μm).

Figure 6. 

The comparisons of the two new species in their outline of basidiospores and cheilocystidia shape a, c basidiospores and cheilocystidia of I. hainanense (FHMU3162, holotype); b, d Basidiospores and cheilocystidia of I. muscarium (FHMU3166, holotype). Scale bars: 10 μm (a–d). Photos by L.-S. Deng

Figure 7. 

The comparisons of the two new species in their dimensions of basidiospores.

Figure 8. 

Representative chromatograms of muscarine.

In Old World tropical clade 2, I. gregarium and I. virosum, both of which described from India, formed a sister lineage with the two new species. They also share fibrillose-rimose pileus, longitudinally striate stipe, crowded lamellae, and elliptic basidiospores (Vrinda et al. 1996; Latha and Manimohan 2016). However, I. gregarium differs from the two new species by its smaller basidiospores (7–8.5 × 5–5.5 μm, Q = 1.3–1.8, Qm = 1.6), versiform and longer cheilocystidia (24–60 × 16–24 µm), the presence of caulocystidia, and an association with Dipterocarpaceae trees (Latha and Manimohan 2016). Inosperma virosum differs in having smaller basidiospores (6.5–8.5 × 5–6 µm, Q = 1.3–1.6, Qm = 1.4), and an association also with Dipterocarpaceae trees (Vrinda et al. 1996; Latha and Manimohan 2017). The remaining species in this subgrouping resemble the two new species to some extent; however, they have appressed-scaly or appressed-fibrillose pileus and different phylogenetic positions (Latha and Manimohan 2017).

There are eight described species in Old World tropical clade 2 so far, three of which were described from China in Fagaceae forest (Deng et al. 2021), and the rest five species were all described from India under Dipterocarpaceae forest or among ginger plants (Pradeep et al. 2016; Latha and Manimohan 2017). By our current knowledge, members in this subgrouping usually have medium-sized basidiomata, gregarious habit, appressed-scaly or fibrillose-rimose pileus, rather crowded lamellae, longitudinally striate stipe, non-changing context, subglobose to elliptic basidiospores, and the lack of distinctive odors (Pradeep et al. 2016; Latha and Manimohan 2017; Deng et al. 2021).

Muscarine toxin in Inosperma

The compound muscarine was initially isolated and identified from Amanita muscaria with the content at about 0.0003% of the fresh weight (Spoerke and Rumack 1994). However, muscarine was more commonly found in Inocybaceae and Clitocybe spp. with significant concentrations reached the highest record of 1.6%. (Lurie et al. 2009). Many Inocybaceae species were well known to contain muscarine (Peredy et al. 2014; Patocka et al. 2021), and various methods have been used to detect this toxin in the past years (Fahrig 1920; Eugster 1957; Brown et al. 1962; Robbers 1964; Kosentka et al. 2013; Latha et al. 2020). Five Inosperma species were reported as muscarine positive, including I. cervicolor (Pers.) Matheny & Esteve-Rav., I. erubescens (A. Blytt) Matheny & Esteve-Rav., I. maculatum (Boud.) Matheny & Esteve-Rav., I. vinaceobrunneum (Matheny, Ovrebo & Kudzma) Haelew. and I. virosum (K.B. Vrinda, C.K. Pradeep, A.V. Joseph & T.K. Abraham ex C.K. Pradeep, K.B. Vrinda & Matheny) Matheny & Esteve-Rav. (Kosentka et al. 2013; Latha et al. 2020). In addition, I. carnosibulbosum (C.K. Pradeep & Matheny) Matheny & Esteve-Rav., a species described from India, is probably a muscarine positive species due to a recent report of poisonous case (Chandrasekharan et al. 2020). Among these muscarine positive species in Inosperma, I. virosum described from India, is more extensively studied in toxin detection, toxicity in vitro using NCM460 colon epithelial cell line, toxic effects in vivo and pharmacokinetics of muscarine (Latha et al. 2020). The muscarine content of I. virosum is 270 or 300 mg/kg reported by separate studies (Sailatha et al. 2014; Latha et al. 2020).

Surprisingly, of the two new species we assayed, both of them have a high content of muscarine that is about 30 to 50 times higher than I. virosum (Sailatha et al. 2014; Latha et al. 2020). For humans, a lethal dose of muscarine is estimated from 40 mg to 495 mg (Pauli et al. 2005). Based on the muscarine concentrations of between 0.1% to 0.33% (dry weight) in Inocybaceae spp., a single mushroom can be lethal (Puschner 2018; Patocka et al. 2021). Consequently, the two new species proposed by the present study were considered to be more dangerous when mistakenly ingested by humans. In particular, for I. muscarium, a species often with a medium-sized basidiomata, a gregarious, large, discrete clusters habitat, and the lack of aposematic coloration make it extremely easily collected by local people as an edible mushroom. The publicity and education of the two new species were essential to prevent mushroom poisoning from tropical areas where they distributed.

The accurate identification of poisonous mushrooms and the knowledge of toxin type and contents are crucial for the treatment of mushroom poisoning patients (Li et al. 2021). However, species identification can usually be difficult for doctors when faced with mushroom-poisoned patients, mainly because of the insufficient identification data of wild poisoning mushrooms (Hall et al. 1987). Our present study provides detailed knowledge for a better prevention of potential Inosperma poisoning from tropical Asia.

Acknowledgements

The authors thank Dr. Shuai Jiang, Mr. Yongqing Fu (Hainan tropical rainforest National Park, China) and Mr. Weiyong Huang (Yangchun Center for Disease Control and Prevention, China) for their kind help in field work, and to Dr. Junqing Yan (Jiangxi Agricultural University, China) and Dr. Yupeng Ge (Ludong University, China) for their kind help in the phylogenetic analysis. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31860009 & 31400024), Hainan Basic and applied research project for cultivating high level talents (2019RC230), The Innovative Research Projects for Graduate Students in Hainan Medical University, Hainan China (HYYS2020-42), and Jilin Provincial Foundation for Excellent Scholars (20180520035JH). We also thank the anonymous reviewers for their corrections and suggestions to improve our work.

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