MycoKeys 35: 37_ | 08 (202 | ) er-reviewed open-access journal doi: 10.3897/mycokeys.85.7 1957 < MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Two new Inosperma (Inocybaceae) species with unexpected muscarine contents from tropical China 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! | Key Laboratory of Tropical Translational Medicine of Ministry of Education, Transgenic Laboratory, Tropi- cal Environment and Health Laboratory, College of Pharmacy, Hainan Medical University, Haikou 571199, China 2. Hainan Institute for Food Control, Haikou 570314, China 3 Jilin Provincial Joint Key Labora- tory of Changbai Mountain Biocoenosis and Biodiversity, Changbai Mountain Academy of Sciences, Yanbian 133613, China 4 Physical and Chemical Department, Ningxia Hui Autonomous Region Center for Disease Control and Prevention, Yinchuan 750004, China § State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China 6 Microbiology Re- search Institute, Guangxi Academy of Agriculture Sciences, Nanning 530007, China Corresponding author: Yu-Guang Fan (mycena@qq.com) Academic editor: T. Lumbsch | Received 23 July 2021 | Accepted 24 November 2021 | Published 15 December 2021 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 Jnosperma (Inocybaceae) species with unexpected muscarine contents from tropical China. MycoKeys 85: 87-108. https://doi.org/10.3897/mycokeys.85.71957 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 mush- room poisoning by /nosperma 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 Jnosperma species from tropical Asia, namely 1. muscarium and I. hainanense. They were described based on mor- phology 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 Z. muscarium and a content of 11.87 + 3.02 g/ kg in LZ. Aainanense, much higher than the known species /. 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 Copyright Lun-Sha Deng et al. 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. 88 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) Introduction Muscarine Cri ING. % CAS number: 300—54—9, is a toxic alkaloid found in Ino- cybaceae, 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; Isiloglu et al. 2009; Zosel et al. 2015). Many species of Inocybaceae are known to contain muscarine (Malone et al. 1962), especially in /nocybe sensu stricto, and Pseudosperma (Kosentka et al. 2013; Matheny et al. 2020). /nosperma, a genus in Inocybaceae, is supposed to contain only a small number of muscarine positive species (Kosentka et al. 2013). However, mush- room poisoning events caused by /nosperma 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. [nosperma was erected as a subgenus of Jnocybe with Inocybe calamistrata (Fr.) Gillet as type (Ktihner 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. [nosperma spe- cies are widespread and there are seventy-one taxa documented globally (http://www. indexfungorum.org, retrieved 7 Oct. 2021). The tropical elements of /nosperma 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; Aignon 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; Aignon 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 /nosperma 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 con- tents, in order to provide an accurate data for the prevention and clinical treatment of potential /nosperma 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 sub- tropical climate. Specimens were photographed in the field using a digital camera and New Jnosperma species with unexpected muscarine contents from tropical China 89 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 Matrcoscopic 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 basidio- spore and basidium were measured, excluding the apiculus and sterigmata respectively «cD (Kobayashi 2009). Numbers in square brackets [n/m/p] represent “n” basidiospores ( 95" percentile), while the ranges “b-c” means 5™ to 95" percentile values. The quotient Q = length/width ratio for individual basidiospore, and Q_ 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 Genom- ic DNA kit (ComWin Biotech, Beijing). The following primers were used: ITS1F/ ITS4 for ITS (Gardes and Bruns 1993), LROR/LR7 for LSU (Vilgalys and Herster 1990), bRPB2-6F/bRPB2-7.1R for 7pb2 (Matheny 2005). The volume of polymerase chain reaction (PCR) mixture solution was 25 wL, containing 9.5 pL dd H,O, 12.5 pL 2xTaq Plus MasterMix (Dye), 1 wL of each primer, and 1 wL of template DNA. PCR conditions for ITS, LSU and rp62 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 [nosperma se- quences 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; Aignon et al. 90 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) 2021; Cervini et al. 2021; Bandini et al. 2021). Then sequences from three genes were aligned respectively using MAFFT online service (https://mafft.cbre.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 par- tition for Bayes analysis (Nylander 2004). The datasets of each locus were combined in MEGA 5.02 (Tamura 2011). Maximum likelihood (ML) was inferred under parti- tioned 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 pow- er 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 cen- trifuged for 5 min with 10000 rpm speed. Total supernatant was collected, using 0.22 uum 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 x 100 mm, 1.7 um). 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 rp62) 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 scor- ing tree is shown in Fig. 1. Three ectomycorrhizal samples (KIC27, KI54, and KIB1) and an environmental sample grouped together with eight specimens of 1. muscarium New Jnosperma species with unexpected muscarine contents from tropical China 91 I. africanum MR00387 Togo : 100) J. africanum HLA0383 (Type) Benin I. africanum I. africanum HLA0353 Benin I. viridipes \153 Australia I. rubricosum PBM3784 Australia I. eyanotrichium 137 Australia I. calamistratoides PBM3384 Australia I. latericium PDD92382 New Zealand I. aff. latericium TR109-02 Papua New Guinea I. proximum 2713015 Thailand Lf. bongardii JV7450F Finland I. calamistratum PBM2351 USA I. apiosmotum PBM3020 USA H 1. subhirsutum JV11950 Latvia sect. Cerv 1 colores I, maximum PBM2222 USA -— I. calamistratum TR74-06 Papua New Guinea L__. |. aff. calamistratum REH8420 Costa Rica ZL mutatum PBM2542 USA I. aff. calamistratum DED8134 Thailand I. cervicolor TURA4761 Finland I. monastichum STU SMNS-STU-F-0901533 Germany 1, mucidiolens DG1824 (Type) Canada L. calamistratum PBM1105 USA I. geraniodorum EL10606 Sweden I. calamistratum EL1904 Sweden ‘ L misakaense PC96234 Zambia L misakaense 1. vinaceobrunneum PBM2951 USA I. vinaceum AMB18747 (Type) Italy 1n0oL. J. rhodiolum EL223-06 France I. adaequatum JV16501F Finland I, erubescens JV9070F Finland I. neobrunnescens var. leucothelotum SAT0427406 USA I. neobrunnescens PBM2452 USA I. sp. PBM2871 USA 1. dodonae STU SMNS-STU-F-0901253 Netherlands L— J. ismeneanum STU SMNS-STU-F-0901561 Germany 1. changbaiense FYG2010156 (Type) China I, maculatum EL12604 Sweden anoor 1. cf. /anatodiscum TURA1812 Finland I. lanatodiscum PBM2451 USA Maculata clade I. aff. lanatodiscum PBM3051 USA I. bicoloratum 2112187 Malaysia I. aff. maculatum PBM2446 USA I. aff. fastigiellum PBM3325 USA I. rimosoides PBM2459 USA I, shawarense ASSE79 Pakistan 1. quietiodor EL11504 Sweden I. cf. reisneri MCA646 Janpan 1. subsphaerosproum FYG5846 China Hainan 1. subsphaerosproum FYG5847 China Hainan 1. subsphaerosproum FYG5848 (Type) China Hainan 1. carnosibulbosum TBGT12047 India I. saragum CAL1360 India I. sp. uncultured L-GN3a Papua New Guinea L. akirnum CAL1358 India 7 1100) J. virosum TBGT753 India ~Old World tropical clade 2 LI. virosum CAL1383 India 199, I gregarium ZT8944 India I, gregarium CAL1309 India I, sp. TR220-06 PNG J. sp. uncultured Inocybaceae China Zeng4737 China Hainan Zeng4736 China Hainan FYG6092 China Hainan /, sp. Agaricales sample China |, FYG6093 China Hainan i FYG6091(Type) China Hainan bat |. muscarium sp. nov. I. sp. K154 China 0.08 wit 1100 1/96 Zeng4720 China Hainan Zeng4719 China Hainan 0.96/88 GDGM76077 China Guangdong I. sp. TJB10045 Thailand 1. sp. NW972 Thailand FYG4386 China Guangdong I, sp. GROP China A FYG4a94 Chine Guangdong |. hainanense sp. nov. FYG4390 China Guangdong Zeng4936 China Hainan Zeng4937 (Type) China Hainan Zeng4935 China Hainan 0.97/89 I. sp. PC96013 Zambia inoo, I. flavobrunneum HLA0372 Benin I. flavobrunneum HLA0367 (Type) Benin TA00| Top peseo73 Zante ~—- Old World tropical clade 1 0.96/88; I. bulbomarginatum MRO00357 (Type) Benin I. bulbomarginatum HLA0417 Benin anoo| |' J. bulbomarginatum HLA0373 Benin L. bulbomarginatum HLA0389 Benin I. bulbomarginatum PC96082 Benin ikl) a A. spiculosa TH9866 Cameroon A. hispida TH10009 Cameroon O utgroup -/80 Figure |. Phylogram generated by Bayesian Inference (BI) analyses based on sequences of a combined data set from nuclear genes (rDNA-ITS, nrLSU, and rp62), rooted with Auritella hispida and A. spiculosa. Bayesian Inference posterior probabilities (BI-PP) >0.95 and ML bootstrap proportions (ML-BP) 270 are represented as BI-PP/ML-BP. 1. muscarium sp. nov. and I. hainanense sp. nov. are two newly described taxa. 92 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) Table |. Taxon sampling information and DNA sequences used for phylogenetic analyses Taxa Auritella hispida Auritella spiculosa Inosperma adaequatum Inosperma aff. lanatodiscum Inosperma aff. calamistratum Inosperma aff. calamistratum Inosperma aff. fastigiellum Inosperma aff. latericium Inosperma aff: maculatum Inosperma africanum Inosperma africanum Inosperma africanum Inosperma akirnum Inosperma apiosmotum Inosperma bicoloratum Inosperma bongardii Inosperma bulbomarginatum Inosperma bulbomarginatum Inosperma bulbomarginatum Inosperma bulbomarginatum Inosperma bulbomarginatum Inosperma calamistratoides Inosperma calamistratum Inosperma calamistratum Inosperma calamistratum Inosperma calamistratum Inosperma carnosibulbosum Lnosperma cervicolor Inosperma cf. lanatodiscum Inosperma cf. reisneri Inosperma changbaiense Inosperma cyanotrichium Inosperma dodonae Inosperma erubescens Inosperma flavobrunneum Inosperma flavobrunneum Inosperma geraniodorum Inosperma gregarium Inosperma gregarium Inosperma hainanense Inosperma hainanense Inosperma hainanense Inosperma hainanense Inosperma hainanense Inosperma hainanense Inosperma ismeneanum Collection number/ Herbaium TH10009 TH9866 JV16501F PBM3051 DED8134 REH8420 PBM3325 TRI109-02 PBM2446 MRO00387 HLA0383 (Type) HLA0353 CAL1358 PBM3020 2112187 JV7450F MR00357 (Type) HLA0417 HLA0373 HLA0389 PC96082 PBM3384 PBM1105 EL1904 PBM2351 TR74-06 TBGT12047 TURA4761 TURA1812 MCA646 FYG2010156 (Type) 137 SMNS- STU-F-0901253 JV9070F HLA0372 HLA0367 (Type) EL10606 Z18944 CAL1309 Zeng4936 Zeng4937 (Type) Zeng4935 FYG4386 FYG4390 FYG4394 STU:SMNS- STU-F-0901561 Locality Cameroon Cameroon Finland USA Thailand Costa Rica USA Papua New Guinea USA Togo Benin Benin India USA Malaysia Finland Benin Benin Benin Benin Benin Australia USA Sweden USA Papua New Guinea India Finland Finland Japan China Australia Netherlands Finland Benin Benin Sweden India India China China China China China China Germany GenBank accession number ITS KT378203 KT378204 JQ801401 GQ892983 JQ801390 JQ801399 JQ801405 DQ241778 MNO096189 MT534298 MT534299 KY440085 JQ801385 GQ892984 MN096190 MT534300 MT534301 MT534302 JQ801412 JQ801393 JQ801386 AM882938 JQ801391 KT329448 JQ801395 JQ408763 MH047251 JQ801396 MW647615 MT534290 MNO096199 FN550945 KX852305 MZ374069 MZ374070 MZ374071 MZ374072 MZ374073 MZ374068 MW647625 LSU KT378207 KT378206 AY380364 JN975026 GQ892937 JN975018 JQ815419 JN975023 AY745700 MNO97881 MT560733 KY549115 JN975021 GQ892938 EU555448 MNO097882 MT560734 JN975027 JQ815415 JQ815409 AM882938 AY380368 JN975020 KT329454 JQ815417 JQ319694 EU555463 MG844976 JN975033 EU569846 MT536756 MT536754 FN550945 EU600903 KX852306 MZ374760 MZ374761 MZ374762 MZ374763 rpb2 KT378215 KT378214 AY333771 JQ846485 JQ846471 JQ846477 JQ846487 EU569863 MT770739 KY553236 JQ846463 JQ846464 MN200775 KJ729949 JQ846466 AY333764 JQ846472 KT329443 JQ846474 JQ846484 MT086755 JQ846476 EU600902 KX852307 MZ388 103 MZ388104 MZ388105 Reference Matheny et al. (2020) Matheny et al. (2020) Matheny et al. (2020) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Aignon et al. (2021) Aignon et al. (2021) Aignon et al. (2021) Matheny et al. (2020) Matheny et al. (2020) Pradeep et al. (2016) Pradeep et al. (2016) Aignon et al. (2021) Aignon et al. (2021) Aignon et al. (2021) Aignon et al. (2021) Aignon et al. (2021) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Pradeep et al. (2016) Bau and Fan (2018) Pradeep et al. (2016) Bandini et al. (2021) Pradeep et al. (2016) Aignon et al. (2021) Aignon et al. (2021) Pradeep et al. (2016) Pradeep et al. (2016) Latha and Manimohan. (2016) ‘The present study ‘The present study ‘The present study ‘The present study ‘The present study ‘The present study Bandini et al. (2021) New Jnosperma species with unexpected muscarine contents from tropical China Taxa Inosperma lanatodiscum Inosperma latericium Inosperma maculatum Inosperma maximum Inosperma misakaense Inosperma monastichum Inosperma mucidiolens Inosperma muscarium Inosperma muscarium Inosperma muscarium Inosperma muscarium Inosperma muscarium Inosperma muscarium Inosperma muscarium Inosperma muscarium Inosperma neobrunnescens Inosperma neobrunnescens var. leucothelotum Inosperma proximum Inosperma quietiodor Inosperma rhodiolum Inosperma rimosoides Inosperma rubricosum Inosperma saragum Inosperma shawarense Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma sp. Inosperma subhirsutum Inosperma subsphaerosproum Inosperma subsphaerosproum Inosperma subsphaerosproum Inosperma vinaceobrunneum Inosperma vinaceum Inosperma viridipes Inosperma virosum Inosperma virosum Collection number/ Herbaium PBM2451 PDD92382 EL12604 PBM2222 PC96234 STU:SMNS- STU-F-0901533 DG1824 (Type) Zeng4720 Zeng4736 Zeng4737 Zeng4719 FYG6091 (Type) FYG6092 FYG6093 GDGM76077 PBM2452 SAT0427406 ZT13015 EL11504 EL223-06 PBM2459 PBM3784 CAL1360 ASSE79 PBM2871 BB3233 L-GN3a TJB10045 TR22006 CROP NW972 KIB1 KIC27 KI54 PC96013 PC96073 JV11950 FYG5848 (Type) FYG5847 FYG5846 PBM2951 AMB18747 1153 TBGT753 CAL1383 Zealand Sweden USA Zambia Germany Canada China China China China China China China China USA USA Thailand Sweden France USA Australia India Pakistan USA Zambia Papua New Guinea Thailand Papua New Guinea China China China Thailand China China China Zambia Zambia Latvia China China China USA Italy Australia India India GenBank accession number ITS JQ408759 GU233367 AM882964 JQ801409 MW647631 HQ201339 MZ373978 MZ373979 MZ373980 MZ373981 MZ373982 MZ373983 MZ373984 MZ520549 JQ801411 EU600839 AM882960 FJ904175 DQ404391 KP308817 KY440103 KY616964 HQ201348 JQ801415 JX316732 KT600658 JQ801416 LS983441 MF532817 LS975930 MN492637 JX456867 JX456949 JX456860 JQ801383 JQ801417 MW403825 MW403826 MW403827 MW561108 KP641646 KT329452 KY440108 LSU JQ319690 GU233413 AM882964 EU569854 EU569875 HQ201340 MZ373988 MZ373989 MZ373990 MZ373991 MZ373992 MZ373993 MZ520550 EU569868 JN975025 EU600840 AM882960 FJ904175 AY702014 KP170990 KY549133 KY616966 HQ201348 EU600885 JX316732 KT600659 JN975017 EU600883 EU600870 EU555452 MW397171 MW397172 MW397173 HQ201353 MW561120 KP171095 KT329458 KY549138 rpb2 JQ846483 AY333767 MZ388089 MZ388090 MZ388091 MZ388092 MZ388093 MZ388094 MZ388095 MZ542730 EU569867 JQ846489 DQ385884 KM406230 KY553249 JQ846475 KT600660 JQ846496 EUG600882 EU600869 AY333763 MW404237 MW404238 MW404239 JQ846478 KM656139 KT329446 KY553253 93 Reference Pradeep et al. 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(2016) Latha and Manimohan (2017) 94 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) 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 [. hainanense with high support (BP = 99%, PP = 0.99). The two new /nosperma 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 J. virosum (K.B. Vrinda, C.K. Pradeep, A.V. Joseph & TK. Abraham ex C.K. Pradeep, K.B. Vrinda & Matheny) Matheny & Esteve-Rav., J. gregarium (K.P.D. Latha & Manimohan) Matheny & Esteve-Rav., and an undescribed specimen /. sp. (TR220- 06) from Papua New Guinea with full support (BP = 100%, PP = 1). Taxonomy Inosperma muscarium Y.G. Fan, L.S. Deng, W.J. Yu & N.K. Zeng, sp. nov. MycoBank: MB840527 Figures 2, 3 Etymology. “muscarium” refers to its high content of muscarine. Holotype. Cuina, 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), Gen- Bank accession number: ITS (MZ373982); LSU (MZ373991) and rp62 (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. Basidiomata. small to medium-sized. Pileus 25—G60 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 re- flexed 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 choco- late brown (5E8) around the center and on the fibrils, yellowish brown (5C6) else- where, 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 x 3-8 mm, central, solid, terete, equal with a slightly swollen apex and base; with sparse fibrils at apex, longitudinally fibril- lose downwards the stipe, with white tomentose hyphae at the base; initially white New Jnosperma species with unexpected muscarine contents from tropical China 95 Figure 2. Basidiomata of [nosperma muscarium a-e basidiomata f-h rimose to rimulose pileus i la- mellae j-k lamellae edge I-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. 96 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) Figure 3. Microscopic features of /nosperma muscarium (FHMU3 162, holotype) a—b basidiospores ¢=d ba- sidia e-h cheilocystidia in clusters i oleiferous hyphae j pileipellis and pileal trama k terminal hyphae at the stipe apex I hymenophoral trama m stipitipellis and stipe trama. Scale bars: 10 um (a—m). Photos by L.-S. Deng New Jnosperma species with unexpected muscarine contents from tropical China 97 (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) x 5-6 (6.5) um, Q = (1.15) 1.42-1.86(2.00), Q_=1.63, mostly ellipsoid to enlongate ellipsoid, occasionally sub-phaseoliform, smooth, thick-walled, yellowish, apiculus small, indistinct, with a spherical to ellip- soid yellowish brown oil-droplet inside. Basidia 17-24 x 7—9 um, clavate to broadly clavate, obtuse at apex, slightly tapering towards the base, 4-spored, sterigmata 2—4 um in length, thin-walled, hyaline or pale yellow, with oily drops in various sizes with age. Pleurocystidia none. Lamella edge sterile. Cheilocystidia 36-50 x 9-14 um, 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 ym thick, sub-regular, color- less to yellowish, composed of thin-walled, smooth, cylindric to mostly inflated, hy- phae 12—25 um 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 um wide. Pileal trama colorless, regular to subregu- lar, hyphae 12—25 um wide. Stipitipellis a cutis, regularly arranged, occasionally with small clusters of terminal cheilocystidoid cells at the stipe apex, cheilocystidoid cells 31-47 x 9-10 um, 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 um 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. Cutna. Hainan Province, Ledong Li Autono- mous 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 Vil- lage, 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). 98 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) Inosperma hainanense Y.G. Fan, L.S. Deng, W.J. Yu & N.K. Zeng, sp. nov. MycoBank: MB840528 Figures 4, 5 Etymology. “hainanense” refers to the its type locality. Holotype. Cuina, Hainan Province, Changjiang Li Autonomous County, Bawan- gling substation of Hainan Tropical Rainforest National Park, under Castanopsis domi- nated 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 /. muscarium by its slender basidiomata, ellipsoid to ovoid basidiospores, and mostly vesiculose cheilocystidia. Basidiomata. small to medium-sized. Pileus 25-53 mm diam., conical to con- vex 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 dry- ing, 2-3 mm in width, edge fimbriate, slightly serrate. Stipe 40-72 x 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) x 5-7 um, Q = (1.18)1.28-1.64 (1.78), = 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 x 6-9 um, clavate, often obtuse at apex, slightly tapered towards the base, thin-walled, 4-spored, sometimes 2-spored, ster- igmata 4—6 um in length, with spherical yellowish brown to golden yellow brown oily inclusions. Pleurocystidia absent. Lamella edge sterile. Cheilocystidia 34-55 x 15-25 um, abundant and crowded, mostly obovoid to balloon-shaped, occasionally broadly clavate, rarely enlongate-clavate, thin- to slightly thick-walled (up to 1 um thick); often rounded or slightly obtuse at apex, colorless to pale yellow, sometimes with golden yellow pigments. Hymenophoral trama 75—138 um thick, sub-regular, hyaline to slightly yellow, composed of cylindric to inflated hyphae 20-33 um wide, slightly constricted at septa. Pileipellis a cutis, hyphae 2.5—10 «um wide, thin-walled, pale yellow to yellowish brown, cylindrical, sometimes slightly encrusted. Pileal trama regular to subregular, hyphae 12—30 um wide, New Jnosperma species with unexpected muscarine contents from tropical China 2) Figure 4. Basidiomata of /nosperma hainanense a-e basidiomata f—g rimose to rimulose pileus h lamellae i lamellae edge j-Kk stipe surface. Cc FHMU3166 (holotype) a—b, dg, i-k FHMU6511 h FHMU3168. Scale bars: 10 mm (a-k). a—b, d-k: photos by L.-S. Deng; ¢: photos by N.-K. Zeng 100 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) Figure 5. Microscopic features of /nosperma hainanense (FHMU3166, holotype) a=b basidiospores c—d basidia e-k cheilocystidia in clusters I pileipellis and pileal trama n hymenophoral trama m, 0 oleif- erous hyphae p stipitipellis and stipe trama. Scale bars: 10 um (a=k). Photos by L.-S. Deng New Jnosperma species with unexpected muscarine contents from tropical China 101 thin-walled, colorless. Stipitipellis a cutis, regularly arranged, walls yellowish to bright yel- low. Oleiferous hyphae 2.5—10 um 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. Cuina. 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 substa- tion 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 & WJ. Yu, FYG4386 (FHMU3168); Shaoguan City, Danxiashan Nature Reserve, 4 June 2019, Y.G. Fan & WJ. 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 1. 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 cali- bration 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, 7 is correlation coefficient). Recovery of mus- carine 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 /nosperma (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). Morpholog- ically, 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 /. hainanense has a relatively more slender habit, more finely rimulose in pileus, and a smoother stipe surface. However, they could 102 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) be easily distinguished by their outlines of basidiospores and cheilocystidia. As is shown in Figs 6-7, Z. muscarium has more elongated basidiospores in outline, as well as narrow- et cheilocystidia (1. muscarium: 36-50 x 9-14 um; I. hainanense: 34-55 x 15-25 um). In Old World tropical clade 2, L. gregarium and I. virosum, both of which described from India, formed a sister lineage with the two new species. They also share fibrillose- Figure 6. The comparisons of the two new species in their outline of basidiospores and cheilocystidia shape a, C basidiospores and cheilocystidia of 1. hainanense (FHMU3162, holotype); b, d Basidiospores and cheilo- cystidia of 1. muscarium (FHMU3166, holotype). Scale bars: 10 um (a=d). Photos by L.-S. Deng New Jnosperma species with unexpected muscarine contents from tropical China 103 rimose pileus, longitudinally striate stipe, crowded lamellae, and elliptic basidiospores (Vrinda et al. 1996; Latha and Manimohan 2016). However, l. gregarium differs from the two new species by its smaller basidiospores (7—8.5 x 5—5.5 um, Q = 1.3-1.8, Q__= 1.6), versiform and longer cheilocystidia (24-60 x 16-24 tm), 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 x 5—6 um, Q. = 1.3-1.6, Q_ = 1.4), and an association also with Dipterocarpaceae trees (Vrinda et al. 1996; Latha and Manimohan 2017). The remaining species in this subgrouping resem- ble 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 stri- ate 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 mus- caria 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 64 I. hainanense Zeng4937 @ FYG4388 average width (um) © FYG4390 ® DLS0043 @ Zeng4935 @ FYG6440 Zeng4936 & m FYG4386 Sd WSW 10286 ® Zeng4737 Zeng4736 ® FYG6091 Zeng4720 I. musearium 8 805 81 815 82 825 83 835 84 845 85 855 86 865 87 875 88 885 89 895 9 905 9.1 9.15 92 925 93 935 94 945 average length (jun) Figure 7. The comparisons of the two new species in their dimensions of basidiospores. 104 Lun-Sha Deng et al. / MycoKeys 85: 87-108 (2021) 10020 030 040 O50 060 O70 080 090 100 110 120 120 140 150 160 170 180 109 200 210 220 230 240 250 260 270 280 290 300 310 320 390 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 Figure 8. Representative chromatograms of muscarine. 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 mus- carine positive, including J. cervicolor (Pers.) Matheny & Esteve-Rav., 1. erubescens (A. Blytt) Matheny & Esteve-Rav., 1. maculatum (Boud.) Matheny & Esteve-Rav., I. vinaceobrunneum (Matheny, Ovrebo & Kudzma) Haelew. and /. 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, 1. 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 pos- itive 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 J. 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 /. 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 2. 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 New Jnosperma species with unexpected muscarine contents from tropical China 105 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 insufhcient identification data of wild poisoning mushrooms (Hall et al. 1987). Our present study provides detailed knowledge for a better prevention of potential /nosperma poisoning from tropical Asia. Acknowledgements The authors thank Dr. Shuai Jiang, Mr. Yongging Fu (Hainan tropical rainforest National Park, China) and Mr. Weiyong Huang (Yangchun Center for Disease Control and Preven- tion, China) for their kind help in field work, and to Dr. Junging 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 Foun- dation 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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