﻿Two new Inosperma (Inocybaceae) species with unexpected muscarine contents from tropical China

﻿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%.


Introduction
Muscarine C 9 H 20 NO 2 + , 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 , 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.

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" < 5 th percentile; "d" > 95 th percentile), while the ranges "b-c" means 5 th to 95 th percentile values. The quotient Q = length/width ratio for individual basidiospore, and Q m 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 H 2 O, 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.

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.

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  Etymology. "muscarium" refers to its high content of muscarine. 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.
Habitat. Gregarious in clusters, usually scattered with numerous clusters under Castanopsis forest, late March to August in tropical China. Etymology. "hainanense" refers to the its type locality.

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%.

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).
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  (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.