Phylogenetic analysis, morphological studies, element profiling, and muscarine detection reveal a new toxic Inosperma (Inocybaceae, Agaricales) species from tropical China

Tropical Asian collections of Inosperma are usually poisonous mushrooms that have caused many poisoning incidents. However, the species diversity and the toxic mechanisms of these Inosperma species are still unclear. In this study, we describe the discovery of Inosperma wuzhishanense sp. nov. from Wuzhishan City, Hainan Province, tropical China. The new species was identified based on morphological and multi-locus (ITS, nrLSU, and RPB2) phylogenetic analyses. The new species is characterized by its reddish-brown pileus, fibrillose stipes with finely protruding fibrils, rather crowded lamellae, smooth and ellipsoid basidiospores, and mostly clavate, thin-walled cheilocystidia. The new species is phylogenetically nested in the Old World tropical clade 2 and is sister to the tropical Indian taxa I. akirnum. Detailed descriptions, color photos of the new species, and comparisons with its closely related species are provided. Additionally, the muscarine content of the new species was analyzed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS). The muscarine contents ranged from 4,359.79 ± 83.87 mg/kg to 7,114.03 ± 76.55 mg/kg, 2,748.37 ± 106.85 mg/kg to 4,491.35 ± 467.21 mg/kg, and 2,301.36 ± 83.52 mg/kg to 2,775.90 ± 205.624 mg/kg in the stipe, pileus, and lamellae, respectively. The elemental composition and concentration were determined using inductively coupled plasma-mass spectrometry (ICP-MS). A total of 24 elements were detected. Among the heavy metals detected, arsenic showed the highest level of toxicity with a concentration of 36.76 ± 0.43 mg/kg.

Poisoning by muscarine-containing mushrooms causes a rapid onset (15-120 min) of classic symptoms of parasympathetic stimulation.The main symptoms are increased sweating, salivation, and lachrymation, and other symptoms include pupil constriction, blurred vision, urgent or painful micturition, nasal discharge or congestion, asthma, bronchoconstriction, hypotension, bradycardia, skin flushing, watery diarrhea, vomiting, abdominal pain, and colic.The more rapid the onset, the more severe the intoxication, and severe poisoning can be lethal, although death is a rare outcome and unlikely to occur with appropriate atropine treatment (White et al., 2019).It has been reported that Inosperma spp.contain muscarine, and certain species are known to have caused many mushroom poisoning incidents (Chandrasekharan et al., 2020;Li W. W. et al., 2021;Parnmen et al., 2021;Deng et al., 2022a;Li H. J. et al., 2023).However, little is known about the muscarine content in most Inosperma species, and only nine species have been confirmed to be muscarine-positive so far.The literature suggests that Inosperma spp.from Old World tropical clade 2 usually have high concentrations of muscarine (Sailatha et al., 2014;Chandrasekharan et al., 2020;Latha et al., 2020;Parnmen et al., 2021;Deng et al., 2021aDeng et al., , 2022a;;Li H. J. et al., 2022Li H. J. et al., , 2023)).In addition, studies have shown that long-term exposure to toxic elements through mushroom consumption may be hazardous to human health (Fu et al., 2020;Nowakowski et al., 2021).However, little is known about the types and concentrations of toxic elements in members of the Inosperma genus (Bizio et al., 2020).
Here, we report a new Inosperma species from Wuzhishan City, in the tropical region of China.This species shares some features with species in Old World tropical clade 2. Comprehensive macro-and microscopic descriptions, color photographs, and illustrations of the new species are provided in this study.In addition, the contents of muscarine and other elements were determined to verify the uniqueness of the new species and to evaluate its potential toxicity.

Sample collections and morphological studies
Fresh basidiomata were photographed, noted, and collected in the field; the color codes of descriptions were derived from Kornerup and Wanscher (1978).An electronic drier was used to dry the collected specimens at 45°C overnight.Microscopic features were observed in these dried specimens rehydrated in KOH solution (5%) or stained with Congo red solution (1%) if needed.The methods of measuring followed Ge et al. ( 2021) with small modifications.At least 20 basidiospores were randomly selected for each individual specimen, and the apiculus was excluded when measured.The abbreviation [a/b/c] represented "a" basidiospores measured from "b" basidiomata of "c" specimens.The size of basidiospores "length × width" was indicated as (d) e-f-g (h) × (i) j-k-l (m), where "d" is the minimum length, "e-g" represents 5 to 95% values, "h" is the maximum value of length, and the bolded "f " means the average value.Alphabets in width "i, " "j-l, " "m, " and "k" are shown with the same meaning.Q refers to the ratio of length/width of basidiospore in side view, and Q m ± SD is the average value of Q ± the standard deviation of all Q values.Total dried specimens were deposited in the Herbarium of Changbai Mountain Nature Reserve (ANTU) with FCAS numbers.

Muscarine detection 2.2.1 Sample preparation
The dried mushroom materials were divided into stipe, pileus (without the lamellae), and lamellae and finely ground into homogenized powder, respectively.Approximately 10-20 mg dry weight was extracted with 2 mL of methanol-water (5:95, V/V).The mixture was vortexed for 30 min at first, then ultrasound-assisted extraction for another 30 min (10°C, 33 Hz).The mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was collected.A 0.22 μm organic filter membrane was used to filtrate the supernatant before UPLC-MS/MS analysis, and acetonitrile-water (7:3, V/V) was applied to a diluted solution if needed.Lentinula edodes (Berk.)Pegler was treated as the blank sample.The analytical results are reported as mean ± SD mg/kg, where the mean is the average content of muscarine and SD represents the standard deviation.

UPLC-MS/MS analysis
The detailed parameter settings of UPLC-MS/MS followed Xu et al. (2020) and Deng et al. (2022a) with some modifications.A Waters ACQUITY UPLC I-Class Plus/Xevo TQ-XS system with an ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 μm) column was applied to the UPCL-MS/MS analysis.The gradient elution was used at a flow rate of 0.3 mL/min as follows (mobile phase A, 0.05% formic acid aqueous solution; mobile phase B, acetonitrile): 0.0-0.1 min 90.0%A, 10.0% B; 0.1-1.5 min 10.0%A, 90.0%B; 1.5-2.0min 10.0%A, 90.0%B; and 2.0-2.1 min 90.0%A, 10.0% B; 2.1-3.0 min 90.0%A, 10.0% B. For UPLC separations, the temperature of the chromatographic column was set at 40°C.The injection volume was 2.0 μL.The [M + H] + ion of muscarine (m/z 174.200) was selected as the parent ion, and ions of 57.000 as well as 97.000 were used for qualitative and quantitative detection, respectively.The rest of the MS parameters were set the same of Xu et al. (2020).

Sample preparation
All experimental samples were carefully cleaned and separated from soil residue.The dried and powdered sample (0.25 g) was 3 http://iqtree.cibiv.univie.ac.at/ digested completely using 7 mL of HNO 3 (67-70%) microwave and it was heated at 110°C on a graphite digestion apparatus for 30 min.After cooling, digested samples were diluted with purified water to a volume of 50 mL.

Chemicals and instruments
All digested reagents were trace metal grade and purchased from Fisher Chemical.The ICP-MS was used for the determination of elements (In, Bi, and Ge are internal standard).The conditions of the ICP-MS are as follows: radio frequency power: 1.60 kw; carrier gas: 99.999% Ar; plasma gas flow rate: 15.0 L/min; auxiliary gas flow rate: 1.2 L/min; nebulizer gas flow rate: 1.02 L/min; detector mode: standard mode; integration time: 1000 ms.The analysis process, triplicate samples, blanks, precision, and rates of recovery were referred to GB5009.268-2016 Chinese National Standard for Food Safety-Determination of Multiple Elements in Food.

Phylogenetic analyses
The final multi-locus concatenated dataset (RPB2-ITS-LSU), which included 96 taxa, had 3,200 nucleotide sites, comprising 860 bp ITS, 1,560 bp LSU, and 780 bp RPB2, of which 1,781 bp were constant sites and 1,004 bp were parsimony informative sites.All gene regions resulted in the GTR + I + G model.Only the phylogenetic tree inferred from the maximum likelihood (ML) strategy is provided because of the topological consistency between ML and BI.The values of maximum likelihood bootstrap (MLB) as well as Bayesian posterior probabilities (BPPs) are presented in Figure 1.A total of 15 newly generated sequences (5 ITS, 5 LSU, and 5 RPB2) have been submitted to GenBank.Auritella hispida Matheny and T.W. Henkel and A. spiculosa Matheny and T.W. Henkel were treated as outgroups.All of our collections of the novel species were nested in Old World tropical clade 2 and were grouped into an independent lineage with strong support (MLB = 100%, BPP = 1).The phylogenetic analysis shows that the new species is sister to I. akirnum (K.P.D. Latha and Manim.)Matheny and Esteve-Rav.(MLB = 100%, BPP = 1), and the distinct lineage that formed by these two taxa is close to I. saragum (K.P.D. Latha and Manim.)Matheny and Esteve-Rav.(MLB = 100%, BPP = 1) and an undescribed species from Papua New Guinea (Inosperma sp.L-GN3a) (MLB = 100%, BPP = 1).

UPLC-MS/MS muscarine analysis
Muscarine in the samples was detected by UPLC-MS/MS.As shown in the representative chromatograms in Figures 4C,D, the muscarine retention time (0.83 min) in the samples was identical to that of the muscarine standard (0.83 min).The calibration curve for muscarine generated during the validation was y = 19621.4x + 33,174 (Figure 4A).The correlation coefficients (r = 0.9995) of linear regression analysis from calibration curves were > 0.99.Linearity was determined for muscarine in the concentration range of 5-200 ng/ mL.The matrix effect (ME) was sufficiently weak (|ME| = 0.019%, 0 ≦ |ME| ≦ 20%) to be ignored.All samples were diluted with 500× before UPLC-MS/MS analysis, and double parallel was made for each sample.The precisions and recoveries of muscarine and the muscarine content of the new species are displayed in Tables 1, 2, respectively.

Element contents
The contents of 24 elements in the new species are summarized in Table 3, provided as means ± standard deviation (mg/kg), as well as the limit of detection (LOD) and the limit of quantification (LOQ).The precisions were performed six times for each element, and the recoveries ranged from 90 to 110%, thereby meeting the specifications of the GB 5009.268-2016Chinese National Food Safety Standard.The elements detected in the new species were ranked, from highest concentration to lowest, as follows: potassium (K) > magnesium (Mg) > aluminum (Al) > iron (Fe) > zinc (Zn) > copper (Cu) > sodium (Na) > titanium (Ti) > arsenic (As) > calcium (Ca) > manganese (Mn) > barium (Ba) > cadmium (Cd) > chromium (Cr) > vanadium (V) > strontium (Sr) > cobalt (Co) > lead (Pb) > mercury (Hg) > (nickel) Ni > molybdenum (Mo) > thallium (Tl) > boron (B) > tin (Sn).Among all the elements, K had the highest concentration (47,237.30± 543.40 mg/kg), Mg was the metal with the second highest concentration (1,298.47± 8.48 mg/kg), followed by Al (891.30± 1.21 mg/kg), and Sn had the lowest concentration (0.01 ± 0.00 mg/kg).Most importantly, the concentrations of five elements (Cr, As, Cd, Hg, and Pb) exceeded limits specified in the Chinese National Food Safety Standard-Maximum Levels of Contaminants in Foods (GB 2762(GB -2022)).Among these five elements, arsenic (36.76 ± 0.43 mg/kg) was the predominant toxic and heavy metal.

Identification of the new species
Inosperma wuzhishanense was collected in a tropical montane fagaceous forest in Hainan Province, tropical China.It can be recognized by its scattered to often gregarious habit, reddish-brown pileus with appressed scales and initially covered with grayish white veilipellis when young, rather crowded grayish lamellae, and fibrilloseflocculose stipe in the field.In some specimens, an indistinct vinaceous tinge could be found in the squamulose on the pileus or in the fibrils on the stipe surface.Microscopically, it has smooth and ellipsoid basidiospores and thin-walled, often clavate to cylindrical clavate cheilocystidia; no caulocystidia was observed, but instead of protruding hyphal elements.Currently, it is only known from the type locality.

Muscarine contents
Species in the Old World tropical clade 2 are frequently reported to contain muscarine and cause numerous muscarine-poisoning events in the tropical regions of Asia, such as the concentrations of 0.58 ± 0.02 to 6.53 ± 1.88 g/kg in I. zonativeliferum (Deng et al., 2022a), 16.03 ± 1.23 g/kg in I. muscarium and 11.87 ± 3.02 g/kg in I. hainanense (Deng et al., 2021a), and 0.27 mg/g to 0.3 mg/g in I. virosum (Sailatha et al., 2014;Latha et al., 2020)   clinical Inosperma samples (Parnmen et al., 2021).The lethal dose of muscarine for humans has been estimated to range from 40 mg to 495 mg (Pauli and Foot, 2005).In the present study, the muscarine content of I. wuzhishanense was determined to be 2,301.36± 83.52 to 7,114.03 ± 76.55 mg/kg, and the muscarine contents differed significantly among the stipe, pileus, and lamellae.Many studies have detected significant differences in the amounts of toxin among different tissues of the mushroom fruit body.For instance, analyses of six different tissues of Amanita fuliginea Hongo (gills, pileus, stipe, annulus, volva, and spores) revealed that the highest concentration of total toxins (amatoxins and phallotoxins) was in the gills (14.46 mg/g, dry weight), and the lowest concentration was in the spores (0.25 mg/g, dry weight) (Zhou et al., 2017).The amount of amatoxin was found to be higher in the pileus than in the stipe in A. phalloides var.alba Costantin and L.M. Dufour, A. exitialis Zhu L. Yang and T.H. Li, and A. phalloides Secr. (Hu et al., 2012;Kaya et al., 2013;Yilmaz et al., 2015), and Lepiota brunneoincarnata Chodat and C. Martín (Yilmaz et al., 2015;Sun et al., 2019).Within the Inocybaceae, in Pseudosperma arenarium Y.G.Fan, Fei Xu, Hai J. Li, and Vauras, the muscarine content was found to be approximately five times higher in the pileus than in the stipe (Yan et al., 2022); whereas the muscarine content was found to be approximately three times higher in the stipe than in the pileus I. zonativeliferum (Deng et al., 2022a).A similar phenomenon was observed in I. wuzhishanense.Our results showed that the muscarine content was much higher in the stipe than in the pileus in the new species.As shown in Figure 4B, the highest muscarine content was in the stipe, followed by the pileus, and the lowest muscarine content was in the lamellae, with significant differences among the three tissues.It is interesting to note that I. wuzhishanense and I. zonativeliferum have the same pattern of muscarine content in the stipe and the pileus.This pattern differs from most of the poisonous mushroom species studied (see above).This includes P. arenarium, a poisonous mushroom also belonging to the Inocybaceae.Future investigations are needed to clarify whether this is a common pattern in the Inosperma genus and the underlying mechanism.

Element contents
Wild mushrooms accumulate toxic and heavy metals (Alzand et al., 2019).Non-essential elements such as Cr, Cd, As, Hg, and Pb are not only poisonous to aquatic organisms but also harmful to human health, even at low concentrations (Balali-Mood et al., 2021;Noman et al., 2022).These harmful heavy metals have been detected frequently in Agaricaceae, Amanitaceae, Boletaceae, Russulaceae, Inocybaceae, etc. (Bizio et al., 2020;Barea-Sepúlveda et al., 2022;Liu et al., 2022).In the present study, five toxic and heavy metals (Cr, As,  GB 2762GB -2022)).Of these three elements, only As was present at a concentration markedly higher than that specified in the GB 2762-2022 standard and in most edible mushrooms (Dowlati et al., 2021;Yu et al., 2021) and most wild mushrooms (Melgar et al., 2014;Fu et al., 2020;Keskin et al., 2021).However, the As concentration in I. wuzhishanense was lower than that reported for some species in the Inocybaceae (Bizio et al., 2020).Exposure to As can result in cardiovascular dysfunction, skin and hair changes, central nervous system injury, gastrointestinal discomfort, and liver damage (Balali-Mood et al., 2021).The concentrations of the metalloid element (B) and other 18 other essential metallic elements (K, Mg, Al, Fe, Zn, Cu, Na, Ti, Ca, Mn, Ba, V, Sr., Co, Ni, Mo, Tl, and Sn) were similar to those reported for other Inosperma's fruit bodies in the literature, with only slight differences (Bizio et al., 2020).

Conclusion
A new Inosperma taxa was found in tropical China and has been named I. wuzhishanense.In this new species, the muscarine contents were ranged from 2,301.36 ± 83.52 to 7,114.03 ± 76.55 mg/kg and differed significantly among the stipe, pileus, and lamellae.Additionally, I. wuzhishanense demonstrated an exceptional ability to accumulate Cr, Hg, Pb, Cd, and As.Therefore, this species poses risks to human health because of the presence of muscarine and the high concentrations of toxic and heavy metals.

FIGURE 1
FIGURE 1Phylogram generated by Bayesian (BI) analyses based on sequences of a combined dataset from nuclear genes, rooted with Auritella hispida and A. spiculosa.Bayesian inference (BI-PP) ≥ 0.95 and ML bootstrap proportions (ML-BPs) ≥ 70 are represented as BI-PP/ML-BP.Inosperma wuzhishanense is the newly described taxa; Scale bar = 0.05.
The author(s) declare financial support was received for the research, authorship, and/or publication of this article.This study was supported by the Hainan Provincial Natural Science Foundation of China (323MS044 and 820RC624), the National Science Foundation of China (32260005 and 31860009), the Key Laboratory of Tropical Fruits and Vegetables Quality and Safety for State Market Regulation (ZZ-2023020), and the Scientific Research Project of Hainan Higher Education Institutions (Hnky2023ZD-8).
was used to manually improve the aligned sequences when necessary.MrModeltest v.2.3 was performed for each locus to select the best-fit evolutionary model with the Akaike Information

TABLE 1
Precisions, and recoveries for muscarine in the new species measured by UPLC-MS/MS.

TABLE 2
Muscarine content in different parts of different specimens in I. wuzhishanense detected by UPLC-MS/MS.

TABLE 3
Determined values, precisions, and recoveries measured by ICP-MS/MS.As shown in Table4, the levels of Cd, As, and Hg in the new species were higher than those specified in the National Food Safety Standard for Maximum Levels of Contaminants in Foods of China ( Cd, Hg, and Pb) and other elements (B, Na, Mg, Al, K, Ca, Ti, V, Mn, Fe, Co, Ni, Cu, Zn, Sr., Mo, Sn, Ba, and Tl) were detected in I. wuzhishanense by ICP-MS.

TABLE 4
Heavy metal concentrations (mg/kg) in targeted species, comparison with guideline values and related studies.
"N" refers to the number of studies; "n" is the sample number; "/" means no data.