Introduction
Inocybaceae is a family of agarics that contains many poisonous species. However, Kosentka et al. (2013) found that the most recent common ancestor of the family did not contain muscarine. Recognising its species diversity and detecting its toxins are essential to control and prevent poisoning incidents (Li et al. 2020; Deng et al. 2021a). According to the latest molecular phylogeny, seven genera were treated in Inocybaceae (Matheny et al. 2020). Pseudosperma, referred to as Inocybe sect. Rimosae sensu stricto (Larsson et al. 2009) or Pseudosperma clade (Matheny 2009), is one of the muscarine-containing genera in the family with numerous cryptic and semi-cryptic species. It is characterised by rimulose to rimose pileus, furfuraceous to appressed furfuraceous stipe with flocculose apex, elliptic to sub-phaseoliform basidiospores, the absence of pleurocystidia and the presence of thin-walled cheilocystidia. Ninety-seven Pseudosperma taxa have been recorded in the IndexFungorum database (www.indexfungroum.org; retrieved 7 May 2022). Of these, more than 40 taxa have been reported or originally described in Europe (Bandini and Oertel 2020). Since the establishment of the genus in 2020, 16 new taxa have been discovered in Asia and Europe in the past 2 years alone (Bandini and Oertel 2020; Cervini et al. 2020; Jabeen and Khalid 2020; Saba et al. 2020; Yu et al. 2020; Bandini et al. 2021; Jabeen et al. 2021). However, the species diversity of Pseudosperma is still poorly explored in East Asia. In China, only six taxa have been verified, including three recently described species, viz., P. yunnanense, P. neoumbrinellum and P. citrinostipes (Bau and Fan 2018; Yu et al. 2020).
Ecologically, Pseudosperma species have an ectomycorrhizal symbiosis with various plants and are commonly found in north temperate forests dominated by Betula, Cedrus, Populus, Pinus, Picea, Quercus, Salix etc. During field surveys in north-western China, a poisonous Inocybaceae mushroom collected under Populus plantations caught the authors’ attention because of its strikingly robust habit. This stout Inocybaceae species has led to three poisoning incidents, with a total of seven patients in north-western China during the past 2 years. Two of these occurred in September in Ningxia and Shanxi in 2020 and another occurred in Ningxia in October 2021 (Li et al. 2021a, 2022). All patients from the three poisoning incidents suffered from classic parasympathetic nervous system stimulation syndromes. After microscopic examinations and molecular analyses, mushroom specimens obtained from poisoning locales, together with a European specimen, were proven as a new Pseudosperma species. Discussions on the distribution, relationships and distinction of the new species and its affinities are also provided. Additionally, to better understand the toxicity of the new species and contribute to their poisoning control and prevention, 11 major mushroom toxins, namely, two isoxazole derivatives (ibotenic acid and muscimol), two tryptamine alkaloids (psilocybin and psilocin), three amatoxins (α-, β- and γ-amanitin), three phallotoxins (phalloidin, phallacidin and phallisacin) and muscarine, were assayed.
Methods
Sampling, morphological observations and descriptions
The Chinese materials were collected in sandy poplar plantations from Ningxia Hui Autonomous Region and Shaanxi Province, where there is a temperate continental climate. The European material JV26578 was collected in a seashore forest from Estonia, in a hemiboreal zone. Macroscopic features were described, based on fresh materials and colour photographs. A small piece of the pileus, lamella or stipe tissue was mounted in 5% aqueous potassium hydroxide (KOH) on the slide and then examined using a light microscope when the tissue was completely rehydrated. Microscopic structures, including basidiospores, basidia, cheilocystidia, hymenophoral trama, caulocystidia, pileipellis and stipitipellis, were examined from rehydrated materials. The measurements of micro-structures follow Fan and Bau (2013) and Yu et al. (2020). The number of measured basidiospores is given as an abbreviation [n/m/p], which denotes n spores measured from m basidiomata of p collections. The measurements and Q values are given as (a)b–c(d), “b–c” covers a minimum of 90% of the measured values, “a” and “d” represent the extreme values; Q means the ratio of length/width in an individual basidiospore, Qm is the average Q of all basidiospores ± sample standard deviation (Ge et al. 2021; Na et al. 2022). Colour designations follow Kornerup and Wanscher (1978). Voucher specimens were deposited in the Herbarium of Herbarium of Changbai Mountain Nature Reserve (ANTU) with FCAS numbers and TUR-A.
DNA extraction, polymerase chain reaction, sequence amplification and data analysis
Genomic DNA was extracted from silica-dried materials using the NuClean Plant Genomic DNA Kit (ComWin Biotech, Beijing). The internal transcribed spacer (ITS) region, the nuclear large subunit (nLSU) and the RNA polymerase II second largest subunit (RPB2) sequences were amplified and sequenced separately by using primer pairs ITS1F/ITS4 (Gardes and Bruns 1993), LR0R/LR7 (Vilgalys and Hester 1990) and RPB2-6F/RPB2-7.1R (Matheny 2005). The PCR thermocycling protocol was 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 (Wang et al. 2021). Sequencing work was done by Sangon Biotech (Shanghai) Co., Ltd. Sequences of related taxa in Pseudosperma, retrieved from previous studies, were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/) for phylogenetic analysis (Suppl. material 1). Mallocybe terrigena (Fr.) Matheny, Vizzini & Esteve-Rav. was used for the outgroup. The sequence data matrix for each locus was aligned by Mafft online service (https://mafft.cbrc.jp/alignment/server/) (Katoh et al. 2019) and manually adjusted by BioEdit 7.0.9.0 (Hall 1999). The aligned datasets were combined with Mega 5.02 (Tamura et al. 2011). MrModeltest v.2.3 was used to determine the optimal substitution model for each locus with the Akaike Information Criterion (Nylander 2004). Bayesian Inference (BI) analyses, executed in MrBayes v.3.2.7a (Ronquist et al. 2012), were run for 1,235,000 generations using four Metropolis-Coupled Monte Carlo Markov chains to calculate posterior probabilities and the standard deviation of the split frequencies was terminated at 0.009977. Maximum Likelihood (ML) analysis was conducted in W-IQ-TREE Web Service (http://iqtree.cibiv.univie.ac.at/) with 1,000 replicates (Trifinopoulos et al. 2016).
Toxin detection
Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was performed for toxin detection. Detailed mushroom sample preparations, analysis of muscarine, amatoxins and phallotoxins (Alta Scientific Co., Ltd., Tianjin, China) referred to our previous works (Xu et al. 2020a, 2020b).
Detailed information for analysis of ibotenic acid and muscimol (Alta Scientific Co., Ltd., Tianjin, China) are as follows: chromatographic separation was conducted on an ACQUITY UPLC C8 column (2.1 × 100 mm, 1.7 μm; Waters, USA). Acetonitrile (A) and 4% formic acid aqueous solution (B) were used as mobile phase solvent flowing at 0.3 ml/min. The column was eluted by 2% A for 1.0 min, followed by 2%–70% A for 1.0 min, then by 70% A for 1.0 min and then by 70%–2% A for 0.5 min, finally by 2% A for 1.5 min. The analytical column was set at 40 °C. The injection volume was 10 μl. The positive MS/MS conditions can refer to muscarine (Xu et al. 2020b). The ion pairs were 115.1 > 68.1 (Cone at 16 V; Collision at 12 V), 159.1 > 113.1 (Cone at 16 V; Collision at 12 V) for ibotenic acid, as well as 115.1 > 98.1 (Cone at 15 V; Collision at 10 V), 115.1 > 68.1 (Cone at 15 V; Collision at 18 V) for muscimol.
For the analysis of psilocybin and psilocin (Alta Scientific Co., Ltd., Tianjin, China), the detailed descriptions are as follows. ACQUITY UPLC T3 column (2.1 × 100 mm, 1.7 μm; Waters, USA) was used as the separation column. The mobile phases were acetonitrile (A) and 10 mmol/l ammonium acetate aqueous solution (B). The flow rate was 0.3 ml/min. The column was eluted by 0% A for 0.5 min, followed by 0%–85% A for 4 min, then by 85% A for 1.5 min and then by 85%–0% A for 1.5 min, finally by 0% A for 2 min. The analytical column was set at 40 °C and the injection volume was 10 μl. The positive MS/MS conditions can refer to muscarine (Xu et al. 2020b). The ion pairs were 285.1 > 85.2 (Cone at 16 V; Collision at 18 V), 285.1 > 240.1 (Cone at 16 V; Collision at 17 V) for psilocybin and 205.1 > 58.2 (Cone at 26 V; Collision at 13 V), 205.1 > 160.1 (Cone at 26 V; Collision at 13 V) for psilocin.
Discussion
The new species is known from three localities in Ningxia and Shaanxi of north-western China and is a locally common mushroom that occurs in late autumn under sandy poplar plantations (Fig. 2). As Populus alba × P. berolinensis plantations are widely distributed over north-western China, the new species may have broader distribution in adjacent areas. Moreover, the European material JV26578 collected in Estonia clustered with the new species with full support in the phylogenetic results. This collection also grew on calcareous fine sand, but under Pinus sylvestris. According to the file notes, the specimen also has robust basidiomata (pileus up to 38 mm broad, stipes 50–55 × 7–9 mm) with ochraceous pileus and stipes and fungoid or slightly spermatic odour. The microfeatures of JV26578 have cylindrical-ellipsoid basidiospores measuring (13.5–)13.9–16.5(–18.2) × (7.1–)7.2–8.5(–8.8) μm (average, 15.3 × 7.7 μm), Q = 1.8–2.2(–2.25) [average, 1.98 (n = 20)] and clavate cheilocystidia measuring (34–)40–62(–70) × 14–20(–22) μm [average, 50 × 18 μm (n = 20)]. Except for the ochraceous tinge in pileus and stipes, no distinct macroscopical difference was observed between the Chinese materials and the Estonian specimen. The European specimen JV26578 is now considered conspecific with the Chinese materials. Accordingly, the new species has a Eurasian distribution.
Pseudosperma arenarium is characterised by its tricholomoid habit, dirty whitish to ochraceous and glabrous pileus, crowded lamellae with fimbriate edges, large cylindrical basidiospores and thin-walled cheilocystidia. The thick and long persistent velipellis gives its pileus a nearly smooth and whitish appearance. In the field, the pileus, stipe and lamellae surfaces are usually covered with humose sands, showing a dirty yellowish or sometimes brownish colour, especially in older individuals. Its mostly large cylindrical basidiospores are microscopically impressive, but cylindrical ellipsoid to elongated ellipsoid basidiospores also exist in the same individual. With the combination of the characteristics listed above, the new species is distinctive. Without examining its microscopic features or molecular sequence analyses, a mycologist or even an Inocybaceae specialist is unlikely to be able to identify it exactly into the genus Pseudosperma. Unexpectedly, the three-gene phylogeny places P. arenarium in the P. rimosum complex, which clusters with the lineage that unified the type material of P. aureocitrinum and a sample labelled as ‘P. cf. rimosum.’ However, P. aureocitrinum has a typical inocyboid habit, yellowish-tinged basidiomata and broadly ellipsoid to subovoid basidiospores and occurs in Mediterranean evergreen oak forests (Esteve-Raventós 2014).
Pseudosperma arenicola (R. Heim) Matheny & Esteve-Rav., a European species also occurring on coastal sandy soils, is similar in having a whitish appearance, long-persisting thick velipellis and long basidiospores, but it has a less robust habit, relatively short basidiospores measuring 11.5–12–18.5 × 6.0–6.4–7.5 μm (Kuyper 1986), different ecological associations and different phylogenetic positions (Heim 1931; Kuyper 1986; Stangl 1989). Pseudosperma pseudo-orbatum (Esteve-Rav. & García Blanco) Matheny & Esteve-Rav. is a whitish species originally described from Spain, resembling the new species in having thick velipellis, non-rimose pileus, large cylindrical basidiospores and clavate cheilocystidia. However, it is distinguished by having pinkish lamellae when young, stockier stipes and an association with Pinus pinaster Ait. and P. pinea L. (Esteve-Raventós et al. 2003). Pseudosperma niveivelatum (D.E. Stuntz ex Kropp, Matheny & L.J. Hutchison) Matheny & Esteve-Rav., a North American species, shares white thick velipellis, elongated large basidiospores and ecological association with Populus tremuloides Michx. and conifers. However, it differs by its sericeous pileus, shorter basidiospores measuring 11.5–13.9–18.5 × 6.0–6.4–7.5 μm, slender cheilocystidia (Kropp et al. 2013) and a clinically insignificant amount of muscarine (Kosentka et al. 2013).
Muscarine is a neurotoxin that causes salivation, sweating, delirium and even coma or death (Işiloğlu et al. 2009; Xu et al. 2020b). In recent years, more and more poisoning cases have been caused by eating Inocybaceae mushrooms containing toxic muscarine (Li et al. 2020, 2021a, 2022; Xu et al. 2020b). According to literature, five Pseudosperma species have been assayed; of these, P. rimosum (Bull.) Matheny & Esteve-Rav., P. niveivelatum, P. sororium (Kauffman) Matheny & Esteve-Rav. and P. spurium (Jacobsson & E. Larss.) Matheny & Esteve-Rav. contain muscarine; only P. perlatum (Cooke) Matheny & Esteve-Rav. was reported lacking muscarine (Kosentka et al. 2013). Xu et al. (2020b) reported that the muscarine content of I. serotina Peck in a poisoning incident was 324.0 ± 62.4 mg/kg wet weight. Li et al. (2021b) reported that the muscarine content in I. squarrosolutea (Corner & E. Horak) Garrido and I. squarrosofulva S.N. Li, Y.G. Fan & Z.H. Chen were 136.4 ± 25.4 to 1683.0 ± 313.0 and 31.2 ± 5.8 to 101.8 ± 18.9 mg/kg dry weight, respectively. Deng et al. (2021b, 2022) found that muscarine content in Inosperma muscarium Y.G. Fan, L.S. Deng, W.J. Yu & N.K. Zeng, I. hainanense Y.G. Fan, L.S. Deng, W.J. Yu & N.K. Zeng and I. zonativeliferum Y.G. Fan, H.J. Li, F. Xu, L.S. Deng & W.J. Yu were 16.03 ± 1.23, 11.87 ± 3.02 and [2.08 ± 0.05 (pileus) and 6.53 ± 1.88 (stipes)] g/kg dry weight, respectively. In this study, results showed that P. arenarium is a muscarine-positive species with middle- and upper-level muscarine content and also led to three poisoning incidents with a total of seven patients in northwest China during the past 2 years (Li et al. 2021a, 2022). Interestingly, the muscarine content in caps was approximately five times higher than in stipes. Although some studies showed that the toxin amount in the cap is higher than in the stipes (Hu et al. 2012; Garcia et al. 2015; Sun et al. 2018, 2019), the mechanism of such difference is still not clear. Li et al. (2021b) reported that the muscarine content of Inocybe squarrosolutea varied a lot in different specimens. However, in this study, the muscarine content in the mushroom samples collected from the same or three different places showed no significant difference. Additionally, no amatoxins, phallotoxins, ibotenic acid, muscimol, psilocybin and psilocin were detected in all samples. This study described P. arenarium as a new species, based on morphological, ecological, molecular and toxic evidence. The publicity and education of the new species are needed to control and prevent mushroom poisoning incidents.