﻿ Pseudospermaarenarium (Inocybaceae), a new poisonous species from Eurasia, based on morphological, ecological, molecular and biochemical evidence

﻿Abstract In this study, Pseudospermaarenarium is proposed as a new species, based on morphological, ecological, molecular and biochemical evidence. The new species grows on sandy ground under Populus and Pinussylvestris in north-western China and northern Europe, respectively. It is characterised by the combination of the robust habit, nearly glabrous pileus, large cylindrical basidiospores, thin-walled cheilocystidia and ecological associations with Populusalba × P.berolinensis and Pinussylvestris and unique phylogenetic placement. Additionally, a comprehensive toxin determination of the new species using ultra-high performance liquid chromatography-tandem mass spectrometry was conducted. Results showed that it was a muscarine-positive species. The content were approximately five times higher in the pilei [4012.2 ± 803.1–4302.3 ± 863.2 mg/kg (k = 2, p = 95%)] than in the stipes [850.4 ± 171.1–929.1 ± 184.2 mg/kg (k = 2, p = 95%)], demonstrating the severity of mushroom poisoning when patients consumed different parts of the poisonous mushroom. Amatoxins, phallotoxins, ibotenic acid, muscimol, psilocybin and psilocin were not detected.


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 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 muscarinecontaining 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. 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.

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, Q m 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 Changbai Mountain Nature Reserve (ANTU) with FCAS numbers and TUR-A.
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 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

Phylogenetic analyses
Nine sequences (three ITS, three LSU and three rpb2) were newly generated and submitted to GenBank. The best-fit model selected by MrModeltest was GTR+I+G for each gene equally. The three-gene data matrix consisted of 104 taxa and 2890 sites. The final multilocus alignment used for phylogenetic reconstruction was submitted to TreeBase (ID29310). The Bayesian Inference and Maximum Likelihood trees were similar in topology; thus, only the BI tree was presented (Fig. 1). In the BI tree ( Fig. 1), all Pseudosperma taxa grouped in a fully supported clade and three Chinese specimens Etymology. refers to its habitat of sandy soils.

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. 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).
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. , 2021aXu et al. 2020b (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. (2021bDeng et al. ( , 2022 found that muscarine content in Inosperma muscarium Y.G. Fan .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. 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. 2018Sun et al. , 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.