Phylogenetic analyses, morphological studies, and muscarine detection reveal two new toxic Pseudosperma (Inocybaceae, Agaricales) species from tropical China

As a result of molecular phylogenetic analyses and morphological studies, two new species of the genus Pseudosperma, namely P. fulvidiscum and P. singulare, were discovered in Wuzhishan Nature Reserve of Hainan Province of China. The two new species are distinct from other known Pseudosperma species in the phylogram inferred from a combined nuclear ribosomal internal transcribed spacer region (ITS), the nuclear ribosomal large subunit (nrLSU), and the nuclear second-largest subunit of RNA polymerase II (RPB2) sequence data. Muscarine contents of the two new species were detected using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach. In addition, a new combination, P. rubrobrunneum, was proposed based on phylogenetic evidence.


Introduction
Inocybaceae is a family of ectomycorrhizal mushroomforming fungi (Matheny and Bougher 2006), and many species in this family are poisonous (Kosentka et al. 2013). Multigene molecular phylogenetic analyses have now culminated in the recognition of at least seven major clades in the family (Matheny et al. 2020). Pseudosperma is a newly established genus in Inocybaceae and is supposed to contain muscarine, a neurotoxin that would cause a series of muscarinic symptoms in humans and animals (Deng et al. 2021b). Mushroom poisoning incidents caused by Pseudosperma species were reported in various regions around China (Li et al. 2022). The recognition of its species diversity, distribution, and toxin type and contents will be helpful to the prevention of poisoning incidents.
Pseudosperma is characterized by the rimulose to rimose pileus, the furfuraceous to appressed furfuraceous stipe with a pruinose apex, spermatic odor, elliptic to subphaseoliform basidiospores, the absence of pleurocystidia, the presence of thin-walled cheilocystidia, and symbiosis ecology with a large number of vascular plants (Matheny et al. 2020). At present, 93 taxa of Pseudosperma were recorded in the IndexFungorum database (www.indexfungorum.org, retrieved 26 Mar. 2022), and 40 of those were reported or originally described from Europe (Bandini and Oertel 2020). The species diversity in other continents was poorly addressed, especially in tropical areas. After the erection of the genus, 16 new taxa were described in the past two years (Yu et al. 2020;Cervini et al. 2020;Saba et al. 2020;Jabeen and Khalid 2020;Jabeen et al. 2021;Bandini and Oertel 2020;Bandini et al. 2021). Currently, seven species of Pseudosperma were reported in China (Bau and Fan 2018;Yu et al. 2020), including four recently described species, viz. P. neoumbrinellum, P. yunnanense (Bau and Fan 2018), P. citrinostipes (Yu et al. 2020), and P. arenarium (Yan et al. 2022, in press).
Hainan is the southernmost provincial administrative region of China and is located on the north edge of the Chinese tropics. Central Hainan harbors the largest contiguous area of tropical rainforest and is a hotspot area of biodiversity in China (Zong 2020). Many new fungal species have been discovered in Hainan province every year (Wang et al. 2021a). In this study, we aim to describe two new Pseudosperma species using combined analyses of molecular, morphological, and ecological data. In addition, a comprehensive method of determination of these two new species using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach has been performed to detect their muscarine contents.

Field sampling and morphological studies
Fresh materials were collected in the Wuzhishan and Yinggeling substations of Hainan Tropical Rainforest National Park and Fujian Province in China. Firstly, basidiomata were photographed using a digital camera in the field. Secondly, fresh specimens were recorded and described macroscopically later. Then, the specimens were dried overnight at 45°C with an electronic drier, and the dried specimens were packed in sealed plastic bags with silica gel (Yu et al. 2020;Deng et al. 2021a). Color codes follow Kornerup and Wanscher (1978).
Microscopical characteristics were observed using an optical microscope (Olympus CX23) in the laboratory. Mushroom tissues from the pileus, the lamellae, and the stipes were cut into thin sections by freehand with the aid of a stereoscope (AV100-240V). Dried materials were rehydrated in KOH (5%) and stained with Congo Red (1%) when necessary. Microscopic structures, including basidiospores, basidia, cheilocystidia, hymenophoral trama, pileipellis, and stipitipellis, were examined and measured. Methods of measurements follow Fan and Bau (2013) and Yu et al. (2020). At least 100 basidiospores for each species were randomly measured from different specimens. The basidiospore measurements are expressed with the [n/m/p], which indicates n basidiospores measured from m basidiomata of p specimens. In addition, the basidiospore size is given with the (a) b-e-c (d) formula, where b-c contains a minimum of 90% of the measured values of the spores, e represents the average values of the spores, and a and d represent the minimum and maximum values of the spores (Ge et al. 2021). Q is the ratio length/width of individual spores (Q value); Q m ± SD is the average Q of all basidiospores ± sample standard deviation (Na et al. 2022;Jean et al. 2022). Examined specimens were deposited in the Herbarium of Changbai Mountain Natural Reserve (ANTU) with FCAS numbers and the Fungal Herbarium of Hainan Medical University (FHMU).

Toxin detection
Dried mushroom samples from holotypes of the two new species (FYG6311 and FYG6363) were grounded into a fine powder; 0.02 g was accurately weighed and put into a 5-mL centrifugation tube with 2 mL of methanol-water (5:95, v/v), respectively. The mixture was vortexed for 30 min at first and then was extracted ultrasonically for another 30 min. After centrifugation at 1000 rpm for 5 min, the total supernatant was collected using a 0.22-μm organic filter membrane to filtrate before UPLC-MS/MS analysis and diluted with acetonitrile-water (7:3, v/v) when necessary. Lentinula edodes was used as the blank sample. The optimal UPLC and MS parameters and other settings followed Xu et al. (2020). The muscarine content was estimated in the mushroom extraction by using standard muscarine (Sigma-Aldrich, Chemical purity ≥98%). The analytical results are reported as mean ± SD g/kg, where the mean is the average content of muscarine, and SD represents the standard deviation.

Phylogenetic analyses
We newly generated 16 ITS, 7 nrLSU, and 7 RPB2 sequences and submitted them to GenBank. The best-fit models selected by MrModeltest for each gene are GTR+I+G equally. The concatenated dataset (Supplementary information) comprises 144 taxa (Table 1) and 3180 nucleotide sites with 958 bp ITS, 1445 bp nrLSU, and 777 bp RPB2, of which 1814 are constant and 884 are parsimony informative. The phylograms resulted from Bayesian inference (BI) and maximum likelihood (ML) analyses are similar with a few statistical differences, and thus, only the ML tree is shown in Fig. 1. All the Pseudosperma taxa were clustered in a full support clade (BP = 100%, PP = 1). Two major subclades were retrieved in Pseudosperma, and one of them was only supported by ML analysis. Thirteen specimens form an independent lineage with full support (BP = 100%, PP = 1), representing P. fulvidiscum, and three specimens cluster together and form an independent lineage representing P. singulare. The two new species are grouped into a full support subclade (BP = 100%, PP = 1) that is sister to all the remainders of the genus. The subclade unifying P. arraneosum (Matheny and Bougher) Matheny and Esteve-R a v . , P . a f f . a r a n e o s u m ( P L 5 8 4 1 0 ) , P. brunneosquamulosum K.P.D. Latha and Manim., P. rubrobrunneum (K.P.D. Latha and Manim) Y.G. Fan, and P. sp. (MCA562) as well as the two new species. In this subclade, P. aff. araneosum (PL58410) is sister to P. fulvidiscum, and P. araneosum is sister to P. singulare. Pseudosperma brunneosquamulosum and P. sp. (MCA562) clustered in a full support lineage that is sister to the lineage unifying P. fulvidiscum and P. aff. araneosum (PL58410); P. rubrobruneum is sister to the lineage unifying P. singulare and P. araneosum.
Diagnosis: Pseudosperma fulvidiscum differs from P. brunneosquamulosum by its relatively larger habit, pallid stipes with brownish tiers of squmules in the lower part, broader lamellae, longer and usually subcapitate cheilocystidia, and its association with Carpinus trees.
Habitat Remarks: Pseudosperma fulvidiscum is a common species under Carpinus forests; it fruits from late April to late August. In the field, it is a typical little brown mushroom with a slender habit and brownish pileus. The pileus status could be fibrillose to rimose with appressed scales or fibrils in different specimens. The stipe surface is usually finely felty or appressedfibrillose with brownish tomentose hyphae toward the lower part, however usually hard to observe after handling. Unlike most species in the genus, the new species has moderately crowded to subdistant lamellae in certain specimens. The differences between individuals or collections were mostly due to the weather conditions or microclimates. In terms of microscopic intraspecific variation in P. fulvidiscum, the basidiospores of FYG6311 are smaller and more spherical (Q m = 1.54, n = 20); however, FYG6327 has larger and narrower basidiospores (Q m = 1.74, n = 20). Phylogenetically, an undescribed Australian specimen (PL58410) labeled P. aff. araneosum, is the closest taxa with moderate support. Pseudosperma brunneosquamulosum, originally described in tropical India, shares similarities in its small and slender habit, brownish pileus color and status, and the size and the shape of the basidiospores; however, it differs by brownish orange stipes, broader basidiospores (Q m = 1.5), shorter and non-capitate cheilocystidia, and an association with trees of Dipterocarpaceae (Tibpromma et al. 2017).
Habitat: Single on clay soils under Carpinus londoniana var. lanceolata.
Geographical distribution: currently known only from the type locality in Hainan Province, China. Remarks: Pseudosperma singulare is encountered under Carpinus forests; it fruits solitarily in late July in Hainan Fig. 1 Phylogram generated by maximum likelihood (ML) analysis based on a combined data set from nuclear genes (rDNA-ITS, nrLSU, and RPB2), rooted with Nothocybe distincta and Mallocybe terrigena. BI-PP ≥ 0.95 and ML-BP ≥ 70 are represented as BI-PP/ML-BP. P. fulvidiscum and P. singulare are two newly described taxa Province, China. Comparing to P. fulvidiscum, its pileus is uniformly brown without discoloring toward the margin, the squamules toward the pileus center are smaller and irregularly erected, the Q value of basidiospores is smaller, and the pileipellis is composed of smooth and encrusted hyphae. Phylogenetically, P. araneosum, originally described from Queensland, is a sister to the new species; however, it has medium-sized basidiomata, non-squamulose and yellowish tinged pileus, longer basidiospores measured (8.0-) 8.5-11.5 × 5.0-7.0 μm (Q m = 1.68), slender cheilocystidia measured 27-53 × 10-17 μm, and is associated with different plants ( M a t h e n y a n d B o u g h e r 2 0 1 7 ) . P s e u d o s p e r m a

Toxin detection
The weights of the tested samples for the two new species were 0.0115 g for FYG6311 and 0.0020 g for FYG6363, respectively. The representative chromatograms of muscarine are shown in Fig. 6. After comparing the retention time (0.95 min) and relative deviation (0.32%) with standard muscarine in the allowance of ±25% relative range, the toxin muscarine was identified. The calibration curve for muscarine generated during the validation was y = 20223.15025x + 18054.61816 (r = 0.99837) for muscarine concentration in the range of 2-100 ng/mL (y means the peak area, and x is the muscarine concentration, r represents correlation coefficient). The muscarine contents are 3.1340 ± 0.4078 g/kg and 0.5505 ± 0.0026 g/kg in P. fulvidiscum and P. singulare, respectively. Precision was performed, injecting six times the standard mixture, and the relative standard deviation (RSD) were 13.01% and 0.48%, respectively. Percentages of recovery was 98.47-100.18%, and the average recovery was 98.78%.

Discussion
In recent years, an increasing number of Pseudosperma species have been discovered, especially after Inocybaceae was re-divided into seven genera by Matheny et al. (2020) and the establishment of the genus. These include P. albobrunneum, (c) Representative chromatograms of muscarine from P. singulare P. brunneoumbonatum, P. flavorimosum, P. pinophilum, and P. triacicularis from Pakistan (Saba et al. 2020;Jabeen and Khalid 2020;Jabeen et al. 2021), P. amabile, P. amoris, P. napaeanum, P. emberizanum, P. huginii, and P. solare from Germany or Austria (Bandini and Oertel 2020;Bandini et al. 2021), P. conviviale, P. melleum, and P. ponderosum from Italy (Cervini et al. 2020), and P. citrinostipes from southwestern China (Yu et al. 2020). These works have greatly facilitated the species diversity of the genus. In the present study, P. fulvidiscum and P. singulare were identified as new species through morphological studies and phylogenetic analyses. They share high similarities in appearance. It would be very difficult to distinguish them by maromorphology or even by most of the microfeatures. However, the appressed scaly pileus and brownish tiers of fibrils in the lower part of the stipe in P. fulvidiscum and the presence of non-encrusted pileipellis hyphae in P. singulare could help discriminate them. Detailed comparisons between the two new species were listed in Table 2.
Tropical Asian elements of Pseudosperma or Inocybaceae spp. are usually encountered in Fagaceae or Dipterocarpaceae forests (Horak 1980;Fan and Bau 2014;Latha and Manimohan 2017). However, the present two new species are both collected under the Carpinus forests. Carpinus is a genus of Betulaceae distributed mainly in temperate and extending to subtropical to northern tropics ). Based on our field observations, the only putative host of the two new species, Carpinus londoniana var. lanceolata, was described from Wuzhishan of Hainan, where there is a tropical mountain climate. One specimen (GN0942) of P. fulvidiscum was collected from Fujian province, where there is a subtropical monsoon climate, but this collection lacks detailed host information.
In our multi-gene phylogeny, the two new species grouped into a full support subclade that is sister to all the remainders of the genus. The subclade unifying P. brunneosquamulosum and P. rubrobrunneum from tropical India, P. arraneosum and P. aff. araneosum (PL58410) from tropical Australia, and Pseudosperma sp. (MCA562) from subtropical Japan. Interestingly, the two Australian taxa, P. aff. araneosum (PL58410) and P. araneosum, are sisters to P. fulvidiscum a n d P . s i n g u l a r e , r e s p e c t i v e l y . P s e u d o s p e r m a brunneosquamulosum and P. sp. (MCA562) clustered in a full support lineage that is sister to the lineage unifying P. fulvidiscum and P. aff. araneosum (PL58410); P. rubrobruneum is sister to the lineage unifying P. singulare and P. araneosum. Matheny et al. (2009) suggested that northern and southern South America, Australia, and New Zealand are primarily the recipients of immigrant taxa during the Palaeogene or later. The present multi-gene phylogeny confirms close relationships between these Pseudosperma taxa from tropical Asia and tropical Australia. Similarly, Inocybe hainanensis T. Bau and Y.G. Fan, a formerly described species from tropical China, has t h e c l o s e s t a f f i n i t i e s t o a n A u s t r a l i a n s p e c i e s I. violaceucaulis Matheny and Bougher based on nrLSU phylogeny (Fan and Bau 2014). With more tropical taxa being discovered and described in the genus, we will reach a better understanding of their distribution pattern and evolutionary history.
The alkaloid muscarine, an ammonium quaternary compound that stimulates the parasympathetic nervous system of animals, is found in Inocybaceae and several mushroom genera (Lurie et al. 2009;White et al. 2019). Among the seven major clades of Inocybaceae, the presence of muscarine was suggested to be a derived trait for an inclusive clade containing Nothocybe, Pseudosperma, and Inocybe s. str. Pseudosperma is supposed to contain many muscarinepositive species, but only five species have been assayed in this genus (Kosentka et al. 2013). Of these, P. rimosum, P. niveivelatum, P. sororium, and P. spurium contain muscarine, and only P. perlatum was reported to lack muscarine (Kosentka et al. 2013). There are still numerous taxa in Pseudosperma waiting for toxin detection. In recent years, cases of poisoning caused by Pseudosperma mushrooms were  (Li et al. , 2022Xu et al. 2020). The present two new species are both toxic mushrooms due to the fact that they contain high concentrations of muscarine. Even though these two new species are just "little brown mushrooms" and may be ignored in the field, the absence of aposematic coloration makes them more easily collected or consumed by mushroom hunters. Accordingly, publicity of the two new toxic mushrooms is undoubtedly needed, especially for the more frequently encountered species P. fulviduscum. Our present work provides scientific details for the identification and publicity of these two toxic species.