Research Article
Research Article
Two new toxic yellow Inocybe species from China: morphological characteristics, phylogenetic analyses and toxin detection
expand article infoSai Nan Li, Fei Xu§, Ming Jiang, Feng Liu§, Fang Wu|, Ping Zhang, Yu Guang Fan, Zuo Hong Chen
‡ Hunan Normal University, Changsha, China
§ Ningxia Center for Disease Control and Prevention, Yinchuan, China
| College of Life Science, Hunan Normal University, Changsha, China
¶ Hainan Medical University, Haikou, China
Open Access


Some species of Inocybe s. str. caused neurotoxic poisoning after consumption around the world. However, there are a large number of species in this genus that have not been studied for their toxicity or toxin content. In this study, we report two new toxic yellow Inocybe s. str. species from China based on morphological characteristics, phylogenetic analyses and toxin detection. Among the two species, Inocybe squarrosolutea is reported as a newly recorded species of China. We also describe a new species, I. squarrosofulva, which is morphologically similar to I. squarrosolutea. The new species is characterized by its ochraceous squarrose pileus, distinctly annulate cortina on the stipe, nodulose basidiospores and thick-walled pleurocystidia. Muscarine in the fruitbodies was detected by UPLC–MS/MS, the content in I. squarrosolutea and I. squarrosofulva were 136.4 ± 25.4 to 1683.0 ± 313 mg/kg dry weight and 31.2 ± 5.8 to 101.8 ± 18.9 mg/kg dry weight, respectively.


Inocybaceae, muscarine, taxonomy


The genus Inocybe (Fr.) Fr. was established as a “tribe” of Agaricus (Fries 1821) and treated as a genus in 1863 (Fries 1863). Recent studies elevated it to the family rank, known as the Inocybaceae (Matheny 2005, 2009; Matheny et al. 2020). The present Inocybaceae (Inocybe sensu lato) is composed of seven genera, namely Auritella, Inosperma, Mallocybe, Nothocybe, Pseudosperma, Tubariomyces, and Inocybe sensu stricto. Inocybe s. str. with about 850 species, turns out to be the largest genus (Matheny et al. 2020), and novel species have continued to be discovered in recent years (Bandini et al. 2020a, 2020b; Fan and Bau 2020; Caiafa 2021; Mešić et al. 2021). Studies on Inocybaceae in China started in the 20th century. From Deng (1963) first reported 15 species of Inocybe s. l. Until 2020 about 140 species of Inocybaceae had been reported, of which about 120 belong to Inocybe s. str. (Fan and Bau 2010, 2013, 2014a, 2014b, 2017, 2018, 2020; Fan et al. 2013, 2018). Wang (1979) described a new species, I. flavobrunnea, which was the first new species of Inocybe s. str. in China. After that, Fan et al. have described seven new species of Inocybe s. str. in China from 2013 to 2020 (Fan and Bau 2013, 2014a, 2014b, 2020; Fan et al. 2018).

Autonomic toxicity disorder, caused by the ingestion of Inocybe s. l. spp., is an important type of neurotoxic mushroom poisoning. Muscarine is the principal toxin in Inocybe s. l. (Chen et al. 2016; White et al. 2018). Based on a review of the literature and their own work on toxin detection, Kosentka et al. (2013) reported whether or not muscarine is present in 98 species of Inocybaceae from 1960 to 2013, including 73 species of Inocybe s. str. Of these 73 taxa, 57 have been reported to contain muscarine. In China, about 21 species of Inocybe s. str. are considered poisonous (Mao 2006; Bau et al. 2014; Xu et al. 2020). However, only three species (I. asterospora, I. aff. ericetorum, I. serotina) of Inocybe s. str., causing typically muscarinic poisoning incidents, could be identified as containing muscarine (Chen et al. 1987; Xu et al. 2020; Li et al 2021). Among them, I. asterospora and I. aff. ericetorum are new toxic Inocybe species reported in China. In summary, toxins have only been reported for 75 species of Inocybe s. str., and ca. 7% (59 of 850) have been identified as muscarine-containing poisonous mushrooms. Hence, the toxicity of a large number of Inocybe s. str. species is still unknown.

In this study, we 1) report I. squarrosolutea as a newly recorded species of China, and redescribed this species based on Chinese materials; 2) describe a new species of Inocybe s. str., based on morphological and phylogenetic evidence; and 3) characterize the muscarine content of these two species by UPLC–MS/MS.

Materials and methods

Specimen collection and drying treatment

Most other specimens were collected from Hunan Province; only one specimen was collected from Huang Mountain, Anhui Province. The fresh basidiomata were dried using an electric dryer EVERMAT operated at 45 °C for 10 h. The dried specimens, along with the holotype of the newly described species, were deposited in the Mycological Herbarium of Hunan Normal University (MHHNU), Changsha, China. A small piece of fresh basidioma was also dried with silica gel for molecular analysis.

Morphological studies

Specimens were photographed in situ using a Sony digital camera (LICE-7, Sony, Tokyo, Japan). The macromorphological characters of fresh mushrooms were recorded as soon as possible after collection. Color codes were described following Kornerup and Wanscher (1978). Microscopic structures were studied from dried materials mounted in 5% aqueous KOH, and Congo red was used as a stain when necessary. All the measurements were performed at 1000× magnification, and a minimum of 20–30 basidiospores from each basidioma were measured in side view. Micromorphological investigations were performed by means of a Nikon Eclipse 50i microscope (Nikon, Tokyo, Japan). The measurement methods followed those of Fan et al. (2013). Dimensions of basidiospores and Q values were given as (a) b–c (d), where “b–c” cover a minimum of 90% of the measured values, and “a” and “d” represent extreme values; Q is the ratio of length to width in an individual basidiospore. Qm is the average Q of all basidiospores ± sample standard deviation. The descriptive terms are in accordance with Fan and Bau (2020), Horak et al. (2015), and Matheny et al. (2012). SEM images of basidiospores were obtained using a scanning electron microscope JSM-6380LV (JEOL Ltd., Tokyo, Japan).

DNA extraction, amplification, and sequencing

DNA was extracted from dried basidiomata using a fungal DNA extraction kit manufactured by Omega Bio-Tek (Norcross, GA, USA). The following primer pairs were used for PCR amplification and sequencing: ITS5 and ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990); LR0R and LR5 for the nuclear ribosomal large subunit (nrLSU) region (Vilgalys and Hester 1990); and bRPB2-6F and bRPB2-7.1R for RNA polymerase II second largest subunit (rpb2) region (Matheny 2005). PCR protocols for ITS and nrLSU were as described in White et al. (1990), and for rpb2, as described in Matheny (2005). PCR products were purified and sequenced by TsingKe Biological Technology Co., Ltd. (Beijing, China).

Sequence alignment and phylogenetic analyses

Thirty-six sequences (12 for ITS, 12 for nrLSU and 12 for rpb2) were newly generated for this study and deposited in GenBank (Table 1). The new sequences were subjected to a BLAST search and relevant related sequences retrieved from GenBank (Table 1).

Table 1.

DNA sequences used in this study and their voucher specimen number, geographic origin, toxin status, and GenBank accession numbers.

Species Voucher Locality Muscarine ITS nrLSU rpb2 References
Inocybe acriolens AU10493 Canada ? NR_153186 NG_057291 N/A Type material
JCS071005D USA ? N/A JN974981 MH577492 Unpublished
I. albodisca PBM1390 USA N/A EU307819 EU307821 Kropp and Albee-Scott (2010)
I. alienospora PBM3743 Australia ? KP171104 KM197209 KM245970 Latha and Manimohan (2017)
REH9667 Australia ? KP171105 KM197210 KM245971 Unpublished
I. chelanensis PBM491 USA ? N/A AY239020 AY337368 Matheny (2005)
PBM2314 USA ? N/A AY239021 AY337369 Matheny (2005)
I. giacomi CLC1321 USA ? N/A MK153655 N/A Unpublished
JV21543 Sweden ? N/A MK153656 N/A Unpublished
EL80-12 Sweden ? N/A MK153657 N/A Unpublished
I. grammata PBM2602 USA N/A JN974977 N/A Unpublished
PBM2558 USA N/A JQ313562 N/A Unpublished
2012038 China N/A KU764690 N/A Fan and Bau (2017)
I. hydrocybiformis TBGT:12318 India ? KP171130 KP170911 KM245987 Latha and Manimohan (2017)
ZT10077 Thailand ? GQ893016 GQ892971 N/A Unpublished
I. lasseroides PBM3749 Australia ? KP171145 KP170924 KM245993 Latha and Manimohan (2017)
PBM3750 Australia ? KP171146 KP170925 N/A Unpublished
I. papilliformis TBGT:10480 India ? KP171131 KP170912 KM245988 Latha and Manimohan (2017)
CAL1372 India ? KY440096 KY549126 N/A Latha and Manimohan 2017
I. relicina JV10258 Finland ? N/A AY038324 AY333778 Matheny (2005)
EL2-05 Sweden ? N/A MN296111 N/A Unpublished
I. sierraensis DED6101 USA ? N/A AY239025 MH249810 Kropp and Matheny (2004)
DED6477 USA ? N/A AY239026 N/A Kropp and Matheny (2004)
I. soluta EL10706 Sweden + N/A FN550878 N/A Unpublished
JV7811F Finland + N/A JN974987 N/A Ryberg and Matheny (2012)
I. sphaerospora 60-774 Japan ? AB509953 N/A N/A Unpublished
ZRL20151281 China ? LT716044 KY418860 KY419006 Unpublished
I. sphaerospora DED8059 Thailand ? GQ892993 GQ892948 MH577472 Horak et al. (2015)
I. aff. sphaerospora DED8153 Thailand ? GQ892994 GQ892949 MH577471 Horak et al. (2015)
PKSR10 India ? KJ411954 N/A KJ411970 Unpublished
I. squarrosofulva MHHNU31548 (holotype) China + MZ050799 MW715814 MW574997 This study
MHHNU31927 China + MZ050802 MW715815 MW729766 This study
I. squarrosolutea MHHNU8536 China + MK250946 MW709445 MW715635 This study
MHHNU8984 China + MK388162 MW709446 MW715636 This study
MHHNU31006 China + MZ050796 MW709457 MW715637 This study
MHHNU31042 China + MZ050800 MW709486 MW715638 This study
MHHNU31173 China + MZ050797 MW715813 MW729760 This study
MHHNU31427 China + MZ050794 MW715804 MW729761 This study
MHHNU 31434 China + MZ050798 MW709488 MW729762 This study
MHHNU31445 China + MZ050801 MW709528 MW729763 This study
MHHNU31875 China + MZ050795 MW709531 MW729764 This study
MHHNU32151 China + MZ050793 MW709532 MW729765 This study
I. stellatospora PRL2716 USA ? N/A EU307840 N/A Kropp and Albee-Scott (2010)
EL3004 Sweden ? AM882747 AM882747 N/A Unpublished
PBM963 USA ? N/A AY038328 AY337403 Matheny (2005)
Auritella dolichocystis Trappe24844 Australia ? N/A AY380371 AY337371 Matheny (2005)
Trappe24843 Australia ? N/A AY635764 AY635780 Unpublished
Inosperma calamistratum PBM2351 USA N/A AY380368 KM245971 Matheny (2005)
JV11950 USA N/A EU555452 KM245971 Unpublished
PBM1105 USA JQ801386 JQ815409 JQ846466 Matheny et al. (2020)
Mallocybe terrigena JV16431 Finland N/A AY380401 AY333309 Matheny (2005)
PBM1563 USA N/A MN178550 N/A Unpublished
Nothocybe distincta ZT9250 India ? N/A EU604546 N/A Matheny et al. (2020)
Pseudosperma sororium ADW0063 USA + JQ408779 JQ319703 JQ421073 Latha and Manimohan (2017)
PBM3901 USA + N/A MH220278 MH249810 Matheny et al. (2020)
Tubariomyces inexpectatus AH20390 Spain N/A EU569855 GU907088 Matheny et al. (2020)
Crepidotus applanatus 420526MF0534 USA N/A AF205694 N/A Kosentka et al. (2013)
420526MF0689 USA N/A AY380406 N/A Matheny et al. (2020)

The sequences were aligned using MAFFT v7.310 (Katoh and Standley 2013) and manually edited using BioEdit v7.0.5 (Hall 1999). Maximum likelihood (ML) analysis was performed using RAxML v7.9.1 (Stamatakis 2006) under the GTR + GAMMA + I nucleotide substitution model and performing nonparametric bootstrapping with 1000 replicates. Bayesian inference (BI) was performed in MrBayes v3.2 (Ronquist et al. 2012). The optimal substitution model was determined using the Akaike information criterion (AIC) as implemented in MrModeltest v2.3 (Nylander 2004). The selected substitution model for the three partitions was as follows: General Time Reversible + Gamma (GTR + G) for ITS, and General Time Reversible + Proportion-Invariant + Gamma (GTR + I + G) for nrLSU and rpb2. The BI analysis was conducted with the following parameters: four simultaneous Markov chains (MCMC), each with two independent runs and trees summarized every 1000 generations. The analyses were completed after 1,000,000 generations when the average standard deviation of split frequencies was 0.009808 for the analysis, and the first 25% generations were discarded as burn-in. The phylograms from ML and BI analyses were visualized with FigTree v1.4.3 (Rambaut 2009).

Analysis of toxins by ultrahigh-performance liquid chromatography tandem mass spectrometry

The procedure of toxin extraction and detection followed Xu et al. (2020) with slight modifications. A 0.05 g powdery sample of dried mushroom pileus was mixed with 2 mL of a methanol-water solution (7:3 v/v) and vortexed for 30 min at room temperature. The mixture was treated in an ultrasonic bath for 30 min. After centrifugation at 10,000 rpm for 5 min, the supernatant was purified using a QuCHERS–PP column. Subsequently, the extract was mixed with acetonitrile to a final volume of 1.0 mL. The obtained sample solution was centrifuged at 21,000 rpm for 2 min before UPLC–MS/MS analysis. Lentinula edodes was used as a blank sample.

UPLC–MS/MS analysis was carried out with a Waters ACQUITY I-Class UPLC system coupled with a Waters Xevo TQ-S MS/MS system (Waters, Milford, MA, USA). The chromatographic separation was conducted using an ACQUITY UPLC Amide column (2.1 × 100 mm, 1.7 μm; Waters). A gradient elution system used the mobile phase A (acetonitrile) and the mobile phase B (0.05% formic acid aqueous solution) at a flow rate of 0.6 mL/min. The gradient program was as follows: (1) 70–10% A for 1 min, (2) 10% A for 0.5 min, (3) 10–70% A for 0.5 min, and (4) 70% A for 3 min. The analytical column was set to 40 °C, and the injection volume was 2.0 μL. The muscarine content was estimated in the mushroom extract by using standard muscarine (Sigma-Aldrich, St. Louis, MO, USA, Chemical purity ≥ 98%).

A protonated molecular ion ([M + H]+ = 174.2) was chosen as the parent ion as well as two daughter ions of 57.0 and 97.0, which were used for qualitative and quantitative detection, respectively. The MS/MS conditions were as follows: ESI+ mode; cone, 18 V; collision, 16 V; capillary, 3.0 kV; desolvation temperature, 500 °C; source temperature, 150 °C; desolvation gas flow, 1000 L/Hr; cone gas flow, 150 L/Hr; and collision gas flow, 0.19 mL/min. All the gases were 99.999% pure. Other parameters were used with default values. The product ion confirmation (PIC) was set as follows: scan function; daughter scan; activation threshold level, 500× background noise; minimum activation threshold, 5000 counts; reset threshold level, 50% of act threshold; mass above parent, 100 Da; minimum mass, 50 Da; centroid; scan speed at 5000 amu/s; PIC duration, 0.5 s; and collision energy, 20 V. The analytical results were reported as X ± U (k = 2, p = 95%), where X is the analytical content and U is the expanded measurement uncertainty (Eurachem 2012).


Phylogenetic data

The combined dataset (ITS, nrLSU, and rpb2) contained 1987 total characters and included 58 sequences. The topologies of ML and BI phylogenetic trees obtained in this study were practically the same and the only ML tree with branch lengths and support values is shown in Figure 1. All members of Inocybe s. str. in the dataset formed a monophyletic lineage with strong support (MLB = 85%, BPP = 1). Ten specimens of I. squarrosolutea from China (MHHNU8536, MHHNU8984, MHHNU31006, MHHNU31042, MHHNU31173, MHHNU31427, MHHNU31434, MHHNU31445, MHHNU31875, MHHNU32151) and two samples labeled as “I. sphaerospora” from China (ZRL20151281) and Japan (60-774) grouped together in a well-supported lineage (MLB = 100%, BPP = 1.0). The new species, I. squarrosofulva, formed a well-supported distinct lineage from I. squarrosolutea (MLB = 100%, BPP = 1.0) and is sister to the lineage of I. squarrosoluta with significant support (MLB = 100%, BPP = 1.0).

Figure 1. 

Phylogenetic relationship and placement of Inocybe squarrosofulva and I. squarrosolutea inferred from the combined dataset (ITS, nrLSU, and rpb2) using ML. Bootstrap values ≥80% and Bayesian posterior probabilities ≥0.95 are reported on the branches. Sequences generated in this study are shown in bold. The new species is indicated in red. The red branch indicates the confirmed presence of muscarine, the gray branch indicates ambiguous for muscarine, and the black branch indicates a lack of muscarine.


Inocybe squarrosolutea (Corner & E. Horak) Garrido, Bibliotheca Mycologica 120: 177, 1988.

Figures 2, 3, 6a

Astrosporina squarrosolutea Corner & E. Horak, Persoonia 10(2): 175, 1979.


Small to medium-sized. Pileus: 30–60 mm in diameter, bell-shaped to convex when young, and then planar with umbonate center; margin strongly in-rolled or deflexed when young, and then gradually straight when mature; center covered with stout, erect, conic squamules (up to 2 mm high, 1–1.5 mm wide), coarsely fibrillose towards the margin; surface dry, primrose yellow (1A6) to bright yellow (2A5), becoming pale brown (3B6) over the disc. Lamellae crowded (ca. 50–70), 3–5 mm wide, adnexed to adnato-decurrent, often subsinuate; light yellow (1A5) turning to pale yellow-fuscous (2B5), edge concolorous, even. Stipe 35–75 × 4–8 mm, cylindrical or attenuated towards apex, stout, base subbulbous to bulbous, up to 16 mm wide; bright yellow (2A5); apex pruinose, covered with bright yellow(2A5) to orange (2A6), longitudinal, floccose-fibrillose fibrils towards base; dry, solid. Cortina conspicuous present in young specimens. Context pale yellow (1A4) in stipe and cuticle.


(5.0) 5.5–9.0 (10.0) µm (av. 7.1 μm, SD 1.1 μm) × (4.0) 4.5–6.0 (6.5) µm (av. 5.3 μm, SD 0.6 μm), Q = (1.00) 1.11–1.67 (1.80), Qm = 1.33 ± 0.19 (n = 200 of 10 coll.), nodulose, 6–8 hemispheric knobs, yellow-brown with 5% KOH. Basidia: 17–26 × 7–9 µm, 4-spored, clavate to broadly clavate. Pleurocystidia: 37–67 µm (av. 46.1 μm, SD 3.0 μm) × 10–18 µm (av. 13.4 μm, SD 1.2 μm), Q = 2.80–4.0 (n = 100 of 10 coll.), abundant, broadly fusoid to lageniform; crystalliferous at apex, base usually truncate to obtuse, occasionally tapered into pedicel; metuloid, hyaline, sometimes contain a few small crystals or resinous inclusions, thick-walled, walls up to 1.5 µm thick, bright yellow with 5% KOH. Cheilocystidia similar to pleurocystidia, 35–62 × 9–17 µm; paracystidia: 12–25 × 5–11 µm, abundant, thin-walled, translucent inside, clavate to broadly clavate. Hymenophoral trama: sub-regularly arranged, yellowish with 5% KOH, composed of thin-walled, cylindrical to inflated hyphae 4–23 µm wide. Caulocystidia: 48–98 × 17–22 µm, present at stipe apex, in clusters, similar to those of hymenial cystidia; cauloparacystidia: 20–35 × 10–13 µm, clavate to broadly clavate, thin-walled, nearly hyaline inside, abundant. Pileipellis a trichoderm, regular to subregular, pale brown with 5% KOH, composed of smooth, thin-walled, cylindrical hyphae, 4–8 µm in diameter. Oleiferous hyphae present in pileus and stipe trama, 3–10 µm in diameter, branched. Clamp connections present and common in all tissues.

Figure 2. 

Basidiomata of Inocybe squarrosolutea a, b MHHNU8536 c MHHNU31006, and d MHHNU31427. Scale bars: 10 mm.


Single to scattered in mixed forest dominated by Pinus and Quercus.

Known distribution

Malaysia (type location) (Horak 1979), China (Hunan Province, Anhui Province).

Specimens examined

China, Hunan Province: Yongshun County, 29 July 2015, MHHNU8536; Yizhang County, 16 September 2016, MHHNU8984; Ningyuan County, 28 May 2017, MHHNU31006; Youxian County, 9 June 2017, MHHNU31042; 18 June 2019, MHHNU31445; Guidong County, 6 July 2018, MHHNU31173; Yongzhou City, 22 May 2019, MHHNU31427; 11 June 2020, MHHNU31875; Qidong County, 2 June 2019, MHHNU31434; Anhui Province, Huangshan City, 11 Aug. 2020, MHHNU32151.

Figure 3. 

Microscopic features of Inocybe squarrosolutea (MHHNU31427) a basidiospores b basidia with probasidium c gill edge d cheilocystidia and paracystidia e pleurocystidia f caulocystidia and cauloparacystidia g, h pileipellis i oleiferous hyphae, and j hymenial hyphae. Scale bars: 10 µm.

Inocybe squarrosofulva S.N. Li, Y.G. Fan & Z.H. Chen, sp. nov.

MycoBank No: 839726
Figures 4, 5, 6b


Squarrosus (Latin), squamous; fulvus (Latin), brown-orange, referring to its pileus.


China. Hunan Province: Zhangjiajie, Badagongshan National Nature Reserve, 29°67.57'N, 109°74.45'E, alt. 1600 m, on ground in subtropical montane forest, 29 July 2019, Z.H. Chen and S.N. Li, MHHNU31548 (GenBank accession no. ITS: MZ050799; nrLSU: MW715814; rpb2: MW574997).


Small to medium-sized basidiomata. Orange-brown to dark brown pileus with squarrose scales. Yellowish brown to brownish , adnexed lamellae. Stipe equal, stout, with distinctly filamentous annulate cortina, pruinose at apex. Odor like raw potatoes. Nodulose basidiospores with six nodules. Hymenial cystidia are broadly fusoid to lageniform, thick-walled. Differs from Inocybe squarrosolutea in its orange-brown to dark brown pileus, distinctly filamentous annulus, and less nodulose basidiospores.

Figure 4. 

Basidiomata of Inocybe squarrosofulva a, b MHHNU31548 c, d MHHNU31927. Scale bars: 10 mm.


Small to medium-sized. Pileus 25–55 mm in diameter, spherical to bell-shaped when young, and gradually flattened to hemispheric or convex; margin strongly in-rolled when young then decurved or slightly uplifted; yellowish (2A5), center covered with yellow ochre (5C7) to brownish yellow (5C8) erect conical fibrillose scales (up to 1.5 mm high, 1–1.5 mm wide), coarsely fibrillose-rimose towards the margin; pileus with crenellated, nonpersisting fibrillose veil remnants at margin. Lamellae adnexed, crowded (ca. 55–70), up to 4 mm wide; yellowish brown (4C7), becoming brownish (5E4) with age, edge concolorous. Stipe 40–80 × 5–8 mm, cylindrical, equal or slightly enlarged at the base, solid; light yellow (2A3) to yellow ochre (5C7); pruinose with few yellowish-brown (4C7) furfuraceous scales at apex; towards the base covered with numerous, yellow-ochre (5C7), woolly-fibrillose, incomplete zones; dry. Cortina conspicuous, annulate, composed of yellow ochre (5C7) fibrils, and remains at the upper part of the stipe. Context: pale yellow (2A5) in pileus and stipe. Odor like raw potatoes.

Figure 5. 

Microscopic features of Inocybe squarrosofulva (MHHNU31548, holotype) a, b basidiospores, c basidia with probasidium d gill edge e pleurocystidia f cheilocystidia and paracystidia g caulocystidia and cauloparacystidia h oleiferous hyphae i hymenial hyphae, and j, k pileipellis. Scale bars: 10 µm.


(4.5) 5.0–7.0 µm (av. 6.6 μm, SD 1.0 μm) × 4.0–6.0 (7.0) (av. 5.3 μm, SD 0.8 μm) µm, Q = (1.00) 1.10–1.67 (1.75), Qm = 1.26 ± 0.16 (n = 80 of 4 coll.), nodulose with six hemispheric knobs, yellowish-brown with 5% KOH, containing a bright yellow oil droplet of uniform size inside. Basidia: 18–24 × 8–10 µm, 4-spored, clavate to broadly clavate. Pleurocystidia: 36–49 µm (av. 43.8 μm, SD 3.9 μm) × 13–18 µm (av. 15.5 μm, SD 2.6 μm), Q = 2.12–3.46 (n = 30 of 2 coll.), mostly hyaline, few with bright yellow oily inclusions, fusiform to broadly fusiform, with crystalliferous apices, obtuse or truncated at base; thick-walled, walls bright yellow with 5% KOH, up to 2 µm thick towards apex. Cheilocystidia: 30–48 × 9–19 µm, similar to pleurocystidia, hyaline. Cheiloparacystidia: 10–23 × 6–12 µm, abundant among cheilocystidia, obovate, elliptic to clavate, thin-walled, hyaline. Hymenophoral trama: regular to subregular, composed of inflated hyphae, up to 18 μm wide, hyaline to lightly yellow with 5% KOH, thin-walled. Pileipellis: a trichoderm, subregular, consisting of cylindrical hyphae 5–13 µm in diameter, walls pale yellow brown with 5% KOH, smooth, thin-walled. Caulocystidia: present at stipe apex, 23–49 × 9–21 μm, in clusters, thick-walled, walls thinner than pleurocystidia, hyaline or with pale yellow intracellular contents. Cauloparacystidia: 8–19 × 3–10 μm, clavate or broadly clavate, hyaline, thin-walled. Oleiferous hyphae present in pileus and stipe trama, 4–11 μm in diameter, branched. Clamp connections seen on all hyphae.

Figure 6. 

SEM images showing basidiospores of a Inocybe squarrosolutea b Inocybe squarrosofulva. Scale bars: 5 µm.


On soil in subtropical montane forest dominated by Fagus lucida.

Known distribution

Known from the type locality.

Other examined specimens

27 July 2020, Z.H. Chen and S.N. Li, MHHNU31927.

Toxin detection

Through UPLC–MS/MS detection, we found that both I. squarrosolutea and I. squarrosofulva contained muscarine (Figs 7, 8). In the qualitative analysis, muscarine was identified by comparing the retention time (0.91 min) and relative deviation (0.6%) within the allowable relative range of 25%. The calibration curve in the matrix blank extract given by Y = 69369X + 6849.33, R2 = 0.9990 (X is injection volume, Y is peak area, and R2 is correlation coefficient) for muscarine concentration was in the range of 0.5–20 ng/mL. The contents of in I. squarrosolutea and I. squarrosofulva were 136.4 ± 25.4–1683.0 ± 313 mg/kg dry weight and 31.2 ± 5.8–101.8 ± 18.9 mg/kg dry weight, respectively (Fig. 9). Recovery of muscarine ranged from 72.2 to 93.6%; the average recovery was 83.0%.

Figure 7. 

Total ion current (TIC) chromatogram of muscarine in Inocybe squarrosolutea (MHHNU31427).

Figure 8. 

Total ion current (TIC) chromatogram of muscarine in Inocybe squarrosofulva (MHHNU31548).

Figure 9. 

Relative muscarine concentrations measured by UPLC–MS/MS.


Species delimitation

Based on the morphological characteristics, the mushroom was identified as I. squarrosolutea, which was first described from Cameron Highlands of Malaysia (Horak 1979). According to the original description, this species is characterized by a large-sized basidiomata, a bright yellow coloration and a scaly pileus and orange fibrillose veil remnants on the stipe. Our Chinese materials fit well with the original description in basidiomata size, outwards appearances, and the shape and size of micro-features. Meanwhile, there are some tiny difference between them. The holotype of I. squarrosolutea has longer scales (up to 4 mm) in pileus, smaller basidiospores (4–8 × 5–6 μm), finer basidia (18–26 × 5–7 μm), thicker hymenial cystidia (30–60 × 14–25 μm) (Horak 1979). This species is a close relative of I. lutea which, by contrast, has a smaller fruiting body, a smooth pileus, and distinctly smaller basidiospores (Kobayasi 1952; Horak 1979). It is easily for people to confuse I. squarrosolutea and I. sphaerospora because of their similar appearance. In fact, they can be easily distinguished by their basidiospores. The basidiospores of I. squarrosolutea are nodulose, while those of I. sphaerospora are globose (Kobayasi 1952; Horak et al. 2015). In phylogenetic analysis (Fig. 1) the specimens of I. sphaerospora identified by Horak et al. (2015) formed a monophyletic lineage with strong support (MLB = 100%, BPP = 1), and was distinct from I. squarrosolutea. However, the two materials labeled as I. sphaerospora from China (ZRL20151281) and Japan (60-774), cluster together with I. squarrosolutea in the phylogenetic tree, indicating an inaccurate identification of these two materials.

Inocybe squarrosofulva is characterized by its orange brown to dark brown pileus with squarrose scales, distinctly filamentous annulate cortina in stipe, stipe pruinose only near the apex, nodulose basidiospores with six hemispheric knobs, and its odor like raw potatoes. Phylogenetic analyses revealed that I. squarrosofulva is an independent lineage in Inocybe s. str. and is sister to I. squarrosolutea. However, I. squarrosolutea differs in having primrose yellow to bright yellow pileus with less squarrose scales, no distinctly filamentous annulus cortina in the stipe, a subbulbous to bulbous stipe base, a less nodulose basidiospores, and smaller hymenial cystidia. Microscopically, I. lutea is similar to new species in shape and size of pleurocystidia and basidiospores, but the pileus of I. lutea covered with radially fibrils and pruinate all over the stipe (Kobayasi 1952; Horak 1979). Lastly, a Papua New Guinea material described as Inocybe luteifolia (E. Horak) Garrido 1988 (non Inocybe luteifolia A.H. Sm. 1941), which is an illegitimate species name, resembles the new species in macromorphology, but it has smaller basidiomata, larger cheilocystidia and pleurocystidia (55–85 × 10–20 μm), no caulocystidia on the stipe, and a fish-like odor (Horak 1979).

Kuyper (1986) recognized two groups on the (informal) level of “supersection”, viz. Cortinatae and Marginatae, according to the different development mode and, hence, absence or presence of a cortina, and the nature of stipe covering. Due to their presence of a cortina and pruinose at the apex of the stipe, both I. squarrosolutea and I. squarrosofulva might be classified in supersection Cortinatae. The morphological characteristics corresponding to the phylogenetic branches are not yet clear (Matheny 2005; Matheny et al. 2020), so the infrageneric framework of Inocybe s. str. is still unknown and its characterization requires more research.

Toxicity in Inocybe

According to the literature, muscarine was first isolated and identified from Amanita muscaria, but the actual muscarine content of A. muscaria is very low (usually around 0.0003% of the fresh weight) (Waser 1961). Conversely, muscarine concentrations are much higher in Inocybe s. l. spp. (Malone et al. 1962). Brown et al. (1962) detected the muscarine contents of 34 species of Inocybe s. l. by paper chromatographic method, ranging from 0.01 to 0.80% in approximately 75% of them. Kosentka et al. (2013) used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine whether muscarine was present in 30 new samples of Inocybe s. l. Of the 30 species they assayed, eleven species tested positive for presence of muscarine, ranging from ca. 0.00006% to 0.5%. Xu et al. (2020) determined the muscarine content of I. serotina by UPLC-MS/MS, and its muscarine content was 324.0 ± 62.4 mg/kg. In our study, the toxin content in each sample was determined using a linear regression equation according to the peak area of the UPLC–MS/MS analysis chromatogram of the test sample (Figs 7, 8). The results showed that both species contained muscarine; the content of muscarine in I. squarrosolutea ranged from 136.4 ± 25.4 to 1683.0 ± 313 mg/kg dry weight and the content in I. squarrosofulva was generally lower, ranging from 31.2 ± 5.8 to 101.8 ± 18.9 mg/kg dry weight (Fig. 9). Calculated on a dry-weight basis, the percentage concentrations were 0.01–0.17% for I. squarrosolutea and 0.003–0.01% for I. squarrosofulva, which is in range of previous reports.

There are some differences in the muscarine content of different poisonous Inocybe spp., even within a particular species. The capacity of Inocybe species to accumulate muscarine may be influenced by certain hereditary (infraspecific races) or environmental factors (Brown et al. 1962). In this study, the differences in muscarine content among specimens of I. squarrosolutea may be related to region and climate. I. squarrosofulva MHNNU31548 and I. squarrosofulva MHNNU31927 were collected in the same place in different years. The weather was sunny at the time of the collection of I. squarrosofulva MHNNU31548, and there was heavy rain at the time of the collection of I. squarrosofulva MHNNU31927, so it is presumed that the difference in muscarine content may be related to rainwater washing.


This research was funded by the National Natural Science Foundation of China (Grant Nos. 31872616 and 31860009); the Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment, China (Grant No. 2019HJ2096001006); and the Ningxia Provincial National Natural Science Foundation (Grant No. 2020AAC03437). The authors are very grateful to Dr. Haijiao Li (National Institute of Occupational Health and Poison Control, Chinese Centre for Disease Control and Prevention, China) and Zhengmi He (Kunming Institute of Botany, Chinese Academy of Sciences, China) for critically reviewing the manuscript.


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