Research Article |
Corresponding author: Yu Guang Fan ( fan@hainmc.edu.cn ) Corresponding author: Zuo Hong Chen ( chenzuohong@263.net ) Academic editor: Marc Stadler
© 2021 Sai Nan Li, Fei Xu, Ming Jiang, Feng Liu, Fang Wu, Ping Zhang, Yu Guang Fan, Zuo Hong Chen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Li SN, Xu F, Jiang M, Liu F, Wu F, Zhang P, Fan YG, Chen ZH (2021) Two new toxic yellow Inocybe species from China: morphological characteristics, phylogenetic analyses and toxin detection. MycoKeys 81: 185-204. https://doi.org/10.3897/mycokeys.81.68485
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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 (
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. (
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.
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 (
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
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 (
Thirty-six sequences (12 for ITS, 12 for nrLSU and 12 for rpb2) were newly generated for this study and deposited in GenBank (Table
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 |
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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 |
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I. alienospora | PBM3743 | Australia | ? | KP171104 | KM197209 | KM245970 |
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REH9667 | Australia | ? | KP171105 | KM197210 | KM245971 | Unpublished | |
I. chelanensis | PBM491 | USA | ? | N/A | AY239020 | AY337368 |
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PBM2314 | USA | ? | N/A | AY239021 | AY337369 |
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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 |
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I. hydrocybiformis | TBGT:12318 | India | ? | KP171130 | KP170911 | KM245987 |
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ZT10077 | Thailand | ? | GQ893016 | GQ892971 | N/A | Unpublished | |
I. lasseroides | PBM3749 | Australia | ? | KP171145 | KP170924 | KM245993 |
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PBM3750 | Australia | ? | KP171146 | KP170925 | N/A | Unpublished | |
I. papilliformis | TBGT:10480 | India | ? | KP171131 | KP170912 | KM245988 |
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CAL1372 | India | ? | KY440096 | KY549126 | N/A |
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I. relicina | JV10258 | Finland | ? | N/A | AY038324 | AY333778 |
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EL2-05 | Sweden | ? | N/A | MN296111 | N/A | Unpublished | |
I. sierraensis | DED6101 | USA | ? | N/A | AY239025 | MH249810 |
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DED6477 | USA | ? | N/A | AY239026 | N/A |
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I. soluta | EL10706 | Sweden | + | N/A | FN550878 | N/A | Unpublished |
JV7811F | Finland | + | N/A | JN974987 | N/A |
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I. sphaerospora | 60-774 | Japan | ? | AB509953 | N/A | N/A | Unpublished |
ZRL20151281 | China | ? | LT716044 | KY418860 | KY419006 | Unpublished | |
I. sphaerospora | DED8059 | Thailand | ? | GQ892993 | GQ892948 | MH577472 |
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I. aff. sphaerospora | DED8153 | Thailand | ? | GQ892994 | GQ892949 | MH577471 |
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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 | |
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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 |
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EL3004 | Sweden | ? | AM882747 | AM882747 | N/A | Unpublished | |
PBM963 | USA | ? | N/A | AY038328 | AY337403 |
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Outgroups | |||||||
Auritella dolichocystis | Trappe24844 | Australia | ? | N/A | AY380371 | AY337371 |
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Trappe24843 | Australia | ? | N/A | AY635764 | AY635780 | Unpublished | |
Inosperma calamistratum | PBM2351 | USA | – | N/A | AY380368 | KM245971 |
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JV11950 | USA | – | N/A | EU555452 | KM245971 | Unpublished | |
PBM1105 | USA | – | JQ801386 | JQ815409 | JQ846466 |
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Mallocybe terrigena | JV16431 | Finland | – | N/A | AY380401 | AY333309 |
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PBM1563 | USA | – | N/A | MN178550 | N/A | Unpublished | |
Nothocybe distincta | ZT9250 | India | ? | N/A | EU604546 | N/A |
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Pseudosperma sororium | ADW0063 | USA | + | JQ408779 | JQ319703 | JQ421073 |
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PBM3901 | USA | + | N/A | MH220278 | MH249810 |
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Tubariomyces inexpectatus | AH20390 | Spain | – | N/A | EU569855 | GU907088 |
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Crepidotus applanatus | 420526MF0534 | USA | – | N/A | AF205694 | N/A |
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420526MF0689 | USA | – | N/A | AY380406 | N/A |
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The sequences were aligned using MAFFT v7.310 (
The procedure of toxin extraction and detection followed
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 (
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
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.
≡ 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.
Single to scattered in mixed forest dominated by Pinus and Quercus.
Malaysia (type location) (
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.
Squarrosus (Latin), squamous; fulvus (Latin), brown-orange, referring to its pileus.
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.
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.
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.
On soil in subtropical montane forest dominated by Fagus lucida.
Known from the type locality.
27 July 2020, Z.H. Chen and S.N. Li, MHHNU31927.
Through UPLC–MS/MS detection, we found that both I. squarrosolutea and I. squarrosofulva contained muscarine (Figs
Based on the morphological characteristics, the mushroom was identified as I. squarrosolutea, which was first described from Cameron Highlands of Malaysia (
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 (
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) (
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 (
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.