Multilocus phylogeny and morphology reveal two new species of Lepiota (Agaricales, Verrucosporaceae) from southwestern China

189 Multilocus phylogeny and morphology reveal two new species of Lepiota (Agaricales, Verrucosporaceae) from southwestern China Xing Li1, Bin Chen2, Yanliu Chen1, Mengya An1, Junfeng Liang1 1 Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou 510520, China 2 Henan Institute of Science and Technology, College of Life Science, Xinxiang 453003, China Corresponding author: Junfeng Liang ([email protected]) Copyright: © Xing Li et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Based on phylogenetic and morphological evidence, two new Lepiota sect. Lepiota species collected from southwestern China are described and illustrated, namely L. brunneophora and L. ochraceosquamea. Lepiota brunneophora has a pileus with yellowish-brown to brown squamules and striations, a reddish-brown to purplish-brown stipe covered with whitish, floccose squamules, and penguin-shaped basidiospores with a narrowed apex. Lepiota ochraceosquamea is characterized by dark yellowish-brown to light ochre squamules on the pileus, a whitish annulus, broadly fusiform basidiospores, and a trichodermal pileus covering composed of subcylindrical terminal elements, narrowing toward the apex and lacking basal short elements. A comprehensive phylogenetic framework within L. sect. Lepiota, including agaricoid and sequestrate species, is also provided. Key words: Lepiota, lepiotaceous fungi, phylogeny, taxonomy Introduction Lepiota (Pers.) Gray is a saprotrophic genus, mainly distributed in (sub)tropical to temperate regions (Vellinga 2004a; Sysouphanthong et al. 2011). The genus was long classified in the Agaricaceae Chevall. (Lange 1935; Pegler 1986; Singer 1986; Candusso and Lanzoni 1990; Vellinga 2001; Liang 2007; Kirk et al. 2008; Yang et al. 2019) until it was recently transferred to the family Verrucosporaceae Jülich (1982) (= Lepiotaceae Kun L. Yang, Jia Y. Lin & Zhu L. Yang, see Yang et al. 2024) (Kalichman et al. 2020; Salichanh et al. 2024; Qu et al. 2025). Some fungi of this genus are non-toxic but of low edible value, such as L. erminea (Fr.) P. Kumm. and L. magnispora Murrill (Dai et al. 2010), while others are highly poisonous, such as L. brunneoincarnata Chodat & C. Martin and L. subincarnata J.E. Lange (Sgambelluri et al. 2014; Sarawi et al. 2022; Bau et al. 2024), whose amatoxins possess significant research and application value in life sciences and medical biology (Wieland and Faulstich 1991). Furthermore, some Lepiota species play an important role in forest ecosystems, particularly in the decomposition of organic matter (Guzmán and Guzmán-Davalos 1992; Vellinga 2004b). Academic editor: Zai-Wei Ge Received: 3 July 2025 Accepted: 7 September 2025 Published: 14 October 2025 Citation: Li X, Chen B, Chen Y, An M, Liang J (2025) Multilocus phylogeny and morphology reveal two new species of Lepiota (Agaricales, Verrucosporaceae) from southwestern China. MycoKeys 123: 189–204. https://doi.org/10.3897/ mycokeys.123.163999 MycoKeys 123: 189–204 (2025) DOI: 10.3897/mycokeys.123.163999 190 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota However, the genus Lepiota is also difficult to distinguish because of its complex taxonomic characters. Vellinga (2001) carried out a comprehensive arrangement of Lepiota based mainly on the structure of the pileus covering and the shape of basidiospores. Her treatment was accepted by most taxonomists (Liang 2007; Yang et al. 2019; Bashir et al. 2020; Niazi et al. 2021; Kaygusuz 2022; Haqnawaz et al. 2022; Rehman et al. 2024), but not supported by her own molecular evidence, which indicated that Lepiota is not monophyletic (Vellinga 2003). Combining morphological and molecular phylogenetic analysis, Hou and Ge (2020) treated the species of the large-spored group within L. sect. Echinatae/Echinoderma s.l. as Echinoderma Bon (1991) s.s. and those of the small-spored group as the basal members of Lepiota, resulting in the monophyly of Lepiota. Sarawi et al. (2025) contributed to the sectional nomenclature, taxonomy, and phylogeny of Lepiota, dividing the genus into seven sections: L. sect. Stenosporae J.E. Lange ex Reschke & Sarawi (Sarawi et al. 2025), L. sect. Helveolae (Bon & Boiffard) Bon (1993), L. sect. Cristatae Kühner ex Wasser (1978), L. sect. Fuscovinaceae Bon & Candusso (Candusso and Lanzoni 1990), L. sect. Lepiota, L. sect. Lilaceae Bon (1981), and L. sect. Eriophorae (Bon) Reschke & Sarawi (Sarawi et al. 2025). During recent investigations, two Lepiota species were collected from southwestern China for further morphological comparison and phylogenetic analysis. The results led to the discovery of two new species of L. sect. Lepiota, which are described and illustrated here. Materials and methods Morphology Specimens in this study, collected from three provinces or autonomous regions, viz., Yunnan, Xizang, and Sichuan, were deposited in the Herbarium of the Research Institute of Tropical Forestry, Chinese Academy of Forestry (RITF), and the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (HKAS). Macromorphological features were directly recorded in the field, with color photos of basidiomata and measurements of the pileus, stipe, etc., taken with a ruler (0.5 mm scale). Fresh basidiocarps were stored in sealed bags with silica gel for DNA extraction. The descriptive terms and herbarium acronyms follow Vellinga (1988) and Thiers (2025), respectively. Color codes follow Kornerup and Wanscher (1981). The material preparation, methods of chemical reactions, and measurement and statistics of micromorphological characters all follow Yang et al. (2019). Sections (20–50 μm) of specimens obtained by manual slicing were fixed in 5% KOH, Congo red, or Melzer’s reagent for microscopic observation. Basidiospores were placed in cresyl blue to test their metachromatic reaction. At least 20 elements were randomly measured from each character per collection. All features and dimensions of basidia, basidiospores, cheilocystidia, and the elements of the pileus covering were observed and measured with a ZEISS Imager M2 microscope at 1000× magnification. The abbreviation [n/m/p] indicates the measurement of n basidiospores from m basidiocarps in p specimens. The length or width of basidiospores is expressed by the notation (a)b–c(d), where 90% of the measured values fall between b and c, and a and d represent the 191 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota minimum and maximum values, respectively. The length-to-width ratio of the basidiospore is denoted by Q, and Q (in bold) represents the average Q and standard deviation of all measured basidiospores. DNA extraction, amplification, and sequencing Using the modified CTAB approach (Li et al. 2013), total genomic DNA was extracted from silica-dried samples. Four DNA loci were amplified with different primers in the polymerase chain reaction (PCR), as follows: ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) for the internal transcribed spacer region (ITS); LR0R and LR7 for the large subunit (LSU) of the ribosomal DNA (Vilgalys and Hester 1990); LR12R and 5SRNA for the intergenic spacer (IGS) regions (White et al. 1990); and MS3 and MS4 for the mitochondrial small ribosomal RNA subunit (mtSSU) regions (Liang et al. 2009). PCR products were delivered to Novo Gene Company (Beijing, China) for purification, sequencing, and editing. Phylogenetic analysis In the phylogeny, a total of 66 representatives (including all sequenced and described species of L. sect. Lepiota reported by previous studies) were sampled, with L. subcastanea Jun F. Liang & Zhu L. Yang and L. mandarina Jun F. Liang & Zhu L. Yang (Liang 2016) from L. sect. Stenosporae as outgroup. The available sequences were uploaded to GenBank and are listed in Suppl. material 1 together with all sampled taxa and voucher information. Using MAFFT v7.490 (Katoh and Standley 2013), four DNA loci were aligned and concatenated into a single data matrix (ITS–LSU–IGS–mtSSU). Phylogenetic reconstructions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) tools implemented in the PhyloSuite v1.2.3 platform (Xiang et al. 2023; Zhang et al. 2020). ModelFinder v2.2.0 (Kalyaanamoorthy et al. 2017) was used to select the best-fit partition model (edge-linked) for ML and BI analyses under the Bayesian Information Criterion (BIC). The best-fit partition models were as follows: IGS (ML: K3Pu+F+I+G, BI: HKY+F+I+G4), ITS (ML: TPM2u+F+G4, BI: HKY+F+G4), LSU (ML: K2P+R2, BI: K2P+I+G4), and mtSSU (ML: TVM+F+R2, BI: GTR+F+I+G4). Maximum likelihood phylogenies were inferred with IQ-TREE v2.2.0 (Nguyen et al. 2015), with 5000 ultrafast bootstrap replicates (Minh et al. 2013) and 1000 SH-aLRT tests (Guindon et al. 2010) to assess branch supports. Bayesian inference phylogenies were conducted in MrBayes v3.2.7 (Ronquist et al. 2012), employing two parallel runs of 40 million generations each (sample frequency once every 1000 generations), with the first 25% discarded as burn-in. ML and BI trees were visualized in FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Results Phylogenetic analysis The combined data matrix (ITS–LSU–IGS–mtSSU) had an alignment (Suppl. material 2) of 4,726 bp (IGS = 1–1210, ITS = 1211–2381, LSU = 2382–3838, mtSSU = 3839–4726), including sequence divergence of 1,641 variable 192 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota sites (34.72%), comprising 641 singleton variable sites (13.56%) and 1,000 parsimony-informative sites (21.16%). The topologies of both ML and BI trees were similar, and the ML tree was chosen for visualization (Fig. 1), with nodal support values from both methods indicated at the corresponding nodes. As shown in the phylogenetic tree, four main clades could be recognized. Two new species each formed a well-supported, monophyletic clade within Clade 1 and were distantly related to known and sequenced species, viz., L. brunneophora (BS = 100%, PP = 1.00) and L. ochraceosquamea (BS = 91%, PP = 0.99). L. brunneophora was sister to L. thrombophora (Berk. and Broome) Sacc., together forming a monophyletic lineage with strong support (BS = 100%, PP = 1.00). L. ochraceosquamea is grouped with some species (BS = 86%), including sequestrate species (L. geocarpa Vellinga & T. Lebel, L. geophana Vellinga & T. Lebel, L. mengei (Kropp & Castellano) T. Lebel & Vellinga) and agaricoid species (L. albofloccosa M. Ahamed, A.K. Dutta, K. Verma & Y.P. Sharma, L. kuehneriana Locq., L. nigrosquamosa Jun F. Liang & Zhu L. Yang). Additionally, sequestrate species with available sequences and formal descriptions all phylogenetically fell into L. sect. Lepiota. These species include L. iberica J.M. Vidal & Juste, L. smurfiorum J.M. Vidal & F. García, L. geogenia T. Lebel & Vellinga, L. viridigleba (Castellano) Z.W. Ge, Castellano & M.E. Sm., L. faiae-bravae Chautrand, A. Paz & Lavoise, and the three sequestrate species mentioned above, most of which have globose to subglobose basidiospores. In addition, L. pallidiochracea Jun F. Liang & Zhu L. Yang was nested within Clade 1 and sister to L. alba (Bres.) Sacc. (BS = 100%, PP = 1.00). Interestingly, L. eurysperma Sysouph., K.D. Hyde & Vellinga and L. pongduadensis Sysouph., K.D. Hyde & Vellinga clustered into a small, strongly supported clade (BS = 100%, PP = 1.00) with equal branch length. Taxonomic treatment Lepiota brunneophora J. F. Liang & X. Li, sp. nov. MycoBank No: 859649 Fig. 2 Holotype. China • Yunnan: Lijiang City, Jinshan Town, Tuanshan Reservoir, ca. 2500 m a.s.l., 18 July 2008, Liang 931 (RITF548). Etymology. ‘brunneophora’ refers to the brown color of the stipe. Diagnosis. Lepiota brunneophora is characterized by reddish-brown to purplish-brown stipe covering whitish floccose squamules, penguin-shaped basidiospores with papilliform apex when shrinking, broadly clavate cheilocystidia, and a pileus covering with a layer of short elements. Description. Basidiomata small to medium (Fig. 2A). Pileus 2.3–6.0 cm in diam, plano-convex or applanate with blunt umbo in the center, surface dry, white to cream, with yellowish-brown (6D5–6D6) to brown squamules, tearing from the center to the periphery into rings, accompanied by growth, margin upturned and striate, fragile and easily breaking off. Context whitish, thin. Lamellae L = 40–60, l = 1–2, free, whitish, moderately crowded, length unequal. Stipe 2.5–8.0 × 0.4–0.6 cm, cylindrical, hollow, thickening towards the base, reddish-brown to purplish-brown (6D6–6D7), covered with whitish and floccose squamules, yellowish-brown (6D5–6D6) squamules at the base. Annulus whitish, membranous, evanescent. Odor not distinct. Taste not recorded. Spore print white. 193 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota Basidiospores (Fig. 2C) [40/2/2] 12.5–14.0(14.5) × 4.0–4.5(5.0) µm [Q = (2.50)2.77–3.5(3.63), Q = 3.08 ± 0.28], penguin-shaped in side view, with suprahilar depression, adaxial side convex, abaxial side nearly straight, apex contracted and narrowed, papilliform, fusiform in front view; colorless, hyaline, Figure 1. Phylogenetic tree derived from both ML and BI methods based on combined ITS-LSU-IGS-mtSSU data, showing the phylogenetic position of two new L. sect. Lepiota species (red font) and sequestrate species (green font). Bootstrap values (≥50%) and posterior probabilities (≥0.90) are indicated at each node. An asterisk indicates the sample for which only the ITS sequence is available. 194 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota smooth, dextrinoid, slightly thick-walled, congophilous, not metachromatic in cresyl blue. Basidia 20–27 × 8–12 µm, clavate, 4-spored. Lamella edge sterile. Cheilocystidia (Fig. 2B) 9–23 × 10–24 µm, mostly clavate to sub-spherical, apex obtuse; colorless, hyaline, thin-walled. Pleurocystidia absent. Pileus covering (Fig. 2D) a trichoderm consisting of elongate, subcylindrical, apically attenuate, terminal elements 120–450 × 8–10 µm, without or rarely with septa at the base, base mixed with a layer of short and clavate elements (35–90 × 7–11 µm), with yellow-brownish intracellular pigment. Clamp connections present in all tissues. Distribution. Known only from Yunnan Province, China. Habitat. Solitary or in small groups, saprotrophic and terrestrial on grasslands under the mixed conifer and broadleaf forest in summer. Additional specimens examined. China • Yunnan: Jinghong City, Dadugang Town, 7 September 2007, alt. 1050 m, Liang 808 (RITF541, paratype). Notes. Lepiota brunneophora is characterized by its pileus with yellowish-brown to brown squamules and striate margin, reddish-brown to purplish-brown stipe covering with whitish and floccose squamules, penguin-shaped basidiospores with a distinctly narrowed apex, clavate to sub-spherical cheilocystidia, and a trichodermal pileus covering intermixed with clavate and short elements. Lepiota brunneophora is similar to L. attenuata Jun F. Liang & Zhu L. Yang in its small basidiomata, the absence of an annulus, a pileus with yellowish-brown squamules and a striate margin, and penguin-shaped basidiospores with a distinctly narrowed apex, but the difference between the two lies in that the latter has whitish stipe covering with fine grayish orange squamules, longer basidiospores, and a pileus covering with inflated elements (Liang et al. 2011). Lepiota metulispora (Berk. & Broome) Sacc. and L. pakistanensis A. Rehman, Afshan, Usman & Khalid have a pileus with pale brown to brown squamules and striations. Both species have some features that serve as diagnostic characteristics separating them from L. brunneophora, such as L. metulispora having a whitish annulus, stipe covering with tomentose squamules at the lower part, penguin-shaped basidiospores without a distinctly narrowed apex (Liang et al. 2011), while L. pakistanensis possesses a whitish annulus, stipe covering with creamy white squamules at the basal part, and penguin-shaped basidiospores without a distinctly narrowed apex (Rehman et al. 2024). Besides, L. cortinarius J.E. Lange could be distinguished from L. brunneophora by larger basidiomata, a pileus without striation on the margin, whitish stipe with pale yellow to dark brown squamules, and basidiospores without a narrowed apex (Lange 1915; Liang 2007). Lepiota clypeolarioides Rea was typically different from L. brunneophora in its whitish to pale ochre pileus without striation, a stipe with whitish upper part and dirty white part, and smaller (6–8 × 4–5 μm) and ellipsoid basidiospores (Rea 1922; Hausknecht and Pidlich-Aigener 2005). Lepiota ampliocystidiata Jun F. Liang shares some identical features with L. brunneophora, such as small basidiomata, pileus with striation, penguin-shaped basidiospores with a distinctly narrowed apex, but is differentiated by its dark brown to black brownish squamules on the pileus, whitish annulus, longer [(13.0) 15.0–20.0 (21.5) × (4.0) 4.5–5.5 (6.0) μm] basidiospores (Liang 2012). Phylogenetic analysis indicated that L. brunneophora is sister (BS = 100% & PP = 1.00) to L. thrombophora. Unlike the former, the latter exhibits 195 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota reddish-brown to dark brown squamules on the pileus, stipe displaying cream and smooth above the annulus and pale brown below, whitish annulus, penguin-shaped basidiospores without a distinctly narrowed apex (Saccardo 1887; Zhou 2010; Liang et al. 2011). Figure 2. Lepiota brunneophora (holotype, RITF548) A. Basidiomata; B. Cheilocystidia; C. Basidiospores; D. Pileus covering. Both the photo and the line drawing are by Junfeng Liang. 196 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota Lepiota ochraceosquamea J. F. Liang & Zhu L. Yang, sp. nov. MycoBank No: 859650 Fig. 3 Holotype. China • Xizang: near Yikang County, 23 July 2004, alt. 3500 m, Yang 4173 (HKAS45559). Etymology. ‘ochraceosquamea’ refers to the color of squamules on the pileus surface. Diagnosis. Lepiota ochraceosquamea is characterized by whitish pileus covering with dark yellowish-brown to light ochre squamules, smooth stipe possessing fine and brown squamules at the base, broadly fusiform or oblong basidiospores (8.5–11.0 × 5.0–6.5 μm), diverse cheilocystidia (clavate, fusiform, or falcate), pileus covering a trichoderm consisting of long, erect elements, without basal short elements. Description. Basidiomata small. Pileus 1–3 cm in diam, initially campanulate, extended gradually, accompanied by development, plano-convex, surface covering with dark yellowish-brown to light ochre (6D7–6D8) squamules on a whitish surface, with blunt and brown (6D8) umbonate center. Context whitish, thin. Lamellae L = 40–60, l = 1–2, free, whitish, then turning dirty white, moderately crowded, ventricose, length unequal. Stipe 1.5–3.5 × 0.2–0.4 cm, tapering upwards, nearly brown, glabrous, with fine and brown (6D6–6D7) squamules at the base. Annulus whitish, margin nearly brown, and evanescent (Fig. 3A). Smell not distinct; taste not recorded. Basidiospores (Fig. 3B) [67/3/3] (8.0)8.5–11.0(11.5) × 5.0–6.5 μm [Q = (1.45) 1.50–2.00, Q = 1.68 ± 0.14], broadly fusiform or oblong in side view, without suprahilar depression, adaxial side convex, apex blunt-round, oblong in front view; colorless, hyaline, smooth, slightly wall-thickened, dextrinoid, congophilous, not metachromatic in cresyl blue. Basidia 24–30 × 9–12 μm, clavate, mostly 4-spored and occasionally 2-spored, sterigmata up to 7 μm long. Cheilocystidia (Fig. 3C) 15–32 × 5–9 μm, diverse, clavate, fusiform, or falcate with flexuous apex, colorless, hyaline, wall-thinned, light pink in Congo red. Pleurocystidia absent. Pileus covering (Fig. 3D) a trichoderm composed of subcylindrical, narrow to apex, mostly flexuous, slightly wall-thickened, terminal elements 70–240 × 6–18 µm, base rarely with short elements, and with yellow to yellow-brownish intracellular pigment. Clamp connections present in all tissues. Distribution. Known only from the Xizang Autonomous Region and Sichuan Province, China. Habitat. Solitary or in small groups, saprotrophic and terrestrial on moist grasslands. Additional specimens examined (paratypes). China • Xizang: Jiangda County, Jila Mountain, 1 August 2008, alt. 4250 m, Yang 4256 (HKAS45635); Sichuan: Daocheng County, alt. 4000 m, 1 July 1998, Yang 1957 (HKAS32150). Notes. The main characteristics of L. ochraceosquamea are ochre-yellow squamules on the pileus, broadly fusiform basidiospores with a blunt-rounded apex, diverse shapes of cheilocystidia, and a trichodermal pileus covering with terminal elements gradually narrowing toward the apex, rarely with short elements at the base. Phylogenetic analysis showed that L. ochraceosquamea clustered with several sequestrate and agaricoid species. Distinct from L. ochraceosquamea, 197 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota the former species displays distinctly different characters, viz., sequestrate fruiting habit and globose basidiospores. The latter, comprising L. albofloccosa, L. nigrosquamosa, and L. kuehneriana, have distinct morphological features that differentiate them from L. ochraceosquamea. Lepiota albofloccosa has larger basidiomata, snow-white to milky white squamules on Figure 3. Lepiota ochraceosquamea (holotype, HKAS45559) A. Basidiomata; B. Basidiospores; C. Cheilocystidia; D. Pileus covering. Photo and line drawing by Zhu L. Yang and Junfeng Liang, respectively. 204 MycoKeys 123: 189–204 (2025), DOI: 10.3897/mycokeys.123.163999 Xing Li et al.: Two new species of Lepiota Supplementary material 1 Table of GenBank accession numbers Authors: Xing Li Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.123.163999.suppl1 Supplementary material 2 Alignment for phylogeny Authors: Xing Li Data type: fas Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.123.163999.suppl2