Taxonomy and Phylogeny of Cystolepiota (Agaricaceae, Agaricales): New Species, New Combinations and Notes on the C. seminuda Complex

Species of Cystolepiota are known as diminutive lepiotaceous fungi with a worldwide distribution. Previous studies revealed that Cystolepiota is not monophyletic and preliminary DNA sequence data from recent collections suggested that several new species exist. Based on multi-locus DNA sequence data (the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2, ITS; the D1–D2 domains of nuc 28S rDNA, LSU; the most variable region of the second-largest subunit of RNA polymerase II, rpb2 and a portion of the translation–elongation factor 1-α. tef1), C. sect. Pulverolepiota forms a distinct clade separating from Cystolepiota. Therefore, the genus Pulverolepiota was resurrected and two combinations, P. oliveirae and P. petasiformis were proposed. With the integration of morphological characteristics, multi-locus phylogeny, and information on geography and habitat, two new species, viz. C. pseudoseminuda and C. pyramidosquamulosa, are described and C. seminuda was revealed to be a species complex containing at least three species, viz. C. seminuda, C. pseudoseminuda, and Melanophyllum eryei. In addition, C. seminuda was re-circumscribed and neo-typified based on recent collections.


Morphological Studies
Macroscopic descriptions are based on field notes and digital images of fresh basidiomata. The color code of Kornerup and Wanscher was used [39]. Measurements and descriptions of microscopic characteristics were performed on dried materials. Sections of specimens were rehydrated in 5% aqueous (w/v) potassium hydroxide (KOH) and stained with 1% Congo red reagent, if necessary. Microscopic features were observed and measured under a Leica DM2500 light microscope (LM). Basidia, basidiospores, cystidia, and pileal structures were measured and depicted using brightfield or phase contrast optics. Chemical reactions of basidiospores were tested in Melzer's reagent and cresyl blue. The lamella fragments of dried specimens were fixed on an aluminum pile with double-sided adhesive tape, then coated with gold palladium and observed under a ZEISS Sigma 300 scanning electron microscope (SEM).
The notations [n/m/p] indicate that the measurements were taken on n basidiospores, from m basidiomata and p collections. The dimensions of basidiospores were provided with the notation (a-)b-c(-d). The range b-c indicates the minimum and maximum values of 90% of the measured structures. The a and d in parentheses indicate the absolute extreme values. Q is used to indicate the length/width ratio of basidiospores in lateral view; Q m means average Q of all basidiospores ± sample standard deviation.
Specimens are deposited in the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (KUN) and in the fungarium of the Senckenberg Museum of Natural History, Görlitz (GLM).

Phylogenetic Analyses
The phylogenetic analyses included 120 ITS, 57 LSU, 44 rpb2, and 42 tef1 sequences. Based on recent phylogenetic studies [18,19], sequences of three Echinoderma species were chosen as the outgroup of the ITS analysis and sequences of two Coprinus species for the combined analysis. Multiple sequences alignments were performed with MAFFT v7.4.95 [48] and ambiguously aligned regions of each sequence were detected and excluded using trimAl v1.4 [49]. The resulting alignments were examined and optimized manually in AliView 1.27 [50].
Firstly, maximum likelihood (ML) phylogenetic trees were reconstructed based on matrices of each respective gene fragment, viz. the ITS matrix (120 sequences), the subsampled ITS matrix with selected sequences, the LSU matrix, the rpb2 matrix, and the tef1 matrix. As no conflicts with high support (>50%) were observed among the ML trees of the five data sets (Figures 1 and S1-S4), three multi-locus concatenated matrices (ITS-LSU-rpb2-tef1, LSU-rpb2-tef1, and rpb2-tef1 matrix) were constructed using PhyloSuite v1.2.2 [51] and analyzed by the ML method and bayesian inference (BI) method.
The best-fit substitution model for each single alignment was estimated by Partition-Finder 2 [52]. The BI analyses were performed with MrBayes v. 3.2.6 [53] by applying the best-fit model for each partition (2 parallel runs, 10,000,000 generations), in which the initial 25% of sampled data was discarded as burn-in. The Bayesian posterior probabilities (BPPs) were calculated from the posterior distribution of the remaining phylogenetic trees. ML estimation was conducted with RAxML 8.2.12 [54] using GTR+I+GAMMA as the best-fit likelihood model. Statistical supports were calculated with 1000 bootstrap replicates. Resulting phylograms were displayed in FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 2 November 2021)). The best-fit substitution model for each single alignment was estimated by Partition Finder 2 [52]. The BI analyses were performed with MrBayes v. 3.2.6 [53] by applying th best-fit model for each partition (2 parallel runs, 10,000,000 generations), in which the in itial 25% of sampled data was discarded as burn-in. The Bayesian posterior probabilitie

Phylogenetic Results
The ITS data set consisted of 120 sequences and 745 sites, including 356 parsimony informative sites. The final matrix of the combined data set included 89 ITS, 57 LSU, 44 rpb2, and 42 tef1 sequences, in which the aligned lengths of the four gene fragments were 651, 862, 705, and 563 bp, respectively, that contained 963 parsimony informative sites. As the ITS phylogenetic tree included the largest number of samples and the ITS-LSU-rpb2-tef1 phylogenetic tree was the second-most supported phylogenetic tree with relatively more species than other phylogenetic trees inferred from the combined data set ( Figure 1, Figure 2, and Figures S1-S6), the phylogenies based on the ITS data set and the combined ITS-LSU-rpb2-tef1 data set were displayed and used for subsequent discussions (Figures 1 and 2).
In the phylogeny inferred from ITS-LSU-rpb2-tef1 ( Figure 2), five main clades can be recognized: the clade including Cystolepiota and Melanophyllum (MLB = 77%, BPP = 0.91), the Echinoderma clade (MLB = 100%, BPP = 1.00), the Smithiomyces clade (MLB = 100%, BPP = 1.00), the Lepiota clade (MLB = 63%, BPP = 1.00), and the Pulverolepiota clade (MLB = 100%, BPP = 1.00). Most of the specimens previously identified as C. seminuda and either sequenced in this study or from previous studies belong to Clade I (MLB = 75%, BPP = 0.98) or Clade II (MLB = 100%, BPP = 1.00) in the first main clade (Cystolepiota/Melanophyllum clade). The two clades of the C. seminuda complex are sister to each other, forming a clade with low support (MLB = 59%, BPP < 0.90). Clade I consists of C. pseudoseminuda, C. aff. pseudoseminuda 1 and 2, M. eyrei, and several further unnamed subclades. Clade II includes C. seminuda (s. str.) and several further unnamed subclades as well. One specimen from China (KUN-HKAS 53985) forms a distinct clade (MLB = 100%, BPP = 1.00) with a specimen from Italy previously identified as C. seminuda and an uncultured Cystolepiota clone from India within the Cystolepiota/Melanophyllum main clade. Two specimens (GLM-F107803 and GLM-F107804) from Germany also form a distinct clade (MLB = 100%, BPP = 1.00) within the Cystolepiota/Melanophyllum clade. A further specimen from China (KUN-HKAS 124759) is grouped with the holotype of C. oliveirae within the Pulverolepiota clade.  The C. seminuda complex formed a well-supported clade in all the resulting phylogenetic trees based on rpb2, tef1, as well as the combined rpb2-tef1 data set ( Figures S3-S5). However, the ITS-LSU-rpb2-tef1 combined data set, and LSU-tef1-rpb2 combined data set support the monophyly of the C. seminuda complex with low supports, while the LSU data set showed the lowest signals for the phylogeny of Cystolepiota. The presence of ITS and/or LSU in the combined data sets seem to decrease the statistical support of certain nodes of the phylogenetic tree inferred from the combined data set (Figures 1, 2 and S1-S6). basidiospores vary strongly between specimens of C. pseudoseminuda and C. seminuda and there is an overlap between the two species in basidiospore size and shape, individuals with high Qm value always belong to C. seminuda and those with low Qm value always belong to C. pseudoseminuda ( Figure 6). In addition, there are 92 (out of 722) nucleotide differences between the ITS sequences of the holotype of C. pseudoseminuda and that of the neotype of C. seminuda.

Taxonomy
Specimens of C. pseudoseminuda form a robust clade with C. aff. pseudoseminuda 1 and C. aff. pseudoseminuda 2 in in both phylogenies (Figures 1 and 2). The ITS sequences of the three groups are 96% to 97% identical. Their geographical distribution pa erns are different: C. pseudoseminuda has so far been collected only in southwestern China and C. aff. pseudoseminuda 1 in Europe and northern China, while all the sequences of C. aff. pseudoseminuda 2 are derived from specimens collected in the Unites States of America (USA). Based on the observation of specimens in this study, no distinct morphological differences were found to distinguish C. pseudoseminuda and C. pseudoseminuda 1 ( Figure 4A-D) and no specimens of C. pseudoseminuda 2 were studied morphologically. Further morphological observations need to be conducted on C. aff. pseudoseminuda 1 and C. aff. pseudoseminuda 2.    Diagnosis: Cystolepiota pseudoseminuda is distinguished from other Cystolepiota species by its slender basidiomata, pulverulent or granulose squamules with a pink to orange tinge that are composed of loosely-arranged inflated cells connected to filamentous hyphae, non-dextrinoid basidiospores with distinct warts on the surface visible under LM and SEM, the absence of cystidia, and the presence of abundant clamp connections. Its ITS, LSU, rpb2, and tef1 sequences differentiate this species from other species.   Description: Pileus 5-18 mm diam, hemispherical to obtusely conical when young, expanding to plano-convex or applanate with a slightly umbonate center with age; surface dry, white to cream, with pulverulent to granulose squamules, fugacious, white, cream, pale orange (5A3), pink (10A3), pinkish-orange to light pink (6A2-5) at the center, turning white to cream towards the margin; margin of the pileus with easily detachable veil remnants. Lamellae up to 2(3) mm broad, free, moderately crowded, unequal, white to light cream, with 1-3 tiers of small lamellulae. Stipe 20-50 × 0.5-2 mm, central, subcylindrical to cylindrical, surface white to cream on the upper portion, with age gradually turning to pale orange (5A3-4), grayish orange (6B5-7), brownish (5D6) to purplish brown (7F7) towards the middle and lower portion with age, with pulverulent to granulose squamules, which are concolorous with those of the pileus; with a fragile and fugacious membranous veil between pileus margin and stipe initially; basal mycelium white. Context thin, whitish. Odorless; taste not recorded.
Basidiospores Notes: Cystolepiota pseudoseminuda is morphologically similar to C. seminuda regarding color and size of the basidiomata. Both species form pulverulent to granulose squamules, non-dextrinoid basidiospores, and clamp connections, while cystidia are lacking. However, the surface of basidiospores of C. seminuda is smooth under LM and SEM, which is in contrast to the warty basidiospores of C. pseudoseminuda. The basidiospores of C. pseudoseminuda are shorter and wider at Q = (1.21-)1.24-1.85(-2.20) and Q m = 1.55 ± 0.19, compared to those of C. seminuda at Q = (1.41-)1.46-2.15(-2.45) and Q m = 1.78 ± 0.22. At least according to the specimens examined in this study, although the characteristics of basidiospores vary strongly between specimens of C. pseudoseminuda and C. seminuda and there is an overlap between the two species in basidiospore size and shape, individuals with high Q m value always belong to C. seminuda and those with low Q m value always belong to C. pseudoseminuda ( Figure 6). In addition, there are 92 (out of 722) nucleotide differences between the ITS sequences of the holotype of C. pseudoseminuda and that of the neotype of C. seminuda.   Specimens of C. pseudoseminuda form a robust clade with C. aff. pseudoseminuda 1 and C. aff. pseudoseminuda 2 in in both phylogenies (Figures 1 and 2). The ITS sequences of the three groups are 96% to 97% identical. Their geographical distribution patterns are different: C. pseudoseminuda has so far been collected only in southwestern China and C. aff. pseudoseminuda 1 in Europe and northern China, while all the sequences of C. aff. pseudoseminuda 2 are derived from specimens collected in the Unites States of America (USA). Based on the observation of specimens in this study, no distinct morphological differences were found to distinguish C. pseudoseminuda and C. pseudoseminuda 1 (Figure 4A-D) and no specimens of C. pseudoseminuda 2 were studied morphologically. Further morphological observations need to be conducted on C. Two ITS sequences from GenBank, JF907983 (from Italy) and KU847887 (from India), are similar to the sequence derived from the type specimen of C. pyramidosquamulosa; both differ by seven nucleotides from C. pyramidosquamulosa, which is considered within the variability of C. pyramidosquamulosa.  Figures 3D-F Description: Pileus 2.5-12 mm diam, first conical, paraboloid to hemispherical, then expanding to plano-convex or convex, often with a broad umbo at the center; surface white, when young, changing to light cream with age, covered with densely powdery to granulose squamules, which become sparse or even disappear with age; light yellowishbrown (6C3-5), pinkish-orange to light pink (6A2-5) at the center of the pileus; white to cream towards the margin, fragile and easily breaking off; pileus margins often appendiculate with veil remnants. Lamellae up to 2(-3) mm wide, free, crowded, white to light cream, with 3-5 tiers of lamellulae. Stipe 10-45 mm long, 0.5-1.5 mm diam, central,  Figure 4L); inamyloid, non-dextrinoid, metachromatic in cresyl blue. Basidia (13.5-)15-18.5 × 5-7 µm, clavate, hyaline, 4-spored; sterigmata 2.5-3.5 µm long. Cheilocystidia and pleurocystidia not observed. Lamellar trama regular, colorless, composed of cylindrical hyphae, 5-7.5 µm in diam. The squamules on pileus and stipe consist of inflated cells and filamentous hyphae; inflated cells abundant, globose to subglobose, rarely fusiform or sphaero-pedunculate, 21-33 × 20-29 µm, smooth-walled, with a slightly thick, chartreuse-yellow wall, usually 2-5 cells forming loosely arranged chains; hyphae rare, 1-3 µm in diam, chartreuse-yellow. Clamp connections present in all tissues (Figure 7).
Habitat and distribution: Presumably saprotrophic. Solitary or scattered on soil. Found in Asia (China, India) and Europe (Italy).
Notes: Pyramidal squamules are the most distinctive macroscopic characteristic of Cystolepiota pyramidosquamulosa. The pilei of P. oliveirae and P. petasiformis (=P. pulverulenta) are also covered with obvious squamules and also lack cystidia. However, the two species can be distinguished from C. pyramidosquamulosa by the rough basidiospores that slowly become red-brown in Melzer's reagent and the squamules composed of irregular, branched, inflated, and oblong elements. In addition, the basidiospores of C. pyramidosquamulosa are narrower than those of the latter two species [5,55,56]. Cystolepiota pyramidalis Sysouph. and Thongkl. forms squamules similar to C. pyramidosquamulosa. However, C. pyramidalis forms cheilocystidia and ellipsoid-ovoid spores and has so far only been collected in Laos and Thailand [57]. In contrast to C. pyramidosquamulosa, C. fumosifolia (Murrill) Vellinga and C. pseudofumosifolia M.L. Xu & R.L. Zhao form warty to granulose squamules and have cystidia [5,58]. Cystolepiota adulterina F.H. Møller ex Bon has warty squamules and somewhat resembles C. pyramidosquamulosa macroscopically. However, it differs in the presence of abundant cheilocystidia. In addition, C. pyramidosquamulosa can be distinguished from other species by its distinct ITS, LSU, rpb2, and tef1 sequences.
Two ITS sequences from GenBank, JF907983 (from Italy) and KU847887 (from India), are similar to the sequence derived from the type specimen of C. pyramidosquamulosa; both differ by seven nucleotides from C. pyramidosquamulosa, which is considered within the variability of C. pyramidosquamulosa.
Cystolepiota seminuda (Lasch) Bon, Documents Mycologiques 6(24): 43,1976. Figures 3D-F forms larger basidiomata [33] and both the pileus and stipe of L. rufipes were described as smooth and glabrous [59], although  inferred that it may be the result of rain washing [36]. Accounting for the difference described above, we temporarily remove L. sororia and L. rufipes from synonyms of C. seminuda until specimens exactly matching the description of the protologue of the two species are found and sequenced.   Description: Pileus 2.5-12 mm diam, first conical, paraboloid to hemispherical, then expanding to plano-convex or convex, often with a broad umbo at the center; surface white, when young, changing to light cream with age, covered with densely powdery to granulose squamules, which become sparse or even disappear with age; light yellowish-brown (6C3-5), pinkish-orange to light pink (6A2-5) at the center of the pileus; white to cream towards the margin, fragile and easily breaking off; pileus margins often appendiculate with veil remnants. Lamellae up to 2(-3) mm wide, free, crowded, white to light cream, with 3-5 tiers of lamellulae. Stipe 10-45 mm long, 0.5-1.5 mm diam, central, subcylindrical to cylindrical; white to cream; glabrous in the upper part, gradually darkening towards the base, central and lower part grayish orange (6B5-7), brownish (5D6), reddish brown (7C6-7) to purplish brown (7F7), with white, light cream to light brownish powdery to granulose squamules, easily breaking off. Context thin (<1 mm), whitish. Smell not distinct; taste not recorded.
Basidiospores Notes: Agaricus seminudus, the basionym of Cystolepiota seminuda, was described in the Margraviate of Brandenburg (not identical with the contemporary state of Brandenburg), Germany [23]. As no holotype specimen of A. seminudus was designated, specimens previously collected in Germany that had been identified as C. seminuda based on morphology were studied. The ITS sequences of these specimens confirmed the placement of this species in the genus Cystolepiota. However, these specimens belonged to four clades, suggesting the existence of four species. Two of them clustered with sequences from GenBank that had been generated from specimens identified as C. seminuda as well and represent two different species. The basidiospores of the specimens of one species were observed to be ornamented under the SEM, while those of another species were smoothwalled. We regard the latter species as C. seminuda because it includes specimens that had been collected closest to the type location and most of the German specimens belonged to it. Moreover, these specimens were presumably growing on soil, two specimens from Estonia were even collected between moss described as the habitat of A. seminudus in the protologue of the species. As the holotype was neither located in ZE Botanischer Garten und Botanisches Museum, Freie Universität Berlin (B), where the type specimens of G. W. Lasch had been deposited, nor in other collections holding specimens collected by Lasch (https://www.sil.si.edu/DigitalCollections/tl-2/history.cfm (accessed on 1 December 2022)), one of the specimens closest to the type location (GLM-F125824) is designated here as a neotype of A. seminudus. In contrast, the two German specimens from the clade with ornamented basidiospores were apparently growing on wood and related to C. pseudoseminuda, which is a species newly described in this study.
Compared with C. seminuda, L. sororia and L. rufipes, which have been reduced to synonyms of C. seminuda by Vellinga in 1987 and 2010 respectively, have larger basidiomata and longer and narrower basidiospores [21,33,36,59]. Furthermore, L. sororia forms larger basidiomata [33] and both the pileus and stipe of L. rufipes were described as smooth and glabrous [59], although  inferred that it may be the result of rain washing [36]. Accounting for the difference described above, we temporarily remove L. sororia and L. rufipes from synonyms of C. seminuda until specimens exactly matching the description of the protologue of the two species are found and sequenced.  [60]. In 1993, he erected a new genus, Pulverolepiota, to accommodate this species [4]. Vellinga and Huijser (1998) subsequently reduced Pulverolepiota to a section of Cystolepiota, C. sect. Pulverolepiota [6]. However, specimens previously identified as C. petasiformes, C. oliveirae, and related species representing Cystolepiota sect. Pulverolepiota form a distinct clade in both phylogenies (Figures 1 and 2). The respective clade in Figure 2 is one of the main clades that represent genera and did not group with the Cystolepiota/Melanophyllum clade, but is sister to the clades formed by Cystolepiota, Melanophyllum, Echinoderma, Smithiomyces, and Lepiota., supporting Pulverolepiota as being a distinct genus rather than a section of Cystolepiota. Therefore, the genus Pulverolepiota Bon (1993) is resurrected here. MycoBank: MB847958 Description: Pileus 14 mm in diam, about 18 mm high, plano-conical, with a distinct umbo, surface whitish to cream, covered with thick flocculose squamules, erect at center, brownish orange (6C8) at the center, fading to yellowish or pale orange (5A2-4) or cream toward the margin; margin appendiculate, easily detachable. Lamellae free, wavy, brownish (5B2), distant, up to 2 mm broad, with 2-3 tiers of lamellulae. Stipe 30 × 1.5 mm, light orange (5A5) at the upper part, reddish-orange (7A6-8) at the base, central, hollow, subcylindrical, with a slightly bulbous base; surface smooth, covered with white, flocculose, easily-detachable squamules on the middle part. Context white in stipe and pileus, thin. Odorless; taste not recorded. Pulverolepiota petasiformis (Murrill)   Habitat and distribution: Solitary or scattered on soil or on a trunk of Dicksonia antarctica in Europe (Portugal), Asia (tropical region of southwestern China) and Australia.
Notes:Pulverolepiota oliveirae was described as C. oliveirae from Portugal [55], characterized by a whitish to cream pileus with erect flocculose squamules, a brown stipe with white flocculose squamules, a lack of cystidia, and rough and slow-staining dextrinoid spores. Our collection represents a new report for China; it matched the protologue of P. oliveirae regarding macro-and micro-characteristics, except for the pileus of the specimen from China that has a distinct umbo. The basidiospores of the specimen from China [(4-)4.5-5.5(-6) × (2.5-)3-3.5 µm] are slightly smaller than those in the protologue of C. oliveirae (5.0-6.4 × 2.9-4.0 µm) [55]. The morphological differences could be explained by population and measurement differences. Furthermore, the ITS sequence is 99.74% identical with the sequence generated from the type specimen (KY472789).
The two closely related species, P. oliveirae and P. petasiformis (=C. pulverulenta) can easily be confused. However, the squamules of C. oliveirae present tightly-arranged globose to subglobose cells among the loosely-arranged cells. Those tightly arranged cells have not been observed in C. petasiformis [5,56]. Paraíso et al. (2016) suggested the size of the basidiospores as a characteristic to distinguish the two species [55]. However, the spores of the collection from China are not significantly larger than those of C. petasiformis. Several Cystolepiota species, including C. fumosifolia, C. pseudofumosifolia, and C. pyramidalis, that are not closely related with P. oliveirae are also similar in the presence of distinct squamules. However, they differ in forming cheilocystidia, inamyloid and non-dextrinoid basidiospores and squamules that are composed of loosely-arranged globose to subglobose cells, while irregular, branched, cylindrical to oblong cells are lacking. Cystolepiota pyramidosquamulosa, a species newly described in this paper, also forms distinct squamules. It can be distinguished from P. oliveirae by having neither amyloid nor dextrinoid basidiospores and forming loosely-arranged globose to subglobose cells in the squamules.
Pulverolepiota petasiformis (Murrill)  MycoBank: MB847957 Notes: Pulverolepiota petasiformis was originally described by Murrill (1912) from Washington State, USA as Lepiota petasiformis [61]. Vellinga (2006) transferred it to Cystolepiota after examining its type specimen. Because of the similar morphology, for example, L. petasiformis also lacks clamp connections, she treated the later described L. pulverulenta, a European species and the type species of Pulverolepiota, as a synonym of C. petasiformis, while morphological differences exist as well [5]. Moreover, in molecular analyses of a previous study, sequences from Europe and North America formed a well-supported monophyletic clade (Figure 1, Figure 2, Figure 3 in Vellinga 2003) [17]. As the clade including C. petasiformis represents a generic level clade named Pulverolepiota (Figure 2), a new combination Pulverolepiota petasiformis is proposed here.
A total of five sequences of P. petasiformis were included in the ITS phylogeny (Figure 1), including three sequences of collections originally identified as C. pulverulenta by Vellinga [62] and two sequences from GenBank labeled as Cystolepiota sp. The five sequences formed a well-supported clade (MLB = 94%, BPP = 1.00) with a subclade (MLB = 97%, BPP = 1.00) formed by two European sequences. As can be seen in Figure 1, there are large differences between the European and the North American collections, indicating them to be distinct populations that may result from geographical isolation and may represent P. pulverulenta and P. petasiformis respectively. However, there is also a large variability within the specimens from North America. Further collections and studies are needed to evaluate, if P. petasiformis is one variable species or a species complex.

Circumscription and Diversity of Cystolepiota and Pulverolepiota
According to the phylogenetic results of this study (Figures 1 and 2), the resurrected genus Pulverolepiota includes P. petasiformis, P. oliveirae and an unnamed species from Hawaii. The main characteristics of Pulverolepiota are: rough basidiospores that are slowly staining reddish-brown in Melzer's reagent, the absence of clamp connections and cystidia, and pileus-covering elements that are composed of loosely-arranged elongate, branched, irregular, sometimes subglobose or ellipsoid cells. Lepiota roseolanata [≡Leucoagaricus roseolanatus (Huijsman) Bon], a species described by Bon (1962) based on a specimen from Limburg, Netherlands, matches the above characteristics and may be a member of Pulverolepiota. Vellinga noted that Cystolepiota pseudogranulosa (Berk. & Broome) Pegler may belong to Pulverolepiota, while further studies are needed as C. pseudogranulosa forms strongly dextrinoid basidiospores and clamp connections [6,9].
With the exclusion of C. sect. Pulverolepiota, two sections remain in Cystolepiota: C. sect. Cystolepiota that include species with non-dextrinoid basidiospores and C. sect. Pseudoamyloideae with species forming dextrinoid basidiospores. In the present study, three species with dextrinoid basidiospores were included in our phylogenetic analyses, namely C. bucknallii (the type species of C. sect. Pseudoamyloideae), C. icterina, and C. rhodella. However, the three species did not form a clade (Figures 1 and 2) and the sequence of C. constricta Singer, the type species of Cystolepiota, is not available. Thus, further sampling and phylogenetic analyses are needed to verify and revise the infrageneric classification of Cystolepiota.
Melanophyllum is distinguished from Cystolepiota by the formation of reddish-brown to greenish spore prints and ornamented spores [10,12,17]. In this study, we demonstrate that some species of the C. seminuda species complex form ornamented spores as well. Phylogenetic results show that the two genera belong to the same main clade (Figure 2). Specimens previously identified as Melanophyllum did not even form a monophyletic clade within Cystolepiota, which confirms that the above-mentioned spore characteristics are not genus specific. It seems reasonable to synonymize the two genera. However, the name Melanophyllum Velen. 1921 has priority as it was published before Cystolepiota Singer 1952, which means that Cystolepiota would become a synonym of Melanophyllum. It requires careful consideration, if the about 50 current species of Cystolepiota should be transferred to Melanophyllum or to conserve Cystolepiota and subsequently combine the six Melanophyllum species in Cystolepiota. A more comprehensive and robust phylogeny is necessary before combining these, in order to confirm that whether they are congeneric.
Four accepted species are included in Melanophyllum according to Species Fungorum (https://www.speciesfungorum.org/ (accessed on 25 April 2023)). Sequences of two of them, M. haematospermum and M. eyrei, were included in the phylogenetic analyses of this study. The resulting phylogenies (Figures 1 and 2), indicate that the species diversity of Melanophyllum was underestimated and several new species are waiting to be described.
Several reasons may lead to this limited knowledge of the species diversity of Cystolepiota. (1) The tiny and solitary basidiomata make them easily overlooked in field investigations and mycologists are not inclined to study and describe species based on only one basidioma. For example, C. oliveirae was first recorded in China in this paper based on one basidioma with a pileus that was 1.4 cm in diam. Several potential new species belonging to the C. seminuda complex, such as KUN-HKAS 56447, KUN-HKAS 84333 and KUN-HKAS 84177, that were included in our ITS phylogeny ( Figure 1) were not described in the present paper, because there is only one mature basidioma each available for description. (2) The collections of many species, e.g., C. seminuda, C. hetieri (Boud.) Singer, and Melanophyllum haematospermum (Bull.) Kreisel, apparently exhibit almost identical morphological characteristics but show a large variability in their ITS sequences as demonstrated by the phylogenies of this study (Figure 1). This indicates the existence of many cryptic species. With further investigations and research from less studied regions such as Africa and Australia, more Cystolepitoa species are expected to be discovered.

Circumscription of Species in the Cystolepiota seminuda Species Complex
We regard the species in Clade I and Clade II (Figure 2) as the members of the C. seminuda species complex that includes three named species: C. pseudoseminuda, C. seminuda, and a species represented by specimens that had been identified as Melanophyllum eryei, as well as ten potential new species according to the sequence similarity. The main characteristics of the specimens belonging to the C. seminuda species complex investigated in the present study are: small basidiomata with a whitish pileus covered by pulverulent or granulose squamules that are whitish, cream to pinkish, rarely brownish; free lamellae that are white to cream in most species and greenish in M. eyrei; a stipe with a whitish upper portion and a brownish lower portion, which turns purplish or reddish-brown with age or when touched; a stipe often covered with whitish to cream pulverulent squamules; basidiospores which are ellipsoid, smooth-walled, or rough-walled; absent cheilocystidia and pleurocystidia; clamp connections present. However, collections may meet the above descriptions, but still show certain morphological differences, especially regarding the basidiospores.
Cystolepiota sistrata has been considered as conspecific with C. seminuda [10,15,[31][32][33]. We follow the view of Vellinga (1987) [15], who did not use the name C. sistrata because the description in the protologue is too general and Fries made the concept of this species even more ambiguous [30,32,34,35]. As with C. sistrata, several names have been treated as synonyms of C. seminuda, namely: Lepiota sororia, L. seminuda f. minima, and L. rufipes. Different treatments of those names and different characteristics used in the circumscriptions of species in the C. seminuda complex are discussed below.
The size of basidiomata varies within the C. seminuda complex. Some authors consider basidiomata size as an important characteristic for distinguishing Cystolepiota species. Lepiota seminuda f. minima and Lepiota sistrata f. minima (both Nom. inval., Art. 39.1, Melbourne) are different from C. seminuda because of the smaller pileus (8 mm in diam) and the more slender stipe (<1 mm in diam) [72]. However, based on this study, these dimensions are within the morphological variability of C. seminuda. In contrast, Huijsman described Lepiota sororia, which was considered a synonym of C. seminuda by Vellinga [21,33], with a pileus of 12-30 mm diam and a stipe measuring 30-70 × 1-3 mm, which is larger than that determined for C. seminuda in this study.
The descriptions of basidiospore size and shape of C. seminuda are inconsistent in different studies. Although the spores are basically elliptical, some authors have noticed variations in spore shape [21,66]. Vellinga (1987) described the spores as ellipsoid to oblong and measured 3.5-5.0(-5.5) × 2.0-3.0 µm with a Q value of 1.35-2.1(-2.2); the spores Yang (2019) described were broadly ellipsoid to ellipsoid and measured 3.5-4.5 × 2.5-3 µm, Q value is 1.25-1.67(-1.80). Thus, the specimens studied in the two articles may belong to different species, C. pseudoseminuda, C. seminuda or further species of the C. seminuda species complex. The two species described in this paper, C. pseudoseminuda and C. seminuda, also vary in size and shape of basidiospores. Based on measurements by Vellinga (1987) [21], the holotype of Lepiota sororia has longer and narrower basidiospores: (4.2-)4.6-5.3(-5.4) × (2.1-)2.3-2.9(-3.0) µm, Q = (1.65-)1.7-2.1(-2.2). Vellinga (2010a) also studied the holotype of Lepiota rufipes [36], a species described from the USA, which she treated as a synonym of C. seminuda. The basidiospores of L. rufipes measured 4.1-4.9 × 2.4-2.8 µm, Q = 1.65-2.05, Q m = 1.85, is larger than the basidiospores of C. seminuda measured in this study. Some collections regarded as C. seminuda formed even larger basidiospores [20,21,73]. Although general differences in shape and size of spores of C. pseudoseminuda and C. seminuda were determined in this study, considerable variations among different fruit bodies were observed in both species ( Figure 6). Thus, when describing new species from the C. seminuda complex, as many specimens as possible from different locations need to be examined.
In most previous studies, basidiospores of C. seminuda are described as smooth-walled, but in some as smooth-walled or with insignificant warts [12,66]. Based on molecular data, we revealed that the C. seminuda complex consists of two clades. Observations of basidiospores with SEM suggest that species of Clade I form ornamented basidiospores, while species of clade II form smooth-walled basidiospores (Figures 1 and 2). Thus, the ornamentation of basidiospores is a key characteristic to distinguish species within the C. seminuda complex. In addition, the basidiospores of the type specimen of L. sororia have a smooth-walled surface ( Figure 4H,I), indicating that this species may belong to clade II.
The basidiospores of all specimens involved in this study are non-dextrinoid. In contrast, basidiospores of the "C. seminuda" collections from South America were reported as being dextrinoid [20,73]. This could be an indication that these collections also represent different species. However, whether the chemical reactions of basidiospores are useful for identification of species in this complex requires further study.
According to the discussion above, which was based on the examination of accessible specimens and the literature review, the species in the Cystolepiota seminuda complex can be differentiated by a combination of the following characteristics: basidiomata size, spore size and shape, ornamentation of spores, and DNA sequence data.
The species in the C. seminuda complex show different geographic distribution patterns. For example, C. seminuda is widely distributed in the temperate and subtropical zones of the Northern Hemisphere, while C. pseudoseminuda has so far only been collected in the subtropical zones of China. Further species in this complex are so far only represented by specimens from China and from North America, respectively. This indicates that geographic distribution may support the species differentiation within the C. seminuda complex.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof9050537/s1, Table S1: Taxa, vouchers and GenBank accession numbers used in the molecular analyses of the ITS data set; Table S2: Taxa, vouchers and GenBank accession numbers used in the molecular analyses of the four-gene data set; Figure S1. Maximum likelihood tree inferred from subsampled ITS sequences of specimens of Cystolepiota and related genera; Figure S2. Maximum likelihood tree inferred from LSU sequences of specimens of Cystolepiota and related genera; Figure S3. Maximum likelihood tree inferred from rpb2 sequences of specimens of Cystolepiota and related genera; Figure S4. Maximum likelihood tree inferred from tef1 sequences of specimens of Cystolepiota and related genera; Figure S5. Maximum likelihood tree inferred from the combined rpb2-tef1 sequence data set of specimens of Cystolepiota and related genera; Figure S6. Maximum likelihood tree inferred from the combined LSU-rpb2-tef1 sequence data set of specimens of Cystolepiota and related genera.