Four new species of Cystolepiota (Agaricaceae, Agaricales) from northeastern China

Cystolepiota is a tiny lepiotaceous fungi. During our 3 years fieldwork, we found four new species of Cystolepiota from northeastern China. A phylogenetic study of a combined dataset of ITS+nrLSU+rpb2+tef1-α revealed that Cystolepiota changbaishanensis and Cystolepiota hetieri are sister clades; Cystolepiota hongshiensis belongs to Cystolepiota seminuda complex; Cystolepiota luteosquamulosa formed a clade not closely related with any other; Cystolepiota nivalis and Cystolepiota sp. (HMJAU68235) formed a sister clade. All new species are provided with descriptions, photos of the basidiomata, and colored illustrations of the microstructures. A key for the identification of Cystolepiota species from China is also presented.


Introduction
The humus layer of the forest harbors a myriad of tiny mushrooms that often go unnoticed, including Cystolepiota Singer.The genus Cystolepiota was erected by Singer in Singer and Digilio (1952) to accommodate small lepiotaceous fungi species with epithelioid squamules and inamyloid, non-dextrinoid basidiospores.Then Singer and Clémençon (1972) divided this genus into two sections: C. sect.Pseudoamyloideae Singer and Clémençon for species showing basidiospores with dextrinoid reactions in Melzer's reagent (e.g., Cystolepiota icterina F. H. Møller ex Knudsen), and C. sect.Cystolepiota Singer with non-reactive basidiospores in Melzer's reagent [e.g., Cystolepiota fumosifolia (Murrill) Vellinga].In addition, Bon (1993) established a new genus, Pulverolepiota Bon, which includes species with the pileus covered by squamules formed by elongated and inflated cells, lacking clamp connections, and basidiospores slowly turning red brown in Melzer's reagent [e.g., C. petasiformis (Murrill) Vellinga = Pulverolepiota petasiformis (Murrill) H. Qu, Damm and Z. W. Ge].However, Vellinga treated this genus as a section of Cystolepiota (Vellinga and Huijser, 1998).Recently, Qu et al. (2023) found that Pulverolepiota formed a unique branch independent of the core members of Cystolepiota, and revived Pulverolepiota as a genus.Nevertheless, much controversy remains in the academic community regarding the boundaries of Cystolepiota.
Like Cystolepiota, the Melanophyllum Velen.(Velenovský, 1921) basidiomata pileus is also composed of loosely arranged spherical cells and hyphae.However, Melanophyllum has basidiomata with lamellae of a distinctive color, reddish or greenish, and it has ornamented basidiospores.Vellinga (2003) observed that Melanophyllum, although it has colored spores, belonged to the same evolutionary branch as Cystolepiota, instead of being related to Agaricus L., as proposed by Singer (1986).Qu et al. (2023) confirmed that Melanophyllum and Cystolepiota form a monophyletic group and that some species in the C. seminuda complex 2 Materials and methods

Morphological studies
Specimens were collected from northeastern China between June and September of 2021-2023.Photos of the basidiomata were taken during field collection and the macroscopic characteristics of the basidiomata were recorded, with color descriptions based on Kornerup and Wanscher (1963).Then specimens were dried using silica gel, and the specimens are currently stored in the Herbarium of Jilin Agricultural University (HMJAU).The colored illustrations are based on photos of the basidiomata in the field collection.Light microscopy (LM: Olympus CX33) was used to observe the microstructure, the samples were rehydrated in 5% KOH, and OPLENIC Pro v1.92 was utilized to measure the microstructure.Among them, in basidiospores, in the notation [n, m, p], n represents the number of basidiospores measured, of m basidiomata of p specimens, and a − b × c − d represents the minimum − maximum value of the length × width of the basidiospores, and Q = a − b represents the minimum − maximum value of the length/width of the basidiospores, Q v = represents the average of the length/width of the basidiospores.Descriptive terminology follows terms proposed by Vellinga (1988) and Clémençon (2012).
To know accurately whether the basidiospore's surface is ornamented or not, we treated the lamellae with gold spray after placing them on a carrier stage and observed the basidiospore surface under a scanning electron microscope (SEM: Zeiss MERLIN, EHT1-5Kv).
In addition, Congo red was used to stain the structures for better observation.To determine whether the basidiospores wall was amyloid or not, Melzer's reagent was employed.Cresyl blue was used to detect the metachromatic reaction, while cotton blue revealed whether the basidiospores were cyanophilous.

Phylogenetic analyses
The ITS phylogenetic tree (Figure 1) included 154 sequences with 693 characters, and the multi-DNA regions phylogenetic tree (Figure 2) 133 sequences with 2,765 characters, including 133 ITS sequences, 54 nrLSU sequences, 30 rpb2 sequences, and 26 tef1-α sequences.BI and ML analysis resulted in a very similar topology, so the ML tree is provided in this study (Figures 1, 2 1, 2), which is not described here for the moment because only one specimen is available for observation.MycoBank number: MB 851389 (Figures 3, 4).Diagnosis: The identifying features of C. changbaishanensis are that the pileus is dirty white to cream, with pulverulent, granulose or subpyramidal squamules, cream, greyish orange, light brown, brown; pileus and pileus context becoming greyish orange to brown after drying; lamellae white to cream, turn grayish orange to light brown when drying; basidiospores obscure small warts visible under SEM; and cheilocystidia lageniform to broadly lageniform.

Taxonomy
Etymology: The species epithet "changbaishanensis" is derived from the name of the mountain where the material was collected.
In the phylogenetic trees (Figures 1, 2), Cystolepiota changbaishanensis and C. hetieri are sister clades, but the lamellae of the latter's basidiomata did not change color after drying and exhibited pleurocystidia.
Diagnosis: C. hongshiensis is distinguished from other Cystolepiota species by its hemispherical to convex pileus, with granulose to warty squamules, white to cream, and rough basidiospores under SEM.Its ITS, LSU, rpb2, and tef1-α sequences are different from those of other species.
Etymology: The species epithet "hongshiensis" is derived from the name of the park where the material was collected.
MycoBank number: MB 849380 (Figures 7, 8).Diagnosis: C. luteosquamulosa is distinguished from other Cystolepiota species by its light yellow to greyish yellow pileus, with greyish yellow to dark yellow warty to subpyramidal squamules, light reddish brown stipe with white to light yellow floccose squamules, and pleurocystidia and cheilocystidia absent.
Key to species of Cystolepiota in China.

Discussion
In both phylogenetic trees, the two species in Melanophyllum belong to Cystolepiota.Because a Melanophyllum (Velenovský, 1921) description was published earlier than that of Cystolepiota (Singer and Digilio, 1952), Melanophyllum should be used as the legal name for these two genera (Turland et al., 2018).However, the number of species in Cystolepiota is significantly higher than that in Melanophyllum.If merged, numerous synonyms can be produced.We thus applied Cystolepiota s.l. to both genera.We also found that no molecular data are available for many of the species in Cystolepiota.In particular, no molecular data is available for the model species C. constricta.For most species, the available molecular data is limited to ITS sequences.Other DNA regions (LSU, rpb2, tef1-α) have been sequenced for very few species.More detailed and comprehensive sampling is required to facilitate further studies of Cystolepiota s.l..The macroscopic and microscopic characteristics of many Cystolepiota species overlap.Molecular data and phylogenetic analyses are thus necessary to identify Cystolepiota species with similar morphological features.For example, the species in Cystolepiota seminuda complex are morphologically similar.Cystolepita hongshiensis is a novel species examined in this study.Morphologically, Cystolepita hongshiensis and C. pseudoseminuda are similar, and require further characterization using molecular data and phylogenetic analyses.Among the Cystolepiota seminuda complex, we also examined C. aff.seminuda 1 and C. aff.seminuda 2. We found no morphological differences between them (Table 2).In two phylogenetic trees (Figures 1, 2), C. aff.seminuda 1 and C. aff.seminuda 2 are genetically distant from C. seminuda.We are thus temporarily treating it as a cryptic species.
We also found that Cystolepiota species morphology did not correspond to phylogeny.Cystolepiota bucknallii, C. rhodella, and C. icterina in Cystolepiota sect.Pseudoamyloideae did not form a clade in the phylogenetic tree.They each formed a distinct long clade.Cystolepiota luteosquamulosa with basidiospore ornamentation did not form a clade with other species displaying basidiospore ornamentation.These require further research.
This study describes four new species belonging to Cystolepiota from northeast China.They are well-supported by molecular phylogenetic and morphological evidence.Thereby enriching the species diversity of Cystolepiota in China.In the phylogenetic trees (Figures 1, 2), Cystolepiota sp.(HMJAU68234, HMJAU68235, HMJAU68257), Melanophyllum sp.(HMJAU68255), and Pulverolepiota sp.(HMJAU68236) are just one specimen.The findings of this study indicate the potential existence of undiscovered species in northeast China needs to be studied further.

FIGURE 1
FIGURE 1Maximum likelihood tree based on ITS sequences.New sequences generated for this study are in bold, new species sequences generated for this study are in purple bold.Bootstrap support (BS) values ≥70%, and Bayesian posterior probability (PP) values ≥0.95 are indicated on branches (BS/PP).Echinoderma asperum and E. flavidoasperum are used as outgroup.

FIGURE 2
FIGURE 2 Phylogenetic tree of Cystolepiota obtained from the maximum likelihood analysis (ML) based on ITS, nrLSU, rpb2, and tefl-a sequence data.New sequences generated for this study are in bold, new species sequences generated for this study are in purple bold.Bootstrap support (BS) values ≥70%, and Bayesian posterior probability (PP) values ≥0.95 are indicated on branches (BS/PP).Coprinus comatus and Cop.sterquilinus are used as outgroup.

TABLE 1
GenBank accession numbers, geographical origins, and voucher numbers of taxa used for the phylogenetic analyses.