A review of Cystoderma (Agaricales/Basidiomycota) from China with four new species and two new records

ABSTRACT Cystoderma comprises the species with heavily universal veil remnants on basidiomes, weakly to strongly amyloid basidiospores, evanescent floccose-scaly ring zone or persistent membranous ring, which were often encountered in forests and grassland. However, they were less studied than other mushroom groups mainly because of its unclearly phylogenetic position. In this study, we gathered 16 specimens from Southwest and Northwest China, where were the richest biodiversity areas in China, and produced their ITS and nrLSU sequences. The related morphological examinations and molecular phylogenetic analysis showed they belonged to eight species, of which four were new species, and named as Cystoderma lilaceum, C. pseudoamianthinum, C. rugosolateritium, C. subglobisporum, and of which two were new records from China, and they were C. granosum, C. subvinaceum. New species and new records were described in details and discussed with other species. This study not only showed the novel geographical distributions as well as high species diversity of Cystoderma in China, but also provided more research data for the further studies in mushrooms systematics.


Introduction
The genus Cystoderma Fayod typified by C. amianthinum (Scop.) Fayod was established in 1889 (Fayod 1889), which belongs to Agaricales. Most species of this genus were saprotrophic and often gregariously growing on mosses, litters, or rotten wood under coniferous trees. Cystoderma species were often reported from temperate region (Smith and Singer 1945;Heinemann and Thoen 1977;Saar 2003;Saar and Laessoe 2006). Up to now there were 27 species of Cystoderma have been reported from worldwide.
Cystoderma species were morphologically distinguished by basidiomes usually with heavily universal veil remnants, with evanescent floccose-scaly ring zone or well-developed persistent membranous ring and weakly to strongly amyloid basidiospores (Saar 2012). In the field, Cystodemella was the most similar genus to Cystoderma in morphology as they were sharing the small to medium sized basidiomes, pileus sometimes with radially wrinkles, granulose to finely scales, and margin usually with veil remnants. Further identifications need micromorphological characteristics and molecular analyses. Cystodermella was characterised by inamyloid basidiospores, an evanescent floccose-scaly ring zone and presence of cheilocystidia and pleurocystidia (Saar 2012).
Historically, this genus was first placed in the tribe Lepiota of Agaricus by Fries (1821). Later, Smith and Singer raised it as a morphologically distinct genus and assigned related taxa into two sections: Granulosa (with inamyloid basidiospores) and Amianthina (with amyloid basidiospores) (Smith and Singer 1945). In 1962, Singer replaced the name of section Amianthina by section Cystoderma (Singer 1962). The first molecular study which related Cystoderma was published in 2002, which focused on the phylogenetic study of Agaricales (Moncalvo et al. 2002). In this study, three Cystoderma speices were involved, and the results showed this genus was not monophyletic. Later, Harmaja considered the results of the phylogenetic analyses in Moncalvo et al. (2002) and divided Cystoderma into two genera, one was kept the name of Cystoderma and another was named as Cystodermella (Harmaja 2002;Moncalvo et al. 2002). Presently the belonging of Cystoderma was still not clearly yet. Based on morphological characteristics, Singer (1986) proposed Cystoderma as a member of Tribus Cystodermateae of family Agaricaceae, which was characterised by free lamellae. However, because none of the species of Cystoderma has free lamellae, some researchers thought it should be placed in family Tricholomataceae, which was with attached lamellae (Heinemann and Thoen 1977;Kühner 1980;Bas 1988). In a molecular phylogenetic study of Agaricales, Cystoderma clustered together with Cyathus and Crucibulum, as a moderately supported sister clade of Agaricaceae in the Agaricoid clade (Matheny et al. 2006). Later, Garnica et al. (2007) found Phaeolepiota had a closer position to Cystoderma, then, they both clustered with Crucibulum and Cyathus in a wellsupported lineage. Unfortunately, both studies only used the type species of Cystoderma. Based on such disagreement, in the most recently outline of Basidiomycota, Cystoderma was placed as incertae sedis in Agaricales (He et al. 2019).
In China, four species of Cystoderma have been reported, C. amianthinum (Scop.) Fayod, C. carcharias (Pers.) Fayod., and C. aureum (Matt.) Kühner & Romagn., C. japonicum Thoen & Hongo. (Tai 1979;Mao 2000;Li et al. 2015;Liu et al. 2021). In this study, together with 16 specimens collected from China, we made a comprehensive phylogenetic study of Cystoderma with the referenced molecular sequences from worldwide specimens Saar et al. 2009;Saar 2016). Based on the phylogenetic and morphological analyses, we introduced four new species and two new recorded species from China in this paper.

Morphological character examination
Specimens were collected in the field after taking photographs. Odour and colour changes on bruising were recorded at the same time. To avoid mixing or crushing, aluminium foil was used for wrapping. Macromorphological features and chemical reactions of fresh specimens were recorded as soon as possible after returning from the field. Specimens were dried completely with a food drier under the temperature of 50°C, sealed in plastic bags, and deposited in the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS).
Anatomical and cytological features including lamellae, pileipellis, veil remnants, basidiospores, basidia and cystidia were observed. Dried specimens were examined following the protocols of Largent (Largent et al. 1986). Melzer's reagent and 5% KOH are used for staining reaction. More than 20 measurements of microscopic features (spores, basidia and cystidia) were recorded, which include x, the mean of length by width ±SD; Q, the quotient of basidiospore length to width, and Qm, the mean of Q-values ±SD (Largent et al. 1986).

DNA extraction and PCR
DNA was extracted from dried specimens using a Broad-spectrum plant Rapid Genomic DNA Kit (Biomed) according to the manufacturer protocol. Primers ITS4 and ITS5 were used for internal transcribed spacer (ITS), LROR and LR5 for large ribosomal subunit (nrLSU) PCR reactions (Moncalvo et al. 2000). The PCR programmesare followed Li et al. (2019). PCR products were sent to a Biomed Biotechnology commercial company for sequencing.

Phylogenetic analyses
Sequences from Chinese specimens and NCBI GenBank database (Zhao et al. 2007;Saar 2012;Saar et al. 2016;) were used in phylogenetic analyses, and the related GenBank accession numbers were listed in Table 1. Sequences of ITS and nrLSU were aligned by Muscle version 3.6 (Edgar 2004) separately, then manually adjusted to remove ambiguous regions in BioEdit version 7.0.4 (Hall 2007). Maximum likelihood (ML) analysis was performed by RAxmlGUI 1.3 (Silvestro and Michalak 2012) under a GTRGAMMA model with one thousand rapid bootstrap (BS) replicates. Bayesian Inference (BI) analysis was performed by MrBayes v3.2.6 (Ronquist and Huelsenbeck 2003). Six Markov chains were run for 2,000,000 generations and trees were sampled every 100th generation. Burn-ins was determined in Tracer version 1.6 with an ESS value higher than 200 and the remaining trees were used to calculate Bayesian posterior probabilities (PP). The trees were displayed in Fig

Results
There were 44 specimens from 21 Cystoderma species were included in the phylogenetic analyses with an outgroup species Crucibulum leave. Thirty-one sequences were newly generated in this study including sixteen ITS and fifteen nrLSU sequences, which were from sixteen specimens from Northwest and Southwest China. The final alignment comprised a total of 1539 base pairs (bp) including 648 bp of ITS and 891 bp of nrLSU. The phylogenetic tree of ML and MrBaye topology were same based on ITS and nrLSU data. The Maximum likelihood tree was showed in Figure 1 with bootstrap values and Bayesian posterior probabilities indicated on the branches.
In the phylogenetic tree (Figure 1), Cystoderma was monophyletic with fully supported by bootstrap and PP values. Two main clades were recognised, and the basal clade comprised by the new species C. lilaceum and known species C. superbum, another was comprised by the rest sixteen species. Both clades were well supported with BS/PP values of 99/1.0 and 97/0.99 respectively. Among them, C. amianthinum, C. andinum, C. rugosolateritium, C. pseudoamianthinum cluster together with full bootstrap and Bayesian posterior probability values (100/1). The specimen HMAS281432 represent C. subglobisporum form a distinct lineage embedded in the phylogenetic tree.  Figure 2 Fungal Names: FN570856  Macroscopic description: Pileus 5-14 mm in diam, at first conical to convex and finally expanded to applanate, surface dry, granulose, off-white to purple grey, finely covered with fibrillose sometimes, margin at first slightly decurved and appendiculate with white to greyish veil remnants. Lamellae adnexed or adnate, with 1-2 lamellulae, less than 2 mm broad, violent to pale purple, subdistant, at times somewhat ventricose. Stipe 20-35 × 2-3 mm, cylindrical, violent, a purple tinge, with evanescent floccose-scaly ring zone, above the ring zone concolorous with the lamellae, silky striate, below the ring zone densely squamulose, covered with evanescent floccose, concolorous with the pileus, the base slightly swollen. Context up to 2 mm, pale purple. Odour not distinctive.
KOH reaction: strongly black on pileus of dry specimen.
Habit: Gregarious on coniferous or moss, usually in a humid environment.
KOH reaction: reddish brown on pileus of dry specimen.
Habit: solitary on a mixture of moss and fallen leaves in forest.
Holotype: CHINA, Gansu Province, Zhangye County, Qilian Mountains, 23 August 2017, collected by Mao-Qiang He. QL20170293 (HMAS291351) Macroscopic description: Pileus 10-28 mm in diam., convex to plano-convex, rusty tawny, cinnamon at the edge, turning into brick-colour towards the disc, deepen in strongly radially winkles, with darker umbo, margin decurved at first, then applanate to plane with age, appendiculate with remnants of partial veil, veil often erected, white to yellowishbrown. Lamellae creamy white to pure white, adnate to adnexed, subdistant, with 1-2 lamellulae of different lengths, ventricose, 3-5 mm broad. Stipe 38-58 × 4-8 mm, cylindrical, hollow, yellowish brown at first, changing to reddish brown when bruised or cut, with evanescent floccose-scaly ring zone, above the ring zone densely covered fribrious striate, white to yellowish. Below the ring zone, attached with small fibrillous squamules which are similar to the ring zone, scanty towards the base, Context thin, less than 2 mm broad, concolorous with stipe, Odour not distinctive.
Habit: Single or scattered on moss, usually in a wet environment.
Known distribution: Northwest China. Notes: Cystoderma rugosolateritium is characterised by brick-red colour of the disc, deep wrinkles on pileus, erected, whitish to yellowish brown remnants of veil, ventricose lamellae, turning reddish brown when bruised or cut. In the phylogenetic analyses, this new species represented by QL20170293 clustered with C. amianthinum, C. andinum and C. pseudoamianthinum with full bootstrap and high Bayesian posterior probability values (100/1). Among them, C. andinum differed by its larger basidiospores (5.0 -)6.0-7.5(-8.5) × (4.0 -)4.5-5.5(-6.0) μm and pileus without wrinkles, furthermore, its brick-red on pileus fading greyish orange, retaining the darker colour at the margin for some time which could easily distinguish them in the field (Saar and Laessoe 2006). Cystoderma amianthinum form the yellowish buff to pale-yellow pileus, narrowly basidiospore (4.0 -)5.0-6.0(-7.0) × (2.5 -)3.0-3.5(-4.0), which features differed from this new species. Cystoderma pseudoamianthinum could be easily distinguished from this new species by the frosted powderies covered on the basidiomes, and a usually umbonate pileus (Saar and Laessoe 2006).  Figure 5 Fungal Name: FN570859 Etymology: referring to the basidiocarps similar to those of Cystoderma amianthinum Holotype: CHINA, Yunnan Province, Zhaotong County, Dashanbao National Park, 2 August 2015, ZRL20150996 (HMAS255932) Macroscopic description: Pileus 24-50 mm in diam., convex to umbonate when young, becoming campanulate to slightly revolute when well developed. Margin moderately decurved at first and applanate to plane with age, usually appendiculate with remnants of partial veil, surface dry, yellow ochre at the edge, but gradually becoming yellowish brown towards the disc, densely covered with granular-furfuraceous, uneven. Lamellae adnexed, with 1-2 lamellulae of different lengths, up to 5 mm broad, white to pale cream, close to subdistant. Stipe 55-70 × 5-6 mm, cylindrical, hollow, enlarged bulbous at the base, surface dry, with evanescent floccose-scaly ring zone, white to yellowish above the ring zone, and silky striate, below the ring zone, light brown to dark brown towards the base, attached with numerous of small squamules similar to the ring zone. Context white to yellow-brown, 2-3 mm broad. Odour not distinctive.
KOH reaction: Reddish-brown on pileus of dry specimen.
Morphologically, C. aureum, C. japonicum, C. jasonis and C. muscicola were similar to this new species because all of them were yellowish pileus, however, those two known species C. japonicum and C. jasonis have inamyloid basidiospores. (Saar 2012). Furthermore, C. jasonis differs smaller basidiocarp (10-25 mm), and more broadly basidiospore (5 -)6.0-7.5(-9.0) × 3.0-4.5 (Saar 2003). Cystoderma muscicola (Cleland) Grgur is the most similar to this new species that both form pileus with frosted granules, stipe covered with yellowish-buff granules and a definite ring. (Grgurinovic 1997) However, this new species C. pseudoamianthinum usually forms annular crack on pileus which is hardly rugose, Besides, C. muscicola is known to be distributed in Australia (Saar 2012). Macroscopic description: Pileus (18-)30-50 mm in diam., convex to campanulate when young, becoming applanate to plane with age, with a conical papilla on central, surface dry, bright orange brown, greyishbrown, or yellowish ochre, finely covered granulose scales, sometimes with small finely powdery, slightly wrinkled, the margin often separates from the partial veil. Lamellae attached, whitish, with 1-2 lamellulae, close to crowded, ventricose. Stipe 30-70 × 6-12 mm, cylindrical, base enlarged, with a persistent membranous ring, coloured with the basidiocarp, above the ring, whitish to yellowish brown, below the ring, concolorous and with the same granular tissue as the cap. Odour not distinctive.
Habit: Gregarious or caespitose on moss, or sometimes on a mixture of pine cones and pine needle.
Known distribution: Northern America (type locality); Northwest China. Notes: Cystoderma granosum is well characterised by its large orange-brown granular basidiocarp and well-developed persistent membranous ring concolorous with the cap, the colour of stipe surface above the ring whitish at first, then becoming brownish yellow with age (Smith and Singer 1945). In the phylogenetic tree, C. granosum were represented by four specimens from Gansu province of China, and they clustered together with fully support. The phylogenetic tree also showed this species might close to C. chocoanum, C. tuomikoskii and C. carcharias. Compared their morphology, C. carcharias was the most similar species, however, it differed from C. granosum by forming pale pinkish or vinaceous coloured basidiocarp and the thinner stipe 30-70 × 4-8 μm (Saar 2003). Generally, characteristics of these four specimens match quite well with the original description of C. granosum (Smith and Singer 1945). Thus, they were identified them as C. granosum and provided the molecular data for this species. This species was firstly recorded for China.
Cystoderma subvinaceum A.H. Smith & Singer Papers of the Michigan Academy of Sciences 30: 120 (1945) Figure 7 Macroscopic description: Pileus 16-28 mm in diam., conical to campanulate, surface dry, granular, finely covered with small scales, light-pink-brown fading light greyish-brown, darken than the background, usually slightly wrinkled. Lamellae adnate to adnexed, light pink to orange white, close to crowded, Stipe 35-65 × 3-5 mm, cylindrical, with evanescent floccose-scaly ring zone, pink-brown to purple wine coloured, below the ring zone, concolorous and with the same granular tissue as the cap, gradually darken towards the base, gradually turning into tan. Odour not distinctive.
Habit: Caespitose on a mixture of pine needles and moss.
Known distribution: Europe (Type locality); Northern America; Northwest China.
Notes: Cystoderma subvinaceum is characterised by light pink-brown to vinaceous red pileus and the stipe, KOH reaction strongly staining olive green to grey olive, present of amyloid basidiospore. (Hausknecht 1994). In the phylogenetic analysis, our specimen HMAS291342 clustered with other two specimens of C. subvinaceum, and formed a clade with highly supports. Then, C. subvinaceum was sister to C. amianthinum, C. andinum, C. pseudoamianthinum and C. rugosolateritium (Figure 1). Cystoderma superbum was the most phenotypically similar species to C. subvinaceum as both having purplish colour of basidiocarps, However, C. superbum has the darker colour on pileus, such as purplish red to vinaceous purple, weakly grey amyloid except for the suprahilar zone strongly amyloid, KOH reaction reddish brown (Saar 2003). Cystoderma subvinaceum the pileipellis colours olivaceus grey in KOH (Saar et al. 2009). For other purplish coloured species, C. haematites and C. lilaceum, the large basidiospore of C. haematites, or the smaller basidiospores and basidiomes of C. lilaceum could be used to distinguished them  (Huijsman 1956). Based on the morphology and mole-10.5-12.5 × 1.2-1.5 cm). Since the type sequences was not obtained, scientific name C. aureum or Phaeolepiota aurea has always been controversial. Liu et al. (2021) did a research of Squamanitaceae, three Cystoderma species and P. aurea clustered together, they thought P. aurea and C. superbum was not well accommodated this genus because inamyloid fusoid and asperulate basidiospores and a small area of the basidiospore amyloid, however, only four species involved. Saar (2016) supported the inclusion of Phaeolepiota in genus Cystoderma, they perferred Cystoderma aureum with more species based on ITS and nrLSU sequence data. In this study, we used 18 Cystoderma species based on ITS and nrLSU sequence data, the specimen HMAS255933 clusted in Cystoderma aureum and nested within Cystoderma, therefore, we continue to recognise Cystoderma aureum.

Discussion
The genus Cystoderma embraces 30 species in the world, and sequences from 19 species of this genus are available in the public sequence repositories presently. (Saar et al. 2009;Saar 2012;Saar et al. 2016). Based on a combination of morphological and phylogenetic analyses, 16 specimens of Cystoderma from Northwest and Northeast China were identified as eight species including of four new species C. lilaceum, C.
pseudoamianthinum, C. rugosolateritium, C. subglobisporum, two new records C. granosum, C. subvinaceum and four known species C. amianthinum, C. carcharias, C. japonicum and C. aureum. Currently, and so that there are eleven species recorded in China, and they are distributed in Yunnan, Sichuan, Gansu, Inner Mongolia, Heilongjiang provinces and Tibet which is a new distribution of Cystoderma. In most cases, they can be found in forest ecosystems, grassland, mosses, litter and rotten wood under coniferous forest and broad-leaved forest. Therefore, Cystoderma has the widely distribution in China and could adapt variable habitats too.
Cystoderma and Cystodermella the two genera are most similar in morphology in the field, such as small to medium-sized basidiomes, pileus covered radially wrinkled and often granulose to finely scales. However, most Cystoderma species have weakly to strongly amyloid basidiospores, sometimes with a persistent membranous ring and none cheilocystidia and pleurocystidia, while Cystodermella has inamyloid basidiospores, evanescent floccose-scaly ring zone and often having cheilocystidia and pleurocystidia. Among Cystoderma, the morphological characters of different species are also variable, for example C. aureum, C. japonicum, C. texense, C. tricholomodies, total those four species have inamyloid basidiospores which is different from the rest Cystoderma species. Besides, there are six species in total constitute membranous ring, C. subglobisporeum, C. japonicum, C.
aureum, C. carcharias, C. granosum, C. tricholomodies, while the rest species only present a floccose-scaly ring zone. Generally, the accurate delimation of each species in this genus need to combine those different morphological features and molecular phylogenetic analysis.