﻿New species, new records and common species of Pluteussect.Celluloderma from northern China

﻿Abstract Wood-rotting fungi are organisms that can decompose wood substrates and extract nutrients from them to support their growth. They play a crucial role in the material cycle of forest ecosystems. The genus Pluteus plays a significant role in wood decomposition. In this study, the morphology and molecular systematics of the sect. Celluloderma of the genus Pluteus were carried out. Pluteusbrunneodiscus was identified as a new species, along with the discovery of two new records, P.cystidiosus and P.chrysophlebius, and a common species, P.romellii. Pluteusbrunneodiscus is characterized by the brown center of the pileus that transitions to white towards the margins, with the surface cracking to form irregular granules. It is typically found in Populus forests growing on decomposing twigs or wood chips. Line drawings, color photographs, and phylogenetic analyses of related species within the genus Pluteus accompany the descriptions of these four species. The analyses are based on ITS + TEF1-α sequence data. Finally, a key for the twenty species within the sect. Celluloderma of the genus Pluteus, which has been documented in China, is provided.


Introduction
The genus Pluteus Fr., which belongs to the Basidiomycota, Agaricomycetes, Agaricales, Pluteaceae, was established by Fries in 1863.The genus Pluteus is characterized by its free lamellae, pinkish spore print, inverse hymenophoral trama, smooth spherical to ellipsoidal basidiospores, various forms of pleurocystidia, and often cheilocystidia.It is predominantly found on decaying wood and has a global distribution (Vellinga and Schreurs 1985;Singer 1986;Justo et al. 2011aJusto et al. , 2011b)).
Singer further subdivided Pluteus sect.Celluloderma into two subsections based on the composition of the pileipellis: subsect.Mixtini Singer, is characterized by elongated elements, and subsect.Eucellulodermini Singer is characterized by the absence of such elements (Singer 1956;Singer 1958).The molecular phylogenetic studies do not divide the Pluteus sect.Celluloderma into two subsections (Justo et al. 2011b).Some species belonged to the sect.Celluloderma (e.g., P. ephebeus (Fr.)Gillet and related species).Based on their characteristics, species composed of non-metuloid cystidia and a pileipellis as cutis should belong to the sect.Hispidoderma.This is not consistent with the phylogenetic results.Thus, the classification of the two subsections of sect.Celluloderma needs to be further justified.Vellinga and Schreurs (1985) proposed a different taxonomic system to distinguish these species (e.g., P. ephebeus (Fr.)Gillet and related species), dividing the Pluteus sect.Celluloderma into three subsects, Mixtini, Eucelullodermini, and Hispidodermini (Fayod) Vellinga and Schreurs.The latter is characterized by a trichodermal pileipellis or a euhymeniderm consisting of cylindrical to fusiform elements, which are similar to some of the characteristics of the sect.Hispidoderma.Additionally, Schreurs and Vellinga proposed a new group sect.Villosi Schreurs and Vellinga, containing species with a cutis-like pileipellis and non-metuloid (Singer 1958;Singer 1986).The proposed new sections and subsections by Singer (1958Singer ( , 1986)), Vellinga, and Schreurs (1985) lack support from molecular systematic studies (Justo et al. 2011a;Justo et al. 2012).
Recent studies (Minnis et al. 2006;Menolli et al. 2010;Justo et al. 2011aJusto et al. , 2011b;;Vizzini and Ercole 2011) have indicated that sect.Celluloderma includes species characterized by the presence of non-metuloid pleurocystidia and a pileipellis that is either euhymeniderm or epithelioid hymeniderm, composed of short elements, which may or may not be intermixed or not with elongate cystidioid elements (corresponding to Pluteus sect.Celluloderma as defined by Singer 1956Singer , 1958Singer , 1986)), refers to species with a cutis-like pileipellis and non-metuloid cystidia (corresponding to Pluteus sect.Villosi or Hispidoderma sensu Singer p.p.).
In the current investigation, a new species, (P.brunneodiscus), two new records to China, (P.chrysophlebius and P. cystidiosus), and a common species, (P.romellii) are described.Detailed descriptions and illustrations are provided for the four species, along with clarification of the phylogenetic relationships of the identified species and related taxa from the genus Pluteus sect.Celluloderma.

Morphology
In the field, photographs of fresh basidiomata were taken to scientifically and adequately reflect the growing environment and characteristics of the basidiomata, including the shape of the pileus, the color of the lamellae, and Munsell Soil Color Chart was followed for color codes (Munsell 2009).For fresh basidi-omata, we promptly determined the size and recorded in detail the shape, size, color, odor, and other macroscopic characteristics of the basidiomata pileus, lamellae, and stipes.About 15 g of fresh context and lamellae were dried in a Ziplock bag with silica gel and returned to the lab for DNA extraction.Fresh basidiomata were dried at 40 ~ 45 °C (Hu et al. 2022), using a plant drying oven and preserved in the fungarium of Jilin Agricultural University (FJAU).
The observation of microstructural features was based on dried specimens.The dry specimens were rehydrated in 94% ethanol for microscopic examination and then mounted in 3% potassium hydroxide (KOH), 1% Congo Red, and Melzer's Reagent, using a light microscope (ZEISS, DM1000, Oberkochen, Germany).Specifically, the following symbols were used in the description: [n/m/p] indicates that 'n' randomly selected basidiospores from 'm' basidiomata of 'p' collections were measured, 'avl' means the average length of basidiospores, except for the extreme values, 'avw' refers to the average width of the basidiospores, except the extreme values, 'Q' represents the quotient of the length and width of a single basidiospore inside view, 'Qm' refers to the average Q value of all basidiospores ± standard deviation.The dimensions for basidiospores are given as (a)b-c(d).The range of b-c contains a minimum of 90% of the measured values.Extreme values (i.e., a and b) are given in parentheses (Qi et al. 2022).

DNA extraction, PCR amplification, and sequencing
According to the instructions, the total DNA of the specimens was extracted by the new plant genomic DNA extraction kit from Jiangsu Kangwei Century Biotechnology Limited Company, P.R. China.Subsequently, sequences of the internal transcribed spacer (ITS) region, and translation elongation factor 1-α (TEF1-α) were used for phylogenetic analyses.The amplification primers of the nr ITS: ITS1-5.8S-ITS2regions were ITS1F and ITS4/ITS4B (White et al. 1990), and TEF1-α regions were EF1-983F and EF1-1567R (Rehner and Buckley 2005).The amplification reactions were carried out in a 25 µL system.The total amount of PCR mixed was as follows: dd H 2 O 13.5 µL, 10 × Taq Buffer 5 µL, 10 mM dNTPs 1 µL, 10 mM upstream primer 1 µL, 10 mM downstream primer 1 µL, DNA sample 2 µL, 2 U/mm Taq Polymerase 1.5 µL.The cycle parameters were as follows: 5 min at 98 °C; 30 s at 98 °C, 30 s at 55 °C, 1 min at 72 °C for 40 cycles; 7 min at 72 °C; storage at 4 °C (Ševcíková et al. 2022).The PCR product was subjected to 1% agarose gel electrophoresis.The purified PCR products were sent to Sangon Biotech Limited Company, P.R.China for sequencing using the Sanger method.The sequencing results were clipped with Seqman 7.1.0(Swindell and Plasterer 1997) and subsequently deposited in GenBank (https:// www.ncbi.nlm.nih.gov/genbank).

Data analysis
The species that were morphologically similar to new species, newly recorded species, and common species, and have high sequence similarity after blast were selected (Justo et al. 2011b(Justo et al. , 2012;;Menolli et al. 2015;Desjardin and Perry 2018;Hosen et al. 2019;Hosen et al. 2021;Ševčíková et al. 2022;Qi et al. 2022;  For obtaining ITS + TEF1-α datasets of related species, sequence alignment was initially performed for ITS and TEF1-α using the "automatic" strategy and normal alignment mode of MACSE V2.03 (Ranwez et al. 2018) and MAFFT (Katoh and Standley 2013), respectively.Subsequently, the alignments were manually adjusted in BioEdit v7.1.3(Hall 1999).Afterward, ITS and TEF1-α sequences were aligned and combined using Phylosuit V1.2.2 (Zhang et al. 2020).Then, ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fit models using the Bayesian information criterion (BIC).In this case, the Maximum likelihood (ML) analyses were performed in IQTree 1.6.8(Nguyen et al. 2015), and the Bayesian inference phylogenies were performed in MrBayes 3.2.6 (Ronquist et al. 2012) (two parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in.The above software was integrated into PhyloSuite 1.2.2 (Zhang et al. 2020).The ML phylogenetic tree was evaluated using the bootstrap method with a bootstrap value of 1,000 replicates; BI determined that the analysis reached smoothness with a variance of less than 0.01 and terminated the calculation.Finally, the evolutionary tree was followed up with Figtree v1.4.

Phylogenetic analyses
This study's nrITS dataset comprises 93 sequences and 650 characters (gaps included).The TEF1-α dataset comprises 41 sequences and 530 characters (gaps included).The combined nrITS + TEF1-α dataset consists of 134 sequences and 1180 characters, including gaps.Of these, 16 sequences (8 nrITS and 8 TEF1-α) were newly generated in this study (Table 1).The overall topologies of the ML and BI trees were nearly identical for all datasets.
For clarity and brevity, we use the term "strongly supported" for a clade/relation that receives a bootstrap (BS) 90 and a posterior probability (PP) = 1, and "well supported" if it receives a BS 70 and a PP of 0.95.The individual support values are shown in Fig. 1.
Diagnosis.Pluteus brunneodiscus differs from P. tomentosulus by its brown pileus in the middle, transitioning to white toward the margins, and the surface cracks to form irregular granules.It grows in poplar forests (Populus alba var.pyramidalis Bge) with decaying wood branches or chips.
Ecology.Scattered on decaying wood in mixed coniferous forests (Pinus koraiensis Siebold and Zucc).
Note.Ševcíková et al. (2023) elevated Pluteus seticeps var.cystidiosus to P. cystidiosus based on specimens from the USA, Canada, Japan, and Russia.The present study reports P. cystidiosus as a new record in China.There was almost complete overlap in morphological variation between those reported in the present study and the holotype specimen.Both grow in temperate/ cold-temperate forests.However, the basidiospores of the species in the present study were slightly larger, measuring (-5.0) 5.5-6.0 (-6.5) × (-4.5) 5.0-5.5 µm, while those of the holotype specimen were smaller, measuring 4.5-5.5 (-6.2) × 3.5-5.0µm.
Note.Pluteus chrysophlebius was first reported in China.It can be distinguished from other yellow-pileus species such as P. admirabilis (Peck) Peck, P. aurantiacus Murrill, P. melleus Murrill, and P. rugosidiscus Murrill by its yellowish pileus and stipe, as well as its bald pileus texture (Minnis and Sundberg 2010;Malysheva et al. 2016).The phylogenetic analysis also supports the differentiation of species.
Note.Initially, the description of Pluteus romellii was rather vague (Britzelmayr 1891), stating that P. romellii was similar to P. nanus (Pers.)P. Kumm, with spores measuring 6-7 μm, and found growing in the soil of Bavaria.It is now widely acknowledged that P. romellii is characterized by a brown pileus, yellow stipe, and the absence of elongated elements in the pileipellis.This species is placed on the phylogenetic tree in subsect.Eucellulodermini under sect.Celluloderma (Orton 1986;Vellinga 1990;Ševcíková et al. 2023).Here, our description of the P. romellii is consistent with the commonly accepted characterization.Phylogenetic analysis shows that it clustered with the epitype (BRNM 761731) with strongly supported (99/0.98).and non-metuloid cystidia.However, in the ephebeus clade, there are P. ephebeus from Europe and P. riberaltensis var.conquistensis from the USA.These species should be placed in sect.Hispidoderma and classified based on the pileipellis, but molecular results indicate that it belongs to sect.Celluloderma.In the phylogenetic tree, it is the sister group to P. fenzlii, P. mammillatus, and some species have a partial veil.P. brunneodiscus in the ephebeus clade in the present study, which has non-metuloid cystidia and pileipellis as a cutis, shares their views with Vellinga and Schreurs (1985).The phylogenetic tree also exhibits a high level of support.Further research is needed to restore these species to sect.Villosi.

Key to the reported species of Pluteus sect. Celluloderma in China
The presence of a partial veil in P. aurantiorugosus, P. aurantiorugosus var.aurantiovelatus, P. fenzlii, and P. mammillatus suggests that the occurrence or nonoccurrence/ lack of the partial veil in the evolutionary history of Pluteus occurred independently.As stated by Singer states (Singer 1958;Minnis and Sundberg 2010;Justo et al. 2011aJusto et al. , 2011b;;Vizzini and Ercole 2011), this characteristic is homoplasic and unsuitable for the natural classification of these fungi at the supraspecific rank.

Figure 1 .
Figure 1.Phylogenetic tree of the sect.Celluloderma of the genus Pluteus.The best tree from the ML and BI analysis of the nrITS + TEF1-α dataset.The two values of internal nodes respectively represent the maximum likelihood bootstrap (MLBP)/Bayesian posterior probability (BIPP).This study species is in bold and red font.

Table 1 .
Names, collection numbers, reported countries and corresponding GenBank accession numbers of the taxa used in this study.Xiang Qi et al.:New species, newly recorded and common species of Pluteus sect.Celluloderma MycoKeys 104: 91-112 (2024), DOI: 10.3897/mycokeys.104.117841Zheng-Bold fonts are the sequences to be determined in this study.
Malysheva et al. 2023;Ševcíková et al. 2023;Xu et al. 2023), and details of the ITS and TEF1-α sequences of these species are shown in Table1.The ITS and TEF1-α dataset comprised 134 representative sequences that exhibited the highest similarity to Pluteus spp., and two sequences of Volvopluteus michiganensis (A.H.Sm.) Justo and Minnis.as an outgroup.
i. Clade I: This includes the clade we consider to represent P. mammillatus (Longyear) Minnis, Sundb.& Methven from the USA, P. fenzlii (Schulzer) Corriol & P.-A.Moreau from Japan, Slovakia, and Russia, P. halonatus from Brazil.ii.Clade II: Includes only the newly described P. brunneodiscus from China.