A new species and new records species of Pluteus from Xinjiang Uygur Autonomous Region, China

Xinjiang Uyghur Autonomous Region in China embraces a unique geographical and ecological environment, and the macrofungi represent a rich resource. However, few studies on the genus Pluteus have been reported from Xinjiang. In 2021, the macrofungal resources in Xinjiang were surveyed, and 10 specimens belonging to the genus Pluteus were collected. Based on the morphological study and molecular analysis, three species were recognized, P. aletaiensis, P. brunneidiscus, and P. hongoi. Pluteus aletaiensis is proposed as a new species. It is characterized by its bright yellow lamellae and stipe, brittle texture, subfusiform to vesicular pleurocystidia, with short pedicels to broadly lageniform to obtuse at apices, a hymeniderm pileipellis, containing dark brown intracellular pigment, and it grows on the ground. Pluteus brunneidiscus, a new record to China, is characterized by uneven, smooth, grayish brown to brown pileus, with an entire margin, and pointed or flatter apices intermediate cystidia, without apical hooks. Pluteus hongoi, a new record to Xinjiang Uyghur Autonomous Region, China, is characterized by the apical hook’s structure (commonly bifid) of pleurocystidia. The nuclear internal transcribed spacer (nrITS) and translation elongation factor 1-alpha (TEF1-a) region were used for the molecular analysis. Phylogenetic trees were constructed using both the maximum likelihood analysis (ML) and Bayesian inference (BI). Detailed descriptions of the three species are presented herein. Finally, a key to the list of eight species of the genus Pluteus knew from Xinjiang is provided.


INTRODUCTION
Genus Pluteus (Fr.) Quél. was established by Fries in 1836 and belongs to Basidiomycota, Hymenomycetes, Agaricales, Pluteaceae. The genus is distinguished from other agarics by the main features of the free and white, pink or pinkish brown lamellae, a pink spores synonym of p. variabilicolor Babos (Ševčíková & Dima, 2021). Xie, Wang & Wang (1986) recorded seven species of the genus Pluteus in the Changbai Mountain area in Illustrations of Agarics from Changbai Mountains, Mao (2000) recognized 12 species of genus Pluteus in China in the Macrofungi in China, Li & Bau (2003) reported five species of genus Pluteus in Changbai Mountain area in Mushrooms of Changbai mountain in China; Zhuang et al. (2005) included 15 species of genus Pluteus in Fungi of Northwestern China. In recent years, many new and newly recorded species in China have also been published (Xi et al., 2011;Hosen et al., 2018Hosen et al., , 2019Hosen et al., , 2021. Although the Xinjiang Uyghur Autonomous Region has a unique geographical and ecological environment, only five Pluteus species have been reported from Xinjiang, and many resources need to be clarified. P. thomsonii (Berk. & Broome) Dennis. was reported from the Jengish Chokusu (Mao, Wen & Sun, 1978). Wang & Zhao (1997) reported P. cervinus (Schaeff.) P. Kumm. from the Central Tianshan Forest Region. Mao (2000) recorded P. umbrosus (Pers.) P. Kumm. from Xinjiang. Zhao (2001) recognized P. cervinus, P. leoninus (Schaeff.) P. Kumm., and P. pellitus (Pers.) P. Kumm. from Xinjiang.
We conducted a preliminary investigation of the taxonomy of Pluteus species in Xinjiang. The goal of the present study was to provide an annotated list of all species recorded, describe one new species, list newly recorded species from China and newly recorded species from Xinjiang, give detailed descriptions and illustrations of three species, and clarify the phylogenetic relationships of the revealed species and related taxa from the genus Pluteus based on morphological and molecular studies.

Site description
Xinjiang Uygur Autonomous Region is located in the hinterland of the Eurasian continent of northwestern China. For example, there are distinctive landforms, including the world's second-highest peak. It is surrounded by high mountains, resulting in early dryness and precipitation varies significantly among regions. There are continental cold temperate zone cold climate, temperate zone continental monsoon climate, temperate zone continental arid climate, and alpine climates from north to south (Yao et al., 2022).

Collection and morphological studies
Photos of fresh basidiomata were taken in the field, scientifically fully reflecting the growth environment and the characters, including the shape of the pileus, the color of lamellae, and color codes followed by Munsell Soil Color Charts (Munsell, 2009). The size of basidiomata was measured when fresh, and detailedly recorded the macroscopic characteristics, such as shape, size, and color of the pileus, odor, color, and density of lamellae. A small portion of fresh context and lamellae was dried on silica gel and used for DNA extraction. Fresh basidiomata were dried at 40-50 C using an electric drier and preserved at the Herbarium of Mycology of Jilin Agricultural University (HMJAU). The observation of microstructure characteristics were based on dry 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). The morphological descriptions follow Largent, Johnson & Watling (1977). As previously described in Rao et al. (2021b), data were collected. 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. 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.
Research methods of molecular systematics DNA extraction, PCR amplification, and sequencing The total DNA of the specimens was extracted by the new plant genomic DNA extraction kit from Jiangsu Kangwei Century Biotechnology Limited Company. The amplification primers of the ITS nrDNA (ITS) regions were ITS1 and ITS4 (White et al., 1990), and TEF1-a regions were EF1-983F and EF1-1567R (Rehner & Buckley, 2005). The amplification reactions were carried out in a 25 ml system. The total amount of PCR mixed was as follows: dd H 2 O 13.5 ml, 10 × Taq Buffer 5 ml, 10 mM dNTPs 1 ml, 10 mM upstream primer 1 ml, 10 mM downstream primer 1 ml, DNA sample 2 ml, 2 U/mm Taq Polymerase 1.5 ml. The cycle parameters were as follows: 4 min at 94 C; 30 s at 94 C, 40 s at 53 C, 1 min at 72 C for 36 cycles; 10 min at 72 C; storage at 4 C (Xu, 2016). The PCR product was subjected to 1% agarose gel electrophoresis. The purified PCR products were sent to Sangon Biotech Limited Company (Shanghai, China) for sequencing using the Sanger method. The sequencing results were clipped with Seqman 7.1.0 (Swindell & Plasterer, 1997) and then submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/).

Data analysis
Based on BLAST results, morphological similarities, and the related articles (Menolli, Asai & Capelari, 2010;Justo et al., 2014;Menolli, Justo & Capelari, 2015;Rao et al., 2021a;Ševčíková et al., 2022), ITS and TEF1-a sequences obtained and related to these samples are listed in For the ITS dataset, the 'auto' strategy and normal alignment mode of MACSE V2.03 (Ranwez et al., 2018) and MAFFT (Katoh & Standley, 2013) were used for sequence alignment, then manually adjusted in BioEdit v7.1.3 (Hall, 1999). ModelFinder (Kalyaanamoorthy et al., 2017) selected the best-fit models using the Bayesian information criterion (BIC). 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 variance <0.01 and terminated the calculation. The evolutionary tree was followed up with Figtree v1.4.
For the ITS + TEF1-a dataset, sequence alignment was performed for ITS and TEF1-a using the "automatic" strategy of MACSE V2.03 (Ranwez et al., 2018) and MAFFT (Katoh  & Standley, 2013) and normal alignment mode, respectively, and then manually adjusted in BioEdit v7.1.3 (Hall, 1999). Afterward, ITS and TEF1-a sequences were combined using PhylosuitV1.2.2 (Zhang et al., 2020). ModelFinder (Kalyaanamoorthy et al., 2017) selected the best-fit models using the Bayesian information criterion (BIC). 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 variance <0.01 and terminated the calculation. The evolutionary tree was followed up with Figtree v1.4.

Nomenclature
The electronic version in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants.
Hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix "http://www.mycobank.org/MycoTaxo. aspx?Link=T&Rec=.". The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.

Phylogenetic analyses
In the dataset, 111 sequences derived from two gene loci (ITS and TEF1-a) from 35 samples were used to build phylogenetic trees; nine of these were newly generated, with six ITS sequences and three TEF1-a sequences. The phylogenetic construction performed via ML and BI analysis for the two combined datasets showed a similar topology (Figs. 2 and 3).
Ecology and distribution. Scattered on the ground in the broad-leaved forest (Populus alba var. Note. In our phylogenetic tree (Fig. 2), P. aletaiensis clustered with similar species of section Celulloderma and together with P. siccus P. parvicarpus, P. vellingae, P. sublaevigatus, and P. globiger with high support (Fig. 2). On phylogenetic trees, P. siccus display a close relationship to P. aletaiensis; morphologically, they are distinctly different, P. siccus (Ševčíková et al., 2022) is distinguishable from P. aletaiensis due to its slightly velvety pileus with a greenish hue, and smaller basidiospores (about 4.6-6.0 × (3.5-) 4.5-5.5 µm), the polymorphic cheilocystidia, and grows on decaying wood and geographically distributed in the Russian Far East. P. parvicarpus (Ševčíková et al., 2022) could be distinguished from P. aletaiensis by its sulcate-striate at the margin of pileus, smaller basidiospores (about 4.5-6.0 × 4.2-5.5 µm), solitary on fallen branches of deciduous trees, and geographical distribution in the Russian Far East. P. aletaiensis and P. vellingae (Ševčíková et al., 2022) are both with yellow-brown to brown pileus and similar basidiospores. P. vellingae has broadly clavate to clavate or ovoid pleurocystidia, and it grows on coniferous or deciduous wood. P. sublaevigatus (Menolli, Asai & Capelari, 2010) differs from P. aletaiensis with its slightly rugulose at the center of the pileus, and translucently striate at the margin, free to subfree lamellae with lamellulae, and gregarious on decaying wood. P. aletaiensis and P. globiger (Singer & Digilio, 1952;Dias & Cortez, 2013) are both smaller pileus (diameter not more than 23 mm), but the lamellae of P. aletaiensis are bright yellow to yellowish, while P. globiger are greyish orange; on the other hand, P. globiger has ventricose of the cheilocystidia.
Ševčíková et al. obtained the molecular sequence of P. sternbergii (ON864116) and verified the position of P. sternbergii in the phylogenetic tree, not in the romellii clade, but in the cinereofuscus clade, therefore, selected collection PRM 154258 as the epitype of P. sternbergii. (Ševčíková et al., 2022). Morphologically, the basidiospores, basidia, pleurocystidia, and stipitipellis of P. aletaiensis and P. sternbergii are similar, while P. sternbergii grows in the stump of Populus.
Ecology. Solitary or gregarious, growing on decayed wood (Betula, Umbellularia, Populus) or the humus layer under hardwoods or conifers in summer.
Distribution. Canada, Russia, USA, Turkey Kaygusuz et al., 2021), Note. Pluteus brunneidiscus and P. washingtonensis Murrill were first reported from USA (Murrill, 1917). However, Singer (1956), and Banerjee & Sundberg (1995) suggested that these two species may be the same. Singer (1986) cited P. washingtonensis as "probably conspecific with P. brunneidiscus" and with only one difference in the size of spores; besides, the spores of P. washingtonensis (about 6.5-9.6 × 5.3-7.1 µm) are slightly larger. Banerjee & Sundberg (1995) described the terminal elements on the pileipellis of P. brunneidiscus and P. washingtonensis as "versiform." However, Justo & Castro (2007a) believe that the terminal elements were difficult to observe in the type collections.
In modern collections, the shape of these elements was variable within the same basidiocarp and with the same range of variation observed. Therefore, they treated P. washingtonensis as synonymous with P. brunneidiscus. P. brunneidiscus in Europe was firstly described by Justo & Castro (2007a). In our study, P. brunneidiscus is reported as a new record in China. In our phylogenetic analysis, P. brunneidiscus gathered into sect. Pluteus, with two other species-P. shikae Justo and E.F., P. kovalenkoi E.F.-in clade brunneidiscus. These three species can be distinguished from molecular data. Morphologically, many of their characters are rather variable, such as basidiospores and pleurocystidia with developed apical hooks. However, P. kovalenkoi can also be distinguished from P. brunneidiscus by the shape of pleurocystidia . We also compared other characters, as detailed in Table 2 Kaygusuz et al., 2021).
Pluteus hongoi was easily confused with P. cervinus. The pileus of P. cervinus (Murrill, 1917;Justo et al., 2014) was very variable in colors (brown, gray-brown, orange-brown, white), aspect of the pileus (with or without conspicuous squamules and radial fibrils) the stipe often had longitudinal and brown, or gray-brown fibers or scales, and the apical hooks structure (commonly entire) of the pleurocystidia. However, these features are variable and cannot be easily expressed on a basidiocarp . But they can be distinguished by sequences analyses.  decaying material of specific tree species or decaying material in all tree species, growing on decaying material of live trees/fallen wood/dead trees/stumps, etc., indirectly having some association with trees, growing on the ground/humus, not associated with trees. In the future, subsequent studies of Pluteus will require additional data to further support it.