Observations on Pluteaceae in Vietnam: Four New Species and New Records of Pluteus

Eighteen specimens of Pluteus collected from the tropical forests of Vietnam were studied using morphological and molecular approaches. Pluteus podospilloides, P. semibulbosus, P. chrysaegis and P. septocystidiatus are registered as additional or new records for Vietnam. Four species (P. conformis, P. lucidus, P. subroseus, and P. ornatus) are proposed as new to science, and several other collections (Pluteus sp. 1, P. aff. septocystidiatus, P. aff. pauperculus and P. cf. velutinus) are given an inconclusive taxonomic status for now. The taxonomic positions of all specimens were confirmed using DNA data (nrITS and tef1). Descriptions of the macro- and microscopic features of the studied collections with a discussion of similar taxa are given.


Introduction
This paper is part of a series dealing with the members of the Pluteaceae family in Vietnam [1][2][3]. The genus Pluteus Fr. is very species-rich and widespread from the Arctic to tropical areas [4][5][6][7][8]. It is characterized by basidiocarps without volva, free lamellae, pink or pinkish brown spore print, smooth and inamyloid basidiospores, and inverse hymenophoral trama. According to recent morphological and molecular studies [6,7], three sections within the genus Pluteus were confirmed: sect. Pluteus characterized by metuloid pleurocystidia and a pileipellis organized as a cutis; sect. Celluloderma which included members with the non-metuloid (or absent) pleurocystidia and a pileipellis as a hymeniderm or epithelium; and sect. Hispidoderma which included species with the non-metuloid pleurocystidia and hymeniderm or trichoderm pileipellis. More than 500 species of the genus have been described worldwide [9]. However, the species diversity of this genus has not yet been comprehensively studied in Vietnam. Prior to our study, only four species of Pluteus-P. cervinus (Schaeffer) Kummer, P. minutus Pat., P. plautus (Weinm.) Gillet and P. semibulbosus (Lasch) Quél.-were previously reported so far from the country [10][11][12]. Our recent studies have shown the presence of ten previously undescribed Pluteus species in the mycobiota of Vietnam [2]. With the addition of the species described below, the number of Pluteus species on the studied territory increased to twenty-three.
In this article four new species are described below, detailed descriptions and illustrations of new taxa and new records are given, and the phylogenetic relationships of the identified species and related taxa from the genus Pluteus are analyzed on the basis of molecular data.

Molecular Techniques
For DNA extraction, small fragments of dried basidiocarps were used. The procedure of DNA extraction completely corresponded to the manufacturer's protocol of the Phytosorb Kit (ZAO Syntol, Moscow, Russia). The following primers were used for amplification and sequencing: ITS1F-ITS4B [15,16] for the ITS1-5.8S-ITS2 fragment; and EF1-983F and EF1-1567R for approximately 500 bp of tef1 [17]. PCR products were purified by applying the GeneJET Gel Extraction Kit (Thermo Scientific, Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequencing was performed with an ABI model 3500 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). Raw data were edited and assembled in MEGA X [18].
All microscopic and molecular studies of specimens were carried out at the Center for collective use of scientific equipment "Cellular and molecular technology of studying plants and fungi" (Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg, Russia).

Phylogenetic Analyses
For this study, 17 new nrITS sequences and 13 tef1 sequences were generated. In addition, 197 nrITS sequences and 52 tef1 sequences, including outgroups, were retrieved from the GenBank database (www.ncbi.nlm.nih.gov/genbank/, accessed on 25 March 2023), using the BLAST application and taxonomic considerations. The sequences from the GenBank database and previous studies (with ≥60% query coverage and ≥85-100% sequence similarity) were selected and listed in Table S1. The taxonomic identities of these sequences are given in phylogenetic trees as they appear in GenBank. The sequences were aligned with the Muscle tool incorporated into the MEGA X program.
Phylogenetic reconstructions were performed for three data sets (sect. Pluteus, sect. Celluloderma and sect. Hispidoderma) with Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Before the analyses, the best-fit substitution models were estimated separately for three data sets based on the Akaike Information Criterion (AIC) using the FindModel web server (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html, accessed on 25 March 2023).
For the sect. Pluteus, we performed a phylogenetic analysis based on two genetic markers (nrITS+tef1). The GTR+G model was chosen for nrITS dataset and K80+G for tef1. Hence, we used a partitioned evolution model for concatenated nrITS+tef1 dataset in ML and Bayesian analyses to reconstruct a robust phylogenetic hypothesis. Maximum likelihood analyses were run on RAxML servers, v.1.0.0 (https://raxml-ng.vital-it.ch/#/, accessed on 25 March 2023) with 1000 bootstrap replicates. BI analyses were performed with MrBayes 3.2.5 software [19], for two independent runs, each with 7 million generations under described models and four chains with sampling every 100 generations. To check for convergence of MCMC analyses and to get estimates of the posterior distribution of parameter values, Tracer v1. 7.1 was used [20]. We accepted the result where the ESS (Effective Sample Size) was above 200 and the PSRF (Potential Scale Reduction Factor) was close to 1. Branches with bootstrap support (BS) and posterior probabilities (PP) values greater than or equal to 70% and 0.95, respectively, were considered to be significantly supported.
For sect. Celluloderma and sect. Hispidoderma, phylogenetic reconstructions were based only on nrITS, because of the lack of a satisfactory data set in the GenBank for the second marker tef1. The same GTR+G model was chosen for both nrITS alignments. The ML and BI analyses were conducted similarly as the pattern for section Pluteus.
Pairwise distances between the nr ITS sequences were calculated using p-distance methods and the maximum composite likelihood model in the MEGA X program.
Newly generated sequences were deposited in GenBank with corresponding accession numbers (Table S1).

Phylogeny
The combined data set of nrITS+tef1 sequences for members of sect. Pluteus contained 1244 characters, including gaps (nrITS: 1-660 and tef1: 661-1244). The nrITS data set for sect. Celluloderma contained total 709 characters and for sect. Hispidoderma there were 787 characters, including gaps. The overall topologies of the ML and BI trees were nearly identical for all data sets. Therefore, we present a single phylogram for each section with bootstrap values ≥ 70% and a posteriori probability ≥ 0.90.
The output ML tree of the phylogenetic analyses for sect. Pluteus comprised 83 specimens of Pluteus and 1 specimen of the outgroup (P. atromarginatus (Konrad) Kühner, LE F-289425). Most of Pluteus species included in the analyses form strongly supported clades, which is in agreement with the earlier studies [6][7][8]21,22]. Our phylogenetic results ( Figure 1) confirmed that three Vietnamese collections (LE F-313663, 313664 and 313665) formed a highly supported (BS = 100% and PP = 1.0) monophyletic clade, which is a sister lineage to the P. concentricus clade, and clearly separated from P. hongoi. We describe them below as a new species, P. conformis. Two collections (LE F-313667, and 313668) belongs to P. septocystidiatus/P. albostipitatus clade, and this placement is strongly supported in the tree ( Figure 1). Another studied Vietnamese collection (LE F-313670) was unexpectedly phylogenetically close to several collections from Vanuatu, which represent a species new to science (under description and publication by Jonathan Andrew del Rosario).
The output BI tree of the phylogenetic analyses for sect. Celluloderma comprised 42 specimens (data set was restricted by all available sequences phylogenetically close to the studied collections) and 1 specimen of the outgroup (P. cervinus, REG 13641). As Figure 2 suggests, the studied Vietnamese collections fall in three lineages: three specimens (LE F-347430, 347431, and 313666), representing additional finds of P. podospilloides E.F. Malysheva et O.V. Morozova and are nested in the same clade as the holotype of the species; newly described species, P. lucidus, occupies a sister position to P. seticeps (G.F. Atk.) Singer; and P. aff. pauperculus is close to P. pauperculus E. Horak and is clustered in a well-supported group that harbors species from the romellii clade [23].
The output BI tree of the phylogenetic analyses for sect. Hispidoderma comprised 83 specimens, including the outgroup (P. cervinus, REG 13641). The nrITS sequences of P. cf. velutinus cluster together with P. velutinus C.K. Pradeep, Justo et K.B. Vrinda on a highly supported (PP = 1.00, BS = 97%) clade in all analyses. Two Vietnamese collections (LE F-313653 and LE F-347436), representing known taxa P. semibulbosus and P. chrysaegis (Berk. et Broome) Petch, are nested in the corresponding monophyletic clades with high support (Figure 3). The collection of newly described species, P. subroseus, is grouped together with a single available sequence of P. albidus Beeli in one clade with high support (PP = 1.00, BS = 86%). However, nrITS sequences of these specimens contain a large number of unique characters (14 differences of 632 b. p., genetic distance is greater than 2%) which could be a reason of considering them as separate taxa. Furthermore, significant morphological differences were found between these two specimens. Another new species, P. ornatus, was recovered as a sister lineage of P. fernandezianus Singer and differ from the latter in 36 b. p. of 656 total b. p. (2.7% difference). Because this genetic distance is seemingly congruous to the morphological differences, we regard it as a sufficient species-specific difference and introduce a new species, P. ornatus, based on studied Vietnamese collections. The output BI tree of the phylogenetic analyses for sect. Celluloderma comprised 42 specimens (data set was restricted by all available sequences phylogenetically close to the studied collections) and 1 specimen of the outgroup (P. cervinus, REG 13641). As Figure 2 suggests, the studied Vietnamese collections fall in three lineages: three specimens (LE F-347430, 347431, and 313666), representing additional finds of P. podospilloides E.F. Malysheva et O.V. Morozova and are nested in the same clade as the holotype of the species; newly described species, P. lucidus, occupies a sister position to P. seticeps (G.F. Atk.)  The output BI tree of the phylogenetic analyses for sect. Hispidoderma compris specimens, including the outgroup (P. cervinus, REG 13641). The nrITS sequences of velutinus cluster together with P. velutinus C.K. Pradeep, Justo et K.B. Vrinda on a h supported (PP = 1.00, BS = 97%) clade in all analyses. Two Vietnamese collections (L 313653 and LE F-347436), representing known taxa P. semibulbosus and P. chrysaegis ( et Broome) Petch, are nested in the corresponding monophyletic clades with high sup (Figure 3). The collection of newly described species, P. subroseus, is grouped together a single available sequence of P. albidus Beeli in one clade with high support (PP = 1.0 recovered as a sister lineage of P. fernandezianus Singer and differ from the latter in 36 b p. of 656 total b. p. (2.7% difference). Because this genetic distance is seemingly congruous to the morphological differences, we regard it as a sufficient species-specific difference and introduce a new species, P. ornatus, based on studied Vietnamese collections.    Notes: Pluteus conformis is introduced here based on three specimens, all collected from the northern Province of Vietnam in montane tropical broad-leaved forest. It is mostly similar to P. hongoi Singer, but the presence of distinct longitudinal fibrils on stipe and smaller basidiospores makes it different from the latter (vs. 5.5-9.0 × 4.5-7.0 µm in P. hongoi [8]). Molecular data ( Figure 1) supports the separation of both taxa.
Pluteus concentricus E. Horak comes very close to P. conformis in the molecular analyses. It differs mainly due to the pigmented lamellae edges, the presence of the characteristic concentric bulges on pileus, smaller basidiospores (less than 6.5 µm), and its distribution range, which is restricted to New Zealand [24]. Pileus 10-20 mm in diam., when young hemispherical or campanulate, expanding to convex with broad low umbo; slightly hygrophanous, translucently striate to the half of the radius; pale brown (RAL 8025) or beige brown (RAL 8024); surface radially fibrillose. Lamellae free, moderately distant, ventricose, pink, with concolorous even edges. Stipe 30-50 × 2.5-4.0 mm, almost cylindrical or slightly broadened towards base but without bulb, white or light ivory (RAL 1015), glabrous or slightly pruinose at base. Smell indistinct, taste not recorded.
Habitat: in small groups, on rotten wood.  MB 848250. Etymology: the specific epithet "conformis" (similar) emphasizes the similarity with P. hongoi and the rest of the species from the P. cervinus complex, for which morphological diversification of species is very difficult.
Diagnosis: differs from P. hongoi as it is characterized by the distinct longitudinal fibrils on stipe and smaller basidiospores.
Holotype Notes: Pluteus conformis is introduced here based on three specimens, all collected from the northern Province of Vietnam in montane tropical broad-leaved forest. It is mostly similar to P. hongoi Singer, but the presence of distinct longitudinal fibrils on stipe and smaller basidiospores makes it different from the latter (vs. 5.5-9.0 × 4.5-7.0 µm in P. hongoi [8]). Molecular data (Figure 1) supports the separation of both taxa.
Pluteus concentricus E. Horak comes very close to P. conformis in the molecular analyses. It differs mainly due to the pigmented lamellae edges, the presence of the characteristic concentric bulges on pileus, smaller basidiospores (less than 6.5 µm), and its distribution range, which is restricted to New Zealand [24].
Habitat: in small groups, on rotten wood. Notes: Pluteus septocystidiatus, originally described based on collections from the Republic of Korea and the USA, is characterized by high variability of both macro-and microscopic morphological characteristics [25]. Nevertheless, the main distinguishing features of the species are recognized to be thick-walled and septate pleurocystidia, sometimes septate cheilocystidia, and strongly striate and concave pileus. The Vietnamese specimen that we studied, being phylogenetically identical to the holotype of P. septocystidiatus and included in the same clade with high support (Figure 1), is morphologically different from the previously studied collections of this species. It is characterized by the presence of campanulate and convex pileus and slightly smaller basidiospores (vs. (5.5-)6.0-8(-9) × 5.5-7.5(-8) µm). Based on the above features, our specimen is also difficult to distinguish from the morphologically close species, P. albostipitatus (Dennis) Singer, which, however, is phylogenetically distinct and occupies a sister position on the phylogenetic tree ( Figure 1).
Thus, our finding extends the geographic distribution of the species and the range of variation in its morphological characteristics.
Habitat: in small groups, on rotten wood. Notes: The appearance of the basidiocarps with characteristically concave pilei, the presence of thick-walled pleurocystidia, and basidiospores measuring Lav = 7.2, Wav = 6.2, allow us to treat this collection as P. septocystidiatus with a high degree of certainty. However, according to the phylogenetic analysis, the studied Vietnamese collection occupies a rather distant position on the tree (Figure 1), falling clearly into neither P. septocystidiatus clade nor the P. albostipitatus clade. As emphasized earlier by authors studying P. septocystidiatus [25], and as we can conclude from our own experience of studying Vietnamese collections, both taxa have quite a large variability of morphological characteristics and require further study in regard to clarification of taxonomic distinctive features and geographical distribution.
Thus, at present we cannot definitively attribute our specimen to one or another species. Pluteus sp. 1 (Figure 9). Pileus 15-30 mm in diam., at first convex, then plano-convex or applanate with slight central depression; hygrophanous; translucently striate to the half of the radius; beige (RAL 1001), often with pinkish tint; surface squamulose, with scattered brown beige (RAL 1011) or nut brown (RAL 8011) small squamules, densely located at the center. Lamellae free, moderately distant, slightly ventricose, pink, with concolorous even edges. Stipe 20-30 × 2-3 mm, almost cylindrical or slightly broadened towards base but without bulb, white, shining, glabrous. Smell indistinct, taste not recorded. Notes: The collection studied is similar to several specimens from Vanuatu on the basis of nrITS sequences. They are all located in the same clade with the highest statistical support (BS = 100% and PP = 1.0). The species identified preliminary as Pluteus sp. nov. 1 by J.A. del Rosario in his recent work [26] is characterized by a greyish-brown to hazel appressed-fibrillose, hygrophanous pileus with a white bulbous base stipe, tissues acquiring a faint bluish-grey color when disturbed or handled, subglobose basidiospores with a mean size of 7.3 × 6.6 µm, clavate to utriform cheilocystidia, fusoid to lageniform thickwalled pleurocystidia with apices varying from 2-5 poorly to well-developed hooks/horns, a cutis pileipellis with clavate to somewhat filiform terminal elements, absent caulocystidia, and the presence of clamp connections.
Habitat: in small groups, on rotten wood. Notes: The collection studied is similar to several specimens from Vanuatu on the basis of nrITS sequences. They are all located in the same clade with the highest statistical support (BS = 100% and PP = 1.0). The species identified preliminary as Pluteus sp. nov. 1 by J.A. del Rosario in his recent work [26] is characterized by a greyish-brown to hazel appressed-fibrillose, hygrophanous pileus with a white bulbous base stipe, tissues acquiring a faint bluish-grey color when disturbed or handled, subglobose basidiospores with a mean size of 7.3 × 6.6 µm, clavate to utriform cheilocystidia, fusoid to lageniform thick-walled pleurocystidia with apices varying from 2-5 poorly to well-developed hooks/horns, a cutis pileipellis with clavate to somewhat filiform terminal elements, absent caulocystidia, and the presence of clamp connections.
The Vietnamese specimen almost completely fits the description given, except for the tissue turning blue, which we did not observe in fresh basidiocarps. However, we admit such a tissue reaction in our specimen, which may have been very slow and therefore was not noticed by us. Thus, additional research is needed to clarify this feature.
The species is currently in the publication phase (Del Rosario, personal communication), but in his earlier work [26], the author provided a very detailed description of this taxon and a comparison of the collections from Vanuatu with closely related species. Therefore, in this article, we leave our collection unnamed for the time being.
According to macro-and microscopic features, P. lucidus most resemble P. seticeps, P. podospileus and P. podospilloides. Recent molecular studies have shown that these species Notes: Pluteus lucidus is characterized by diminutive basidiocarps, dark brown granulose-squamulose pileus, totally white stipe, the absence of pleuro-and caulocystidia, utriform or broadly lageniform cheilocystidia, and mixed pileipellis consisting of two types of elements.
According to macro-and microscopic features, P. lucidus most resemble P. seticeps, P. podospileus and P. podospilloides. Recent molecular studies have shown that these species apparently belong to a species complex [2,6], and similar conclusions have been made before [27]. P. lucidus can be considered as a part of P. seticeps species complex, and it occupies a sister position to the P. seticeps clade on the phylogenetic tree ( Figure 2). However, P. lucidus is distinguished from the latter species by the lack of brown fibrils on stipe and utriform or lageniform cheilocystidia, which are sphaeropedunculate or clavate in P. seticeps [27]. Other similar species, P. podospileus and P. podospilloides, all differ from P. lucidus in the possession of pleurocystidia.
The collections analyzed in this study are new findings of this species in Vietnam. Moreover, each time the specimens were found on rotten wood in the mountainous regions. Although the ecology and geographical distribution of the species still need to be clarified, we can already conclude that it is quite common and widespread in the territory of Vietnam.
Pluteus aff. pauperculus (Figures 12 and 13). Pileus 6-8 mm in diam., initially hemispherical or campanulate, becoming convex with broad umbo; not hygrophanous; surface minutely granulose, strongly venose across the entire surface, with a distinct reticulate pattern in the center, sometimes cracked near edge, brown beige (RAL 1011) with honey yellow tint (RAL 1005), ochre brown (RAL 8001), with clay brown (RAL 8003) center; margin not striate, slightly serrated. Lamellae free, distant, ventricose, whitish, becoming pink, with concolorous even edges. Stipe 10-12 × 0.5-1.0 mm, almost cylindrical or slightly broadened towards swollen or subbulbose base, in the upper half pale sulfur yellow (RAL 1016), at the bottom zinc yellow (RAL 1018) or colza yellow (RAL 1021), smooth or longitudinally fibrillose. Smell indistinct, taste not recorded.  Notes: The studied Vietnamese collections match P. pauperculus in almost all characters, except that they are considerably more diminutive than the New Zealand and Western Australian collections on which the species is based [24]. In addition, our specimens have caulocystidia, but they are rare.
From descriptions of modern collections of the species currently available on the Internet (http://iucn.ekoo.se/iucn/species_view/531297/; https://www.gbif.org/es/species/5241379, accessed on 1 March 2023), it is obvious that the variation in macro-characters and substrate preferences may indicate that in this case we are dealing not with a single taxon, but with a complex of species. An additional confirmation of this assumption is the phylogenetic analysis performed (Figure 2). There are two nrITS sequences of this Notes: The studied Vietnamese collections match P. pauperculus in almost all characters, except that they are considerably more diminutive than the New Zealand and Western Australian collections on which the species is based [24]. In addition, our specimens have caulocystidia, but they are rare.
From descriptions of modern collections of the species currently available on the Internet (http://iucn.ekoo.se/iucn/species_view/531297/; https://www.gbif.org/es/species/ 5241379, accessed on 1 March 2023), it is obvious that the variation in macro-characters and substrate preferences may indicate that in this case we are dealing not with a single taxon, but with a complex of species. An additional confirmation of this assumption is the phylogenetic analysis performed (Figure 2). There are two nrITS sequences of this taxon in the GenBank database (MN738621, MN738636), and our specimens do not form a single clade with either of them. Moreover, all sequences of "P. pauperculus" occupy an independent position on the tree, close to species from the /romellii group (P.  (Figure 14).
While comparing P. chrysaegis with a very similar species, P. conizatus var. africanus E. Horak, originally described from Equatorial Africa [32], which was previously differentiated from P. chrysaegis based on the thickened wall of the hymenial cystidia and the size of pileipellis elements, Pradeep et al. [30] concluded that these characteristics were not reliable for separating the two taxa, because they were highly variable even in one specimen. Based on a detailed morphological study of the specimens of both taxa, including the type collection of P. conizatus var. africanus, the aforementioned authors began to consider the latter as synonymous to P. chrysaegis.
Pluteus chrysaegis, in its new conception, is now known from India [28][29][30], Equatorial and West Africa [31,32], the United States and China [33], and from Vietnam. These data indicate that this species has a very wide geographic distribution, correlating with a wide range of variability in micromorphological characteristics. Thus, the Vietnamese specimens we studied were characterized, in contrast to previous observations, by predominantly ellipsoid basidiospores, cheilocystidia with subglobose and globose apexes, the presence of apical projections in pleurocystidia, as well as the presence of quite numerous caulocystidia on the stipe surface. The phylogenetic analysis (Figure 3) showed that the studied collection belonged to P. chrysaegis clade with high statistical support (PP = 1.00, BS = 96%).
Holotype Pileus 80-100 mm in diam., hemispherical at first, then convex, with low broad umbo; not hygrophanous; surface densely squamulose, with erect tapered squamules, densely located at the center and forming pattern of veins radiating in a network from the center to the margin, showing whitish background between; squamules signal brown (RAL 8002), copper brown (RAL 8004) or fawn brown (RAL 8007) at margind, and mahogany brown (RAL 8016), chocolate brown (RAL 8017) or grey brown (RAL 8019) at center; margin even, striate. Lamellae free, moderately crowded, ventricose, pink, with brown, serrulate edges. Notes: Pluteus ornatus is morphologically most similar to known species P. umbrosus (Pers.) P. Kumm. and P. umbrosoides E.F. Malysheva, both distributed in Eurasia. However, detailed morphological studies indicate that P. ornatus can be distinguished from P. umbrosus by its smooth, not squamulose stipe, larger basidiospores (vs. 5.5-6.5 × 4.0-5.0 µm, [5]), and smaller terminal elements of pileipellis. P. umbrosoides differs in lamellae without brown edges, capitate pleurocystidia and smaller basidiospores [22]. Our phylogenetic analyses indicate that all the three species form well-supported distinct clades (Figure 3). The nrITS sequence of the new species is grouped together with one designated as P. aff. fernandezianus from Brazil (OM060370, [34]). However, the latter species, originally described from Chile [35], differs significantly from P. ornatus in its very small basidiocarps (with pileus not exceeding 2 cm), smaller and differently shaped pleurocystidia, and shorter elements of pileipellis [34,35]. A comparison of the structure of the nrITS sequences also showed their strong difference from each other (see phylogeny section).
Two nrITS sequences (MN738654 and MN738677), labeled as P. decoloratus E. Horak from New Zealand, form a highly supported close sister clade with the new species and P. albidus ( Figure 3). P. decoloratus, originally described from New Zealand [24], differs in trichoderm pileipellis with much longer terminal cells, shape of pleurocystidia and the absence of caulocystidia. Notes: Pluteus cf. velutinus was recently found in Vietnam [2], and we have now updated the description of the Vietnamese material based on additional collection from a neighboring province in the southern part of the country.
Compared to the previously studied specimen, the new collection differs in the shape of the pleurocystidia, which are predominantly broadly utriform, broadly clavate or spatuliform, usually without apical protrusions, however, other microscopic characters are generally the same. Here, we give an illustration of the new collection ( Figure 19), but provide a combined description based on two studied specimens.
The nrITS sequences of both Vietnamese specimens are almost identical to each other, and on the resulting phylogenetic tree (Figure 3) they form a monophyletic clade, sister to the P. velutinus clade, which includes the holotype. Compared to the previously studied specimen, the new collection differs in the shape of the pleurocystidia, which are predominantly broadly utriform, broadly clavate or spatuliform, usually without apical protrusions, however, other microscopic characters are generally the same. Here, we give an illustration of the new collection ( Figure 19), but provide a combined description based on two studied specimens.
The nrITS sequences of both Vietnamese specimens are almost identical to each other, and on the resulting phylogenetic tree (Figure 3) they form a monophyletic clade, sister to the P. velutinus clade, which includes the holotype.
A more detailed discussion and justification of the inconclusive taxonomic interpretation of the studied material is provided in a previously published article [2].

Discussion
According to our current molecular and morphological studies on the specimens collected from Vietnam, the species diversity of Pluteus in the region is underestimated and many more new species remain to be described. This tropical mountain region of Southeast Asia harbors a rich fungal diversity due to the diverse ecological niches, as revealed by recent studies focusing on other groups of larger fungi in this region, such as the boletoid genera and Entoloma [42][43][44][45][46]. This is probably also true for the genus Pluteus.
Previously, only four species of the genus Pluteus have been reported from Vietnam [10][11][12]. However, research in the last few years has increased this diversity to seventeen species [2]. This study makes it possible to add six more species to the total diversity of the genus.
In this paper, twelve species of Pluteus are detected based on molecular and morphological data. Four species (P. conformis, P. lucidus, P. ornatus and P. subroseus) are described as new to science. Several taxa (P. aff. septocystidiatus, P. aff. pauperculus and P. cf. velutinus) are recognized as separate lineages from known species. The phylogenetic data presented above and inferred from nrITS and tef1 sequences generally support the recognition of species circumscribed by macro-and micromorphological characteristics, and in many cases can be used to delimit clades of taxa with shared morphological features.
Many of the studied species occur in montane tropical broad-leaved forests, mainly in the southern (Dak Nong, Dong Nai, Lam Dong) and northern (Lao Cai, Nghe An) provinces of the country, but there are also some collections found in the central region (Quang Nam Province). Most species described here inhabit decomposed wood (stumps, trunks, and branches) of angiosperms, sometimes sawdust or wooden chips; only P. chrysaegis was found on a living tree in a coffee plantation. Ecology and geographical distribution of all species considered require further study, as many species are known so far only from their type localities, or some species are known due to few scattered finds.
Interestingly, based on phylogenetically confirmed results, P. chrysaegis is widely distributed but rare in the United States, Equatorial and West Africa, India, China and Vietnam. We expected to find P. septocystidiatus, which was originally described from Korea. At the same time, however, many Pluteus collections, emphasizing the high uniqueness of the Vietnamese mycobiota, leave many questions about the origin of species and the biogeographic relationships of the region. Thus, two collections (P. aff. velutinus and P. aff. septocystidiatus) are phylogenetically distinct from the known species, while the findings of Pluteus sp. 1 and P. aff. pauperculus suggest a common origin of some taxa from Vietnam, Australia, and Melanesia. Hence, the historical migration pathways remain to be studied. Therefore, to assess the real species diversity of the genus Pluteus, it is necessary to continue intensive research in this biodiversity-rich region.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof9050584/s1, Table S1. List of species used in the phylogenetic analyses.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Publicly available datasets were analyzed in this study. Those data can be found here: https://www.ncbi.nlm.nih.gov, accessed on 1 April 2023; https://www.mycobank. org, accessed on 1 April 2023.