Holarctic Species in the Pluteus podospileus Clade: Description of Six New Species and Reassessment of Old Names

We studied the taxonomy of Pluteus podospileus and similar species using morphological and molecular (nrITS, TEF1-α) data, including a detailed study of the type collections of P. inflatus var. alneus, Pluteus minutissimus f. major, and P. granulatus var. tenellus. Within the P. podospileus complex, we phylogenetically confirmed six species in Europe, five in Asia, and eight in North America. Based on our results, we recognize P. seticeps as a separate species occurring in North America, while P. podospileus is limited to Eurasia. We describe six new species and a new variety: P. absconditus, P. fuscodiscus, P. gausapatus, P. inexpectatus, P. millsii, and P. notabilis and its variety, P. notabilis var. insignis. We elevate Pluteus seticeps var. cystidiosus to species rank as Pluteus cystidiosus. Based on the holotype of P. inflatus var. alneus, collections of P. inflatus identified by Velenovský, and several modern collections, we resurrect the name P. inflatus. Based on molecular analyses of syntypes of Pluteus minutissimus f. major and a holotype of Pluteus granulatus var. tenellus, we synonymize them under P. inflatus. We also increase our knowledge about the morphology and distribution of P. cutefractus.

For DNA extraction, small fragments of dried basidiomata were used. Sequences of collections deposited in fungaria BRNM, PC, and PRM were generated by M. Sochor following the molecular methods described by Ševčíková et al. [33]. For Pluteus tenellus var. notabilis (holotypus), the extraction and sequencing were performed following Eyssartier et al. [34]. For collections in LE F, DNA was extracted according to the manufacturing protocol of the Phytosorb Kit (ZAO Syntol, Moscow, Russia); the Estonian lab protocol for TUF collections followed Voitk et al. [35]. The total genomic DNA of the OK and GF collections was extracted in accordance with Kaygusuz et al. [36] and Dovana et al. [37], respectively. The following primers were used for amplification and sequencing: ITS1F-ITS4/ITS4B [38][39][40] for the internal transcribed spacer (nrITS: nrITS1-5.8S-nrITS2) fragment, and EF1-983F and EF1-1567R for part of the translation elongation factor 1-alpha (TEF1-α) [41]. PCR products were purified using the GeneJET Gel Extraction Kit (Thermo Scientific, Thermo Fisher Scientific Inc., Waltham, MA, USA). Raw data were edited and assembled in MEGA 10 [42]. Collections studied by AJ were sequenced using the methodology described in Justo and Hibbett [43]. In the sequence comparisons, the term "evolutionary changes" is used to refer to the number of individual nucleotide differences between sets of sequences being compared to each other. The number of evolutionary changes is given as the minimum number of events, including indels (multiple base indels treated as one change), transitions, and transversions. Only differences shared by all specimens of the same species were counted (missing data for sequences of different lengths were not taken into account). datasets (nrITS, TEF1-α, and nrITS + TEF1-α), two separate phylogenetic analyses were run: maximum likelihood (ML) and Bayesian inference (BI). The ML analyses were run in RaxML 8.2.12 under a GTRGAMMAI model as recommended [49] with 1000 rapid bootstrap (BS) replicates. The BI analyses were run using MrBayes 3.2.7 [50] for 10 million generations under a GTRGAMMAI model with four chains and trees sampled every 1000 generations. The initial burn-in phase was set to 2.5 million generations, and this value was confirmed to be adequate by checking the graphic representation of the likelihood scores of the sampled trees. Additionally, we also confirmed that the standard deviation of split frequencies was <0.05 and that potential scale reduction factor (PRSF) values were close to 1, as detailed in [51]. All analyses were run using resources at the CIPRES Science Gateway [52].

Phylogeny
The nrITS dataset comprises 135 sequences and 741 characters (gaps included). The TEF1-α dataset comprises 60 sequences and 805 characters (gaps included). The combined nrITS + TEF1-α dataset consists of 136 combined sequences and a total of 1546 characters (gaps included). There were no major differences, or strongly supported conflicts, in the overall topologies of the best tree from the ML analysis and the consensus tree from the BI analysis for any of the datasets.
In Figure 1, we present the best tree from the ML analysis of the nrITS + TEF1-α dataset, which will be the main reference point for the taxonomic discussion. In the Supplementary Figures, we provide the 50% majority rule consensus tree from the BI analyses of the combined dataset (Supplementary Figure S1), as well as the individual ML trees from the analyses of the nrITS dataset (Supplementary Figure S2) and the TEF1-α dataset (Supplementary Figure S3).   Figure S3) analyses, these same four lineages are recovered, but the relations between them are not strongly supported in any of the analyses. For clarity and brevity, we use the term "strongly supported" for a clade/relation that receives bootstrap (BS) ≥ 90 and posterior probability (PP) = 1, and "well supported" if it receives BS ≥ 70 and PP ≥ 0.95. Individual support values are shown in Figure 1 The relations between these four lineages receive no support in the ML tree ( Figure 1 Figure S3) analyses, these same four lineages are recovered, but the relations between them are not strongly supported in any of the analyses.
The topological relations within each of these four lineages are overall similar in the trees from the analyses of the combined nrITS + TEF1-α dataset ( Figure 1) and the individual nrITS (Supplementary Figure S2) and TEF1-α (Supplementary Figure S3). In general, support values for all the species recognized here are higher in the combined nrITS + TEF1-α trees, with the exception of P. cystidiosus, which gets higher support in the TEF1-α trees (100/1) than in the combined dataset (99/-).

Taxonomy
Here we present the descriptions of the eleven species and one distinct variety currently known to occur in the Holarctic region. We consider that the P. podospileus group contains two well-known species, Pluteus podospileus and P. seticeps; the recently described P. cutefractus; P. seticeps var. cystidiosus elevated to species rank as P. cystidiosus; the forgotten P. inflatus, which is rediscovered and molecularly confirmed; and six new species (P. absconditus, P. fuscodiscus, P. gausapatus, P. inexpectatus, P. millsii; and P. notabilis with its new variety Pluteus notabilis var. insignis). Moreover, there are at least four other undescribed species that occur in the Holarctic region.
Distribution: Eurasia: Europe (molecularly confirmed from Spain, the Czech Republic, and Estonia), and as far east as Siberia. It is likely distributed all over Europe e.g., [4,11], but is apparently less frequent than Pluteus inflatus. Pluteus podospileus has been cited from Japan [15], but it is possible the Japanese record corresponds to Pluteus cystidiosus. Confusion with similarly-looking taxa makes it hard to interpret older records in the absence of well-annotated voucher specimens.
Collections examined: CZECH REPUBLIC: Vysoké Chvojno, decaying deciduous trunk, 13 August 2015, leg. P. Včelička (BRNM825840). ESTONIA: Võru Co., Varstu Comm., Krabi, Paganamaa Landscape Reserve, 57.59608 • N, 26. Notes: In the concept accepted here, Pluteus podospileus agrees well with the original description by Berkeley and Broome (1881, as Agaricus spilopus): a relatively small species with a brown, rugose pileus and a stipe covered with brown squamules. Modern authors [2,4] have accepted P. podospileus as a species with a hymenidermal pileipellis composed of both short and long elements. Collections of P. podospileus studied here have brown-squamulose stipes and were collected directly on wood.
The exact relationship between Pluteus podospileus and P. minutissimus Maire is not currently clear, and different interpretations of these taxa exist in the literature. Pluteus minutissimus was originally described as a very small species, growing solitary under Quercus in northeastern Spain [19]. The following characters were specifically mentioned in the original description: small basidioma (pileus 5 mm diam, stipe 10 × 1 mm), pileus without rugulosity and striation, basidiospores subglobose 5 × 4.0-4.5 µm, pleurocystidia rare but present near the lamellar edge, clavate cheilocystidia, and pileipellis composed of two types of elements (short and long). Traditionally, the main character separating P. minutissimus from P. podospileus (as accepted here) is the glabrous stipe surface without dark squamules. Kühner and Romagnesi [10] attributed to P. minutissimus (as forma "typicus") a cap of 9-11 mm in diameter, a glabrous white stipe, and a preference for growing terrestrially, without direct connection to wood. At the same time, they described P. minutissimus f. major as differing from the typical morphology in its larger size (cap 12-37 mm), stipe dotted with brown squamules, and growth directly connected to decayed wood of angiosperms. Orton [2] recognized P. podospileus as a species with brown squamulose stipe growing on wood and P. minutissimus as a species with glabrous stipe growing terrestrially, dismissing differences in size as useful characters to separate them. Vellinga and Schreurs [1] reduced P. minutissimus to a form of P. podospileus, with the lack of brown squamules on the stipe as the only separating character. It should be noted that in the original description of P. minutissimus, the only information on habitat is "under Quercus", but there are no specifics as to whether the species was growing terrestrially or connected to wood. The original collection of P. minutissimus exists, but it is in poor condition and not available for study. However, curator Caroline Loup kindly revised the holotype. The conserved material consists of a single basidioma with a stipe 10 mm tall and a pileus 4 mm in diameter with only two visible pieces of protruding lamellae, less than 0.5 mm each. By her revision, basidiospores are globose, about 5-6 µm, a few clumps of cystidia are present, mostly clavate to (sub)fusiform; and basidia are collapsed. In the absence of modern collections from the type locality (Vallvidrera, Spain) or from similar habitats nearby, we are not in a position to accept or refute the synonymy of P. podospileus and P. minutissimus. It is possible that the taxon described by Kühner and Romagnesi as P. minutissimus f. typicus and by Orton as P. minutissimus is indeed a separate species from P. podospileus by virtue of the terrestrial habitat and the non-squamulose stipe. Whether P. minutissimus would be the correct name for such a species would have to be confirmed. In the present study, we recognize six species in the P. podospileus complex occurring in Europe, five of them molecularly confirmed from Spain or southern France: Pluteus podospileus (NBM-F-009808), P. cutefractus (BRNM825872, GM 3784, GM3458), P. inflatus (BRNM 825873), Pluteus sp. (GM3751), and P. notabilis var. notabilis (BRNM825867). Two of these species (P. inflatus and Pluteus sp. GM 3751) can have white, non-squamulose stipes and also grow terrestrially. Pluteus sp. GM 3751 differs from the original description of Pluteus minutissimus, at least in the strongly rugose-venose pileus. Pluteus inflatus is a geographically widespread species also confirmed to occur in Spain, and some collections of P. inflatus match the P. minutissimus protologue. It should be noted that P. minutissimus does not have priority over either P. podospileus or P. inflatus, the two species accepted here that more closely match its original description. However, we prefer to verify the taxonomic value of P. minutissimus strictly by molecular analysis of the holotype collection in the future when less invasive methods become available.       30, subglobose to broadly ellipsoid, rarely globose or ovoid, thick-walled. Basidia 15-34 × 6-12 µm, 4-spored, rarely 2-spored, mostly clavate, less frequently almost cylindrical or subfusiform. Pleurocystidia absent or rare, rarely common all-over lamellar faces, 36-73(-80) × (10-)15-30 µm, narrowly clavate to broadly clavate or oviform, some ellipsoid, narrowly utriform or inflated-fusiform, hyaline, thin-walled. Lamellar edges heterogeneous and fertile, with cheilocystidia, basidia, and basidioles, with cheilocystidia crowded in places in clusters but not forming a continuous strip. Cheilocystidia 20-60(-65) × (10-)15-30 µm, numerous, narrowly to broadly clavate or cylindrical, ovoid or rarely narrowly utriform to utriform or subfusiform, hyaline, thin-walled. Pileipellis a hymeniderm, or rarely an epithelioid hymeniderm, made up of two types of elements: (i) spheropedunculate or broadly clavate to clavate, rarely subovoid 28-63(-66) × 16-42 µm; (ii) narrowly to broadly fusiform or cylindrical, rarely narrowly clavate to clavate or narrowly utriform, 72-130(-190) × (10-) 16-30(-34) µm; all elements with pale brown to brown or yellow-brown intracellular pigment often aggregated into spots, thin to thick-walled. Stipitipellis a cutis of 5-12 µm wide cylindrical hyphae, slightly thick-walled, hyaline, rarely with pale brown pigment. Caulocystidia absent to numerous, often in clusters, narrowly to broadly fusiform, rarely clavate or flexuose, some with 2-3 basal septa, 40-145 × (7-)8-20 µm, colorless or with pale brown to brown, unevenly distributed intracellular pigment. Clamp connections absent in all studied tissues.  Pileus 10-30 mm in diameter, hemispherical or campanulate when young, expanding to convex to plano-convex, rarely plane, with or without indistinct, low and broad umbo, only rarely with distinct umbo, not or very rarely hygrophanous, smooth or slightly rugose at center, without striation or rarely translucently striate from margin up to half the radius of pileus, mostly not cracked, rarely with some cracks revealing the white context towards the margin, one collection cracked almost to the center. Surface dry, slightly to strongly velvety, becoming entirely minutely to distinctly granulose-squamulose (RAL 040 40 30), root brown (RAL 040 30 30), or wild brown (RAL 040 20 19), brown (S00 C00 Y60-S00 C00 Y70), red brown or chestnut brown, dark brown to almost back in the center (S00 C00 Y50). Lamellae free, crowded to moderately distant, mostly ventricose, up to 4 mm broad, white (Y10 M00 C00) or grey-white, later pinkish (Y10 M00 C00) to pink, with a concolorous, even, slightly pubescent to distinctly flocculose edge. Stipe 25-45(-60) × 1-2(-4) mm, cylindrical, sometimes slightly broadened at base, sometimes (finely) longitudinally fibrillose, whitish or greyish, entirely white pubescent or only in the lower half, sometimes with minute distinct brown scales in the lower part, tomentum at the base white. Context in stipe and pileus white or grey-white. Smell and taste indistinct.   30, subglobose to broadly ellipsoid, rarely globose or ovoid, thick-walled. Basidia 15-34 × 6-12 µm, 4-spored, rarely 2-spored, mostly clavate, less frequently almost cylindrical or subfusiform. Pleurocystidia absent or rare, rarely common all-over lamellar faces, 36-73(-80) × (10-)15-30 µm, narrowly clavate to broadly clavate or oviform, some ellipsoid, narrowly utriform or inflated-fusiform, hyaline, thin-walled. Lamellar edges heterogeneous and fertile, with cheilocystidia, basidia, and basidioles, with cheilocystidia crowded in places in clusters but not forming a continuous strip. Cheilocystidia 20-60(-65) × (10-)15-30 µm, numerous, narrowly to broadly clavate or cylindrical, ovoid or rarely narrowly utriform to utriform or subfusiform, hyaline, thinwalled. Pileipellis a hymeniderm, or rarely an epithelioid hymeniderm, made up of two types of elements: (i) spheropedunculate or broadly clavate to clavate, rarely subovoid 28-63(-66) × 16-42 µm; (ii) narrowly to broadly fusiform or cylindrical, rarely narrowly clavate to clavate or narrowly utriform, 72-130(-190) × (10-) 16-30(-34) µm; all elements with pale brown to brown or yellow-brown intracellular pigment often aggregated into spots, thin to thick-walled. Stipitipellis a cutis of 5-12 µm wide cylindrical hyphae, slightly thick-walled, hyaline, rarely with pale brown pigment. Caulocystidia absent to numerous, often in clusters, narrowly to broadly fusiform, rarely clavate or flexuose, some with 2-3 basal septa, 40-145 × (7-)8-20 µm, colorless or with pale brown to brown, unevenly distributed intracellular pigment. Clamp connections absent in all studied tissues.

Holotype of
Habit, habitat, and phenology: mostly solitary, on very decayed wood of deciduous trees, on litter, or on soil. May-October.
Collections Notes: Collection PRM 154569 was labeled as a holotype of P. inflatus var. alneus on a fungarium sheet but was collected after the publication of the protologue. The curator of PRM, J. Holec, confirmed (pers. comm.) that this collection was not labeled as a holotype by Velenovský but was labeled as such by a later worker. The original material of Pluteus inflatus mentioned in the protologue was also collected by Velenovský from the village of Mnichovice on an Alnus trunk, but earlier (7 August 1939, PRM 154261). This collection is the only material that perfectly matches the protologue. Thus, we recognize PRM 154261 to be the true holotype. The holotype of P. inflatus does not exist. The holotype of Pluteus inflatus var. alneus (PRM 154261) as well as PRM154569 and Pluteus inflatus PRM154566 fall into the same/inflatus clade, while one collection (PRM154563 as Pluteus inflatus) falls into the/cutefractus clade. Velenovský [20] described P. inflatus var. inflatus with a non-cracked pileus without striation, growing on deciduous (Carpinus) stumps. This description confirms the/inflatus clade, supported by three collections, as the best candidate for the original concept of P. inflatus. Velenovský [21] described a variety of Pluteus inflatus var. alneus only with the short note that it grows on the trunk of Alnus and that it is easily recognized. Because species of the genus Pluteus often grow on substrates of various deciduous trees (e.g., [1,2,7,53]), we do not believe that growth on Alnus versus Carpinus is sufficient to describe a separate variety. Thus, we synonymize Pluteus inflatus var. alneus under Pluteus inflatus.
Both sequenced syntypes of Pluteus minutissimus f. major (G00126660 and G00126659) belong to P. inflatus, which has priority. Thus, Pluteus minutissimus f. major is only a later synonym of P. inflatus. Surprisingly, our molecular studies show that the holotype of Pluteus granulatus var. tenellus J. Favre (G K9933, G00126179) also represents P. inflatus, even though Favre [54] described this variety as having a pileipellis composed of three types of elements, while there are only two significant element types in a typical basidioma of P. inflatus. Unfortunately, the size and condition of the Pluteus granulatus var. tenellus holotype do not allow a precise verification of the pileipellis structure in numerous parts of the pileipellis, but, based on our studies, there are various elements that can be interpreted as representing two or three types by their similarities. Pluteus inflatus is a highly variable species that is difficult to distinguish from related species and has a broad ecology, being recorded from both soil, plant litter, and decaying wood.
Pluteus inflatus is widely distributed in Eurasia, and even two North American collections have been molecularly confirmed. Both collections were made in disturbed, man-made habitats (urban gardens, parking lots), which could indicate a recent, humanmediated introduction to North America, but more collections are needed to confirm this hypothesis.
Pluteus absconditus Justo, Kalichman and S.D. Russell sp. nov. Figures 8 and 9. MB 849337. Etymology: absconditus, meaning hidden or concealed, because of the difficulty separating this species from P. inflatus without molecular data.
Habit, habitat, and phenology: solitary to subgregarious, growing on soil, often among leaves, twigs, and other plant matter, but without apparent connection to wood. June-August.
Distribution: Eastern North America: known from the USA (Indiana, Tennessee Notes: Pluteus absconditus is recovered in the phylogenetic analysis as a sister to Pluteus inflatus. Their nrITS sequences differ in 5-8 evolutionary changes, and their TEF1-α sequences in 5. Morphologically, both species are very similar, with P. inflatus differing in the rare or absent pleurocystidia and the more abundant and slightly larger cheilocystidia (20-60(-65) × (10-)15-30 µm). P. absconditus has 2-spored and 1-spored basidia, which is not a common character for Pluteus species, where most taxa have exclusively 4-spored basidia. The pileus of Pluteus absconditus seems to be paler than typical P. inflatus; however, the latter is an extremely variable species, and the variability of P. absconditus has not been well investigated yet. Original diagnosis. Basidiomata with a markedly cracked pileipellis, globose to broadly ellipsoid basidiospores, predominantly ovoid to broadly clavate cheilocystidia and pleurocystidia, and trichohymeniderm pileipellis consisting of broadly utriform and fusiform elements.
Basidiopores Notes: Pluteus absconditus is recovered in the phylogenetic analysis as a sister to Pluteus inflatus. Their nrITS sequences differ in 5-8 evolutionary changes, and their TEF1α sequences in 5. Morphologically, both species are very similar, with P. inflatus differing in the rare or absent pleurocystidia and the more abundant and slightly larger cheilocystidia (20-60(-65) × (10-)15-30 µm). P. absconditus has 2-spored and 1-spored basidia, which is not a common character for Pluteus species, where most taxa have exclusively 4-spored basidia. The pileus of Pluteus absconditus seems to be paler than typical P. inflatus; however, the latter is an extremely variable species, and the variability of P. absconditus has not been well investigated yet.  Original diagnosis. Basidiomata with a markedly cracked pileipellis, globose to broadly ellipsoid basidiospores, predominantly ovoid to broadly clavate cheilocystidia and pleurocystidia, and trichohymeniderm pileipellis consisting of broadly utriform and fusiform elements.
Habit, habitat, and phenology: solitary or gregarious, growing on well-decayed wood of angiosperms (e.g., Acer). In temperate or transitional boreal/temperate forests. July-August. According to Minnis and Sundberg [13], also June and September.
Distribution: North America: widely distributed in eastern North America. Molecularly confirmed from Canada (Québec) and the USA (Illinois, Indiana, Missouri, New Jersey, Ohio, Pennsylvania, Tennessee, and Wisconsin). Notes: Pluteus seticeps has been considered by some authors to be a synonym of P. podospileus [2,4], but detailed revision of the type collection, modern North American collections, and molecular data clearly indicate these are separate species [7,13]. Both species have non-overlapping geographical ranges, with P. podospileus occurring only in Eurasia and P. seticeps only present in eastern North America. Minnis and Sundberg [13] confirmed the usage of the name Pluteus seticeps for North American collections lacking pleurocystidia and established Pluteus seticeps var. cystidiosus Minnis and Sundb. for North American collections similar to P. seticeps with pleurocystidia. Molecular data from the type collection of P. seticeps var. cystidiosus indicate that this taxon is not closely related to P. seticeps but instead represents a sister taxon to the European P. podospileus. The new combination P. cystidiosus is adopted here for this species. Morphologically, P. seticeps can be distinguished from P. cystidiosus by the smaller basidiomata, pileus surface more markedly rimulose, stipe surface not covered in brown squamules, absence of pleurocystidia, and smaller caulocystidia that mostly do not occur in clusters.
Pluteus nanellus Murrill was described from New York as a species with small basidiomata, pale bay (pale reddish brown), minutely tomentose (visible only under the lens) pileus turning castaneous on drying, and a snow-white stipe [25]. Singer [22] and Homola [24,56] synonymized Pluteus nanellus and P. seticeps mostly based on the similarity of the pileipellis elements. Minnis and Sundberg [13] revised the type collections of both taxa and considered them to be identical. The only significant difference in the original descriptions of these species is the presence of some brown fibrils at the base of the stipe of P. seticeps, while this character is not mentioned in P. nanellus. Unfortunately, stipes are absent in both types of collections. Many of the specimens examined here have some brown fibrils at the base, but completely glabrous stipes also occur. We agree with Minnis and Sundberg [13] and consider P. seticeps and P. nanellus synonymous. Pileus 14-35(-40) mm in diameter, hemispherical or campanulate when young, expanding to convex or plano-convex, often with a low, broad umbo; surface pruinose, slightly to strongly velvety, becoming granulose-squamulose especially around center, smooth or strongly rugose all over when young, in older specimens smooth or rugose at center, for the most part not cracked, or with some cracks revealing the white context towards the margin, especially in older specimens; with predominant brown or red-brown colors (Munsell: 2.5YR 4/6-4/8, 3/4-3/6; 5YR 5/4-5/8, 4/4-4/6; when young 5YR 3/3, 2.5/2; RAL color chart: chestnut brown (RAL 040 40 30), red brown (RAL 020 20 20) or wild brown (RAL 040 20 19), usually darker at center; dry, hygrophanous; margin entire, serrate, slightly rimose or translucently striate. Lamellae crowded, free, ventricose, up to 5 mm broad, white when young, later pink, with even, serrated, or flocculose edges, white or concolorous. Stipe 15-40 × 1-5 mm, cylindrical, with a slightly broadened base; surface white, entirely minutely squamulose with dark or black-brown squamules, more densely so in the lower half. Context in stipe and pileus white. Smell and taste none.    (ii) broadly fusiform, inflated-fusiform, lanceolate, narrowly utriform, often mucronate, 66-121.5 × 9-17(-32) µm; all elements with brown or yellow-brown intracellular pigment, often aggregated in spots, slightly thick-walled. Stipitipellis a cutis of cylindrical, slightly thick-walled, 7-10 µm wide hyphae, hyaline, or some with pale brown pigment. Caulocystidia common, often in clusters, 36-125 × 6-25 µm, cylindrical, narrowly clavate, narrowly fusiform, spheropedunculate, often septate; most with brown or yellow-brown pigment, sometimes aggregated. Clamp connections absent in all studied tissues.

Pluteus cystidiosus (Minnis and
Habit, habitat, and phenology: often gregarious, growing on well-decayed wood of angiosperms (e.g., Acer, Betula), also on still-standing trees. In temperate or transitional boreal/temperate forests. July-October. Based on current data, P. podospileus is not expected to occur in North America, and P. cystidiosus is not expected to occur in Europe, but it is not known if their geographical ranges overlap in Asia. Pluteus cystidiosus occurs in Japan and the Russian far east, while P. podospileus has been confirmed to occur as far east as Siberia (Krasnoyarsk Territory).
Habit, habitat, and phenology: isolated or gregarious, growing on well-decayed wood or wood chips of angiosperms. Thus far, it has been collected in forests or open areas of the temperate zone in September and October.
Notes: Pluteus podospileus differs from P. inexpectatus in its subglobose to broadly ellipsoid basidiospores and distribution restricted to Eurasia; some collections of P. podospileus have larger basidiomata, but the variability in size of P. inexpectatus is not well known yet. Pluteus cystidiosus can have a more robust habit, a more or less velvety pileus, and subglobose to broadly ellipsoid basidiospores. In North America, confusion is most likely with Pluteus seticeps, which can be distinguished by the absence or very rare pleurocystidia even close to the lamellar edge and caulocystidia mostly not occurring in clusters. Another North American species, P. absconditus, differs by its pulverulent-powdery and/or white-translucent pubescent pileus, larger basidiospores, heterogeneous lamellar edge, and growth on soil.  Diagnosis: Closely related to Pluteus necopinatus, differing in the presence of pleurocystidia, shorter cheilocystidia, occurrence in the Northern Hemisphere, and   Pileus 5-10 mm in diameter, hemispherical when young, expanding to convex or plano-convex, with a low, broad umbo; surface strongly granulose-squamulose all over, with cracks revealing the white context underneath, except at the center; with predominant brown colors (Munsell: 5YR 4/6, 3/4; 7.5YR 5/6-5/8, 4/6), sometimes gray-brown (7.5YR 6/3-6/6), darker at center; dry, not hygrophanous; margin entire, rimose, or very slightly translucently striate, Lamellae crowded, free, ventricose, up to 2 mm broad, white when young, later pink, with white flocculose edges. Stipe 10-20 × 1-2 mm, cylindrical, with a slightly broadened subbulbous base (up to 3 mm wide); surface white, silvery-white, shiny, and entirely hairy-woolly, covered with white hairs, sometimes grouped in definite mats with pale brown tips. Context in stipe and pileus white. Smell and taste none.
Habit, habitat, and phenology: subgregarious, growing on well-decomposed wood and other plant matter. June-September.
Distribution: Eastern North America: so far, only known from the USA (Connecticut, Kentucky, and Tennessee).
Additional Notes: Pluteus millsii stands out macroscopically by the densely hairy-woolly surface of the stipe. Pluteus absconditus can also have a relatively hairy stipe, but this species differs in the presence of 2-spored and 1-spored basidia, slightly larger pleurocystidia (up to 66 µm long), a heterogeneous lamellar edge, and common caulocystidia.
Molecularly, P. millsii is quite distinct from all other taxa in the/podospileus clade: its nrITS sequences differ by 19 evolutionary changes from those of its closest relative, Pluteus necopinatus (96.9% sequence similarity). All other Pluteus nrITS sequences sampled in this study are less than 90% similar to P. millsii.
Pluteus necopinatus differs from P. millsii by the non-hairy-woolly stipe surface, the absence of pleurocystidia, the larger cheilocystidia (37-74 × 12.5-30 µm) that are predominantly lageniform or utriform, and a pileipellis with shorter elongated elements (up to 125 µm long). Both taxa have similar tufts of hyphal elements above the stipitipellis, which we do not consider to be caulocystidia in the sense the term is applied in Pluteus, as they are loosely arranged bundles of hyphae that lay on top of the tightly arranged hyphae of the stipitipellis. In other taxa of Pluteus with true caulocystidia, these arise directly from the stipitipellis. Pluteus necopinatus was originally described and is thus far only known from the Atlantic Forest of Brazil, growing directly out of a concrete wall [46]. Figures 24 and 25. MB 849804. Etymology: gausapatus refers to the plush pileus and stipe surface. "Gausapatus" also means "happy", which well expresses the authors feelings about enjoying this beautiful species.

Pluteus gausapatus Ševčíková and Antonín sp. nov.
Diagnosis: Pluteus gausapatus notably differs from Pluteus podospilloides by the absence of the following features: khaki, dark brown, or olive-brown squamules and white short hairs on a pileus surface; dark brown squamules of the stipe; utriform or broadly lageniform colored pleurocystidia with a short neck; and by a distinct phylogenetic position (nrITS, TEF1-α) among the Pluteus species in the/podospileus clade.
Distribution: Known only from Asia: Republic of Korea. Notes: Pluteus gausapatus is characterized by its small basidiomata with a plush, brown pileus, brown lamellar edges, a whitish, finely pubescent to plush stipe, mostly fusiform pleurocystidia, and growth on decayed deciduous trees.
Pluteus podospilloides E.F. Malysheva and O.V. Morozova, originally described from Vietnam, differ by their pileus surface covered with minute khaki, dark brown, or olivebrown squamules and white short hairs; a stipe with a distinct dark brown squamule along its entire length; utriform or broadly lageniform colored pleurocystidia with a short neck; slightly thick-walled cheilocystidia; and pileipellis elements [28]. Pluteus delicatulus C.K. Pradeep and K.B. Vrinda is somewhat similar to P. gausapatus by its small basidiomata and pileipellis, but it differs by its lamellae without brown edges, glabrous stipe without caulocystidia, slightly broader basidiospores, and vesiculose to clavate pleurocystidia [57]. Pluteus stigmatophorus (Berk. and Broome) Sacc. has similar pileipellis elements to P. gausapatus, but its pileus is larger and depressed at the center, its stipe is brownish yellow and black-dotted, its basidiospores are slightly larger, and its pleurocystidia are clavatepedicellate to submucronate [8,16,18]. Pluteus stigmatophorus is known from Sri Lanka [17]. Pluteus extremiorientalis E.F. Malysheva and Malysheva also have a brownish-colored and tomentose-squamulose pileus, brownish to fuscous lamellar edges, and a stipe (lower part) covered by dark brown squamules (or fibrils). However, this Russian Far East species differs by having a fibrillose-squamulose pileus margin, larger basidiospores, predominantly lageniform or utriform pleurocystidia, and a pileipellis consisting of only two types of shorter elements [58]. Pileus 18-45 mm broad, convex or hemispherical when young, later plano-convex; surface completely and remarkably felted, often fairly coarsely radially wrinkled, not striate or only shortly translucently striate on overmature specimens, with fimbriate or denticulate margin, not or very weakly hygrophanous, warm date brown to almost blackish brown when young (RAL 8022), paler and with chestnut brown to reddish brown tinges on adult specimens, dark brown with distinctly paler margin on very old specimens. Lamellae moderately distant, free, ventricose, white when young, then pinkish, with an eroded-denticulate edge, with a whitish, less frequently brownish flocculose edge, especially near the pileus margin. Stipe 17-48 × 2-5 mm, cylindrical, with Pileus 18-45 mm broad, convex or hemispherical when young, later plano-convex; surface completely and remarkably felted, often fairly coarsely radially wrinkled, not striate or only shortly translucently striate on overmature specimens, with fimbriate or denticulate margin, not or very weakly hygrophanous, warm date brown to almost blackish brown when young (RAL 8022), paler and with chestnut brown to reddish brown tinges on adult specimens, dark brown with distinctly paler margin on very old specimens. Lamellae moderately distant, free, ventricose, white when young, then pinkish, with an erodeddenticulate edge, with a whitish, less frequently brownish flocculose edge, especially near the pileus margin. Stipe 17-48 × 2-5 mm, cylindrical, with a slightly enlarged base, whitish to cream, with a very pale, indistinct ochre tinge on very old specimens, densely covered with distinct brown and very fine floccules, often aligned in longitudinal stripes. Context white to whitish; smell and taste indistinct; basal mycelium white. Spore print pink. J. Fungi 2023, 9, 898 4 250) × 15 µm, thin-walled on exsiccata, giving a fluffy appearance to areas of the where they are abundant. Stipitipellis composed of cylindrical and thin-walled h hyphae (3-)5-10(-15) µm wide. Caulocystidia numerous, in tufts, (40-)45-145 × 15 35) µm, thin-walled, predominantly long, subcylindrical, and reminding the elements of the pileipellis, but also clavate or (sub-)utriform. Clamp connections abs all studied tissues.  Habit, habitat, and phenology: on soil with woody debris and sawdust of conifers and on strongly decomposed wood near fallen trunks of Abies alba. Pileus 20-35 mm broad, convex to campanulate when young, later plano-convex to almost plane, with obtuse center without or with low umbo, dull; surface finely granulose to granulose-velvety, some finely tomentose in the center, not or indistinctly wrinkled or sparsely veined radially up to the edge, without striation, with slightly inflexed margin, not hygrophanous, warm date brown, reddish brown, dark brown to almost warm blackish brown when young (RAL 8017 or darker, RAL 8022), then slightly paler. Lamellae L = 36-48, l = 1-5, free, crowded to moderately distant to almost distant, ventricose, white when young, then pinkish to flesh pink, with an eroded-denticulate, concolorous, finely flocculose edge. Stipe 38 × 1.5-2.5 mm, cylindrical, slightly broadened toward the lower part, with a slightly enlarged base, solid, innately fibrillose, twisted or not, hyaline, whitish, or cream, graying with age, entirely minutely to densely covered with distinct brown fine floccules, sometimes aligned in short longitudinal stripes, at least on the lower part. Context whitish in pileus, whitish or hyaline in stipe. Smell and taste indistinct. Spore print pinkish.
Basidiospores  Notes: fusiform to utriform pileipellis elements with a mucronate apex up to 8 µm are confirmed only for Pluteus notabilis var. notabilis and for Pluteus seticeps. The later pleurocystidia are absent or extremely rare, and this species occurs in North America. Pluteus notabilis var. insignis differs from Pluteus notabilis var. notabilis by its concolorous lamellar edges, somewhat larger basidiospores on average, pleurocystidia only up to 58 µm long, and pileipellis without elements displaying a distinct, long mucronate apex. Moreover, Pluteus notabilis var. notabilis has a distinctly radially wrinkled pileus, while some basidiomata lack this feature and some are less distinctly wrinkled. However, the variability of this feature needs to be confirmed. Phylogenetically, based on nrITS and TEF1-α, both taxa are similar.

Discussion
In this study, we show the P. podospileus complex to be considerably more diverse than previously believed. Although some overlap in the morphological variation of the species does occur, the distribution patterns and macroscopic aspects often include characters that allow identification at the species rank. Macroscopically, P. gausapatus differs from all similar species by the combination of a plush pileus surface and a dark lamellar edge. P. notabilis can also have some darker lamellar edges, but its pileus is granulose-velvety rather than plush with a wrinkled center. Furthermore, the whole stipe is densely covered with distinct, fine brown floccules, often aligned in longitudinal stripes. P. millsii is remarkable for its densely hairy-woolly stipe surface and strongly granulose-squamulose pileus all over, with cracks that reveal the white context underneath. P. podospileus was originally described with a rugulose pileus center. We keep the original concept of P. podospileus and use this name for a clade in which a strongly rugulose pileus center is a typical feature. However, P. notabilis, some collections of P. inflatus and P. cutefractus, and the North American P. seticeps, P. cystidiosus, and P. absconditus can also have a rugulose pileus, at least when young. Typical basidiomata of P. fuscodiscus have a distinct darker disc in the pileus center. However, several species commonly possess a darker pileus center. Thus, microscopical examination is needed to identify P. fuscodiscus.
Pluteus cutefractus is characterized by a markedly cracked pileus, but as the pileus of several species of the P. podospileus group may be cracked, a combination of features must be used to identify this species. The pileus margin of P. seticeps is often translucently striate up to half the radius. However, P. cystidiosus may also share this feature. The most macroscopically variable species is P. inflatus. P. absconditus is often macroscopically indistinguishable from P. inflatus, but a pileus with a pulverulent-powdery aspect and/or a white-translucent pubescence can be useful if present.
Spores of P. inflatus, P. absconditus, and P. cutefractus are commonly slightly longer (up to ± 7 µm), while basidiospores of P. inexpectatus, P. cystidiosus, P. millsii, and P. notabilis do not exceed 6 µm. However, this is a relatively subtle difference. Basidiospores of P. inexpectatus seem to often be almost globose to subglobose (Q* = 1.03-1.07), but more collections are needed to confirm the stability of this feature.
Pleurocystidia are an important feature for the delimitation of Pluteus species. Pleurocystidia are absent or extremely rare in P. seticeps and absent or present in P. inflatus and P. fuscodiscus. They are present (from scattered to common) in the other species described here.
The lamellar edge is sterile in several species, while a heterogeneous and partially fertile lamellar edge is typical for P. inflatus and P. absconditus and also occurs in some collections of P. cutefractus. Cheilocystidia are numerous in all species of the P. podospileus complex and variable across almost all species. Cheilocystidia tend to be utriform to spathulate in P. fuscodiscus, while they are more often subglobose, ovoid, or ellipsoid in P. inexpectatus and mostly clavate or narrowly clavate in P. millsii, but this unstable feature is not sufficient for a definitive identification of individual species.
Further investigation will confirm whether the colorful cheilocystidia of P. gausapatus can be a stable character similar to, for example, P. umbrosus (Pers.) P. Kumm. [4,63] or, on the contrary, if it is variable and unreliable as in P. keselakii Ševčíková, P.-A. Moreau, and Borovička [31].
In most species, the pileipellis is composed of two types of elements (short and elongate). Favre [54] described P. granulatus var. tenellus as having a pileipellis composed of three types of elements: (i) short, broadly clavate to spheropedunculate and (sub)fusiform, (ii) elongate cylindrical, and (iii) long, elongate (sub)lanceolate to subfusiform or subcylindrical. However, there are several options on how to classify these elements as being of two or three types. In fact, in some collections, there is a fine line between whether we can describe two or three types of elements. Fusiform to (sub-)utriform elements with a distinctly mucronate apex up to 8 µm long in the pileipellis are a unique feature of P. notabilis var. notabilis.
Caulocystidia are present in most species but might be lacking in some particular collections. Pluteus millsii and P. necopinatus have a layer of loosely arranged hyphae on top of the stipitipellis that we do not consider caulocystidia here, as they are not terminal elements that rise from the stipitipellis. Some authors have considered the presence/absence of caulocystidia a reliable feature, distinguishing Pluteus nanellus from P. seticeps [2]. P. inflatus frequently has long caulocystidia up to 145 µm, while the caulocystidia of P. cutefractus and P. gausapatus are shorter. Our studies indicate that this feature could be used as an additional character to help define species on morphological grounds, but it might have limited usefulness to identify individual collections due to its inconstant presence within collections of the same species.
Based on phylogenetically confirmed collections, the distribution area of P. podospileus may be presumed to be limited to Eurasia, or at least Europe and Siberia. In Europe, six species are phylogenetically confirmed: Pluteus podospileus, P. inflatus, P. cutefractus, P. fuscodiscus, P. notabilis, including P. notabilis var. insignis, and an undescribed species represented by only one collection (GM 3751) in our tree. The range of at least three species, P. podospileus, P. inflatus, and P. fuscodiscus, extends to northwestern Asia. Because both P. cystidiosus and P. fuscodiscus were also found in the Russian Far East, we expect these species to have wider distributions than currently documented. P. gausapatus is only known from the Republic of Korea, where an additional, probably undescribed species, P. aff. podospilloides, was also collected. Eight North American species are recognized in our tree (Figure 1). Only P. seticeps and P. cystidiosus were previously described. P. millsii, P. absconditus, and P. inexpectatus are described in this article as new, while more collections will be necessary to describe the remaining species.
The present paper represents a big step forward in our understanding of the taxonomy of P. podospileus and related taxa, but it also shows that more work is needed to fully describe the species diversity of this group. Four single-collection taxa sampled during this study are included in the phylogenies (Figure 1). Molecularly, they seem to represent separate species from the ones described here, but we await additional collections to formally describe them. It should be noted that these yet-undescribed species come from areas that are heavily sampled in this study, like Europe (Pluteus sp. GM3751) and Eastern North America (Pluteus sp. MO270622; Pluteus sp. iNaturalist 13936381; Pluteus sp. iNaturalist 27406926).
Most of the species described here appear on decayed wood (stumps or trunks) of angiosperms or on woody debris. P. inflatus, P. cutefractus, and P. notabilis var. insignis can have a terrestrial growth habit, but they can also grow on decayed wood. In the protologue of P. granulatus var. tenellus, which represents P. inflatus, an association with Filipendula ulmaria is mentioned. P. absconditus grows on soil, often among leaves, twigs, and other plant matter, but without connection to wood. However, more collections are