Pluteus insidiosus Complex, Four New Species Described and Pluteus reisneri Resurrected

We studied the taxonomy of Pluteus insidiosus and similar species using morphological and molecular (nrITS, TEF1-α) data, including a detailed study of the type collection of P. insidiosus. Based on our results, we recognize five species in this group: P. insidiosus sensu stricto and four other taxa: P. assimilatus; P. farensis; P. flavostipitatus; and P. pseudoinsidiosus; described here as new. All these taxa are distinct from each other based on molecular data, but some of them are semi-cryptic based on morphology and co-occur in the Palaearctic region. An additional molecular lineage, phylogenetically separates from the P. insidiosus complex, but with many morphological similarities, was recognized in the molecular phylogenies. Based on the revision of available type collections, the name Pluteus reisneri Velen., was adopted for this Clade. Pluteus reisneri was validly published in 1921, but it has barely been used since its original description. A modern epitype, with molecular data, was selected for P. reisneri.


Introduction
The agaricoid genus Pluteus Fr. belongs to the family Pluteaceae Kotl. and Pouzar [1] and is characterized by producing basidiomata with free lamellae and without volva or universal veil, a pinkish spore print, smooth thick-walled basidiospores, an inverse hymenophoral trama, the presence of cheilocystidia and often also pleurocystidia [2,3]. It has 2.2. Molecular Phylogeny 2.2.1. DNA Extraction, Amplification, Sequencing and Sequence Alignment For DNA extraction, small fragments of dried basidiomata were used. Collections deposited on the herbaria L, BRNM and LIP were performed by M. Sochor and followed the molecular methods described by Ševčíková et al. [25]. For collections on LE, the procedure of DNA extraction completely corresponded to the manufacturing protocol of the Phytosorb Kit (ZAO Syntol), and for collections on GDOR the DNA was extracted with NaOH following the procedures reported in Dovana et al. [26]. The following primers were used for amplification and sequencing: ITS1F-ITS4/ITS4B [27,28] 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) [29]. PCR products were purified applying the GeneJET Gel Extraction Kit (Thermo Scientific, Thermo Fisher Scientific Inc., Waltham, MA, USA). Raw data were edited and assembled in MEGA 10 [30].

Phylogenetic Analyses
We assembled an nrITS dataset of all available sequences phylogenetically close to P. insidiosus and P. thomsonii ("thomsonii clade" in Menolli et al. [9]). This includes 24 newly generated nrITS sequences for this study, and 35 sequences generated in previous studies (see Table 1; [5,9,14,[31][32][33][34][35][36]) or available in public databases and biodiversity repositories (GenBank, UNITE, iNaturalist; see Table 1). A total of 59 nrITS sequences were used in the final dataset, including voucher-based and environmental sequences. We assembled a TEF1-α dataset of 17 sequences, 16 of them newly generated for this study and an additional sequence previously available in GenBank (see Table 1, [36]). In all datasets we included sequences of P. phlebophorus and P. rugosidiscus as outgroup taxa, based on previous phylogenetic work on Pluteus [8,9]. Sequences were aligned using MAFFT version 7 [37] and the strategy FFT-NS-i. The alignment was inspected and manually corrected in AliView [38]. No topological conflicts were detected in the phylogenetic analyses of the nrITS and TEF1-α datasets (detailed below), so a combined dataset was created by concatenating the nrITS and TEF1-α matrices.  [40] 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 PRSF values were close to one, as detailed in Ronquist et al. [41]. All analyses were run using resources at the CIPRES Science Gateway [42]. In order to best understand the relationships between the different species within the Clade I, an Intra-and Inter-specific patristic distances for each region were calculated in Geneious R11 [43] using RaxML 8.2.11 with GTR GAMMA model. Intra-and Inter-specific distances were represented by using boxplots drawn in Rstudio Version 1.1.453 using ggplot2 library [44].

Phylogeny
The nrITS and TEF1-α datasets comprised 719 and 574 characters, respectively, and the final combined nrITS + TEF1-α dataset consisted of 59 sequences and a total of 1293 characters (gaps included). All individual and combined datasets and their respective ML and BI trees have been deposited at TreeBASE (S28792).
There were no major differences in the overall topologies of the best tree from the ML analysis and the consensus tree from the BI analysis. In Figure 1, we present the best tree from the ML analysis of the nrITS + TEF1-α dataset, with bootstrap values ≥ 70% and posterior probabilities ≥ 0.90.
Four distinct clades can be recognized in the analyses: (i) Clade I includes P. insidiosus and the newly described P. pseudoinsidiosus, P. assimilatus, P. farensis and P. flavostipitatus. All taxa, except P. insidiosus received high support in the ML and BI analyses. None of the sister-taxa relationships received significant support in any of the analyses; (ii) Clade II includes thirteen sequences: eleven sampled collections assignable to P. thomsonii in the broad sense; one of the public sequences in GenBank (coll. MCVE15120) has been originally named P. insidiosus, but it represents an incorrect identification and a second sequence (coll. HATFD14-10) reported as "Uncultured Pluteus". This Clade includes five phylospecies that will be treated separately in a subsequent paper; (iii) Clade III includes two tropical taxa, P. dominicanus var. hyalinus from Brazil and a likely undescribed species from the US Virgin Islands; (iv) Clade IV includes P. reisneri and a likely undescribed species from the USA (New York).
Only the sister-clade relationship between Clades II and III received good support. Intra-and interspecific patristic distances between the different species within Clade I showed variable values within the different regions.
The boxplots of patristic distances of nrITS1, nrITS2, complete nrITS, translation elongation factor 1 alpha exon, intron and complete regions are reported in Figure 2. The nrITS intraspecific patristic distances ranged from 0.0000 to 0.0100, and interspecific distances from 0.0134 to 0.047. The nrITS2 was the most variable sub-region among those considered (intraspecific distances from 0.0000 to 0.0146 and interspecific distances from 0.0295 to 0.0772), highlighting it to be the best single region to separate species within the Clade I. nrITS1 intraspecific distances ranged from 0.0000 to 0.0082, and interspecific distances from 0.0082 to 0.0509, P. insidiosus showed hypervariability in the nrITS1 region, and its maximum intraspecific distance corresponded to minimum interspecific distances. TEF1-α interspecific patristic distances ranged from 0.0106 to 0.0216 (TEF1-α-exon: 0.0065 to 0.0156; TEF1-α-exons: 0.0282 to 0.0475).   Best tree from the ML analysis of the nrITS + TEF1-α dataset. Bootstrap values ≥ 70% and posterior probabilities ≥ 0.90 are indicated on or below the branches. The root length has been reduced to facilitate graphical representation.

Taxonomy
Here we present the descriptions of the six species phylogenetically and/or morphologically related to Pluteus insidiosus currently known to occur in the Palaearctic region. Generally, there are only a few distinct morphological or ecological differences between the treated taxa, and in many cases, identification of individual basidiomata without molecular data will prove difficult. Despite these difficulties, we do interpret these phylogenetic lineages as separate species, and therefore they have to be described and named. Without a correct understanding of the natural history of species in the Pluteus insidiosus complex and a transparent taxonomy and nomenclature, it will be impossible to obtain more accurate data about the distribution, ecology, morphology and conservation status of these taxa. Regional endemics in this group need further studies to establish their possible conservation status. Morphological features are discussed below, and the differences are presented in the key. Pluteus insidiosus Vellinga and Schreurs ( Figure 3) Vellinga, E.C.; Schreurs, J. 1985. Notulae ad Floram Agaricinam Neerlandicam-VIIl. Persoonia. 12(4): 337-373 Type L0053623 Protologue [12]: Pileus 25-40 mm, planoconvex, applanate, with low umbo, slightly hygrophanous, when moist very dark brown to black in centre, pallescent towards margin to dark brown or brown (Munsell 7.5 YR 2/1-3/3), with translucently striate margin, on drying pallescent to brown (Munsell 7.5 YR 4/4), in centre venulose or smooth. Lamellae (L = 48-72/1 = 0-3) fairly crowded, free, slightly ventricose, up to 5 mm broad, first pink, later incarnate, sordid or brownish pink, with an even concolorous edge. Stipe 35-45 mm, cylindrical or slightly broadening downwards, solid, whitish or silverish grey, innately fibrillose, glabrous and shiny. Context in pileus hygrophanous, when moist grey, pallescent on drying to whitish; in stipe concolourous with surface. Smell absent or bitterish; taste absent or slightly unpleasant.
Holotype revision: basidiospores [60/1/1] (5.6-)6.1-7.  Notes: Vellinga and Schreurs [12] described the cheilocystidia of P. insidiosus as being not crowded, but later, Vellinga [3] mentioned the lamellar edge as being sterile. Our examination of the holotype showed that in some places, cheilocystidia form clusters rendering the lamellar sterile, while in other portions, the lamellar edge was heterogeneous, with basidioles, and in some places even basidia intermixes with the cheilocystidia. However, these fertile sites were not predominant, and it remains uncertain if the nature of the lamellar edge is a stable separating character. Although the phylogenetic analysis presented in Figure 1 does not statistically support the /P. insidiosus Clade, excluding the nrITS1 region and the TEF1-α introns from the previously used dataset, ML analysis supports the /P. insidiosus Clade (ML bootstrap = 76%) (tree reported Notes: Vellinga and Schreurs [12] described the cheilocystidia of P. insidiosus as being not crowded, but later, Vellinga [3] mentioned the lamellar edge as being sterile. Our examination of the holotype showed that in some places, cheilocystidia form clusters rendering the lamellar sterile, while in other portions, the lamellar edge was heterogeneous, with basidioles, and in some places even basidia intermixes with the cheilocystidia. However, these fertile sites were not predominant, and it remains uncertain if the nature of the lamellar edge is a stable separating character. Although the phylogenetic analysis presented in Figure 1 does not statistically support the /P. insidiosus Clade, excluding the nrITS1 region and the TEF1-α introns from the previously used dataset, ML analysis supports the /P. insidiosus Clade (ML bootstrap = 76%) (tree reported in Supplementary Figure S1). Within Clade I, P. insidiosus is characterized by -TC-deletion in nrITS1, -T-insertion in nrITS2 and a non-silent mutation in TEF1-α-exon (GTC/Valine instead of GCC/Alanine). Notes: Based on our current knowledge, Pluteus pseudoinsidiosus is macroscopically almost indistinguishable from P. insidiosus, except for a somewhat paler pileus with ochraceous or olivaceous tones, which may however not be a stable feature; as well as a greenish blue stipe if present. Microscopically, these two taxa differ only by inconspicuous features. Basidiospores of P. pseudoinsidiosus are smaller than those of P. insidiosus and many pleurocystidia of P. pseudoinsidiosus have rostrum, which has never been observed in P. insidiosus. However, pleurocystidia are scarce and difficult to find in both taxa. They might even be absent in some collections of both species (DMS-10194422, Vellinga [3]). The sterile lamellar edge seems to be another distinguishing feature, but a stability of this feature is uncertain. Based on known collections, P. pseudoinsidiosus seems to have a more southern distribution than P. insidiosus, but the sample size is too small to judge if this tendency is reflecting a real difference. Further experience with both taxa is needed, and at present these species may be regarded as cryptic and only separable by sequence data. Basidiomata of P. pseudoinsidiosus with a bluish stipe may resemble Pluteus cyanopus Quél. However, by its protologue [45], this species has a black or purplish pruinose pileus, greyish or lilac lamellae and basidiospores about 6 µm. Vellinga [3] interpreted this species based on recent collections with a bluish stipe by having a brown pileus, (narrowly) utriform to pedunculate and ovoid-conical pleurocystidia and narrowly utriform to ovoid cheilocystidia without rostrum. A discussion on the true identity of Pluteus cyanopus falls out of the scope of the present paper, but it will be discussed in another prepared article about the taxa placed in the /cinereofuscus and /phlebophorus clades sensu Menolli et al. [9] and Malysheva et al. [14]. Pluteus phaeocyanopus, Minnis and Sundb., also has a bluish stipe, but has larger basidiospores (6.2-8.4 × 5.7-7.9 µm), (narrowly) lageniform pleurocystidia with pedicel, long neck and obtuse apex with brown intracellular pigment and subglobose to pyriform cheilocystidia with brown intracellular pigment [46]. This species is known from Western North America (San Francisco, CA, USA), and molecular data based on Californian collections show that this species belong in the/cinereofuscus Clade, not related with the P. insidiosus complex (Data not shown). greenish blue stipe if present. Microscopically, these two taxa differ only by inconspicuous features. Basidiospores of P. pseudoinsidiosus are smaller than those of P. insidiosus and many pleurocystidia of P. pseudoinsidiosus have rostrum, which has never been observed in P. insidiosus. However, pleurocystidia are scarce and difficult to find in both taxa. They might even be absent in some collections of both species (DMS-10194422, Vellinga [3]). The sterile lamellar edge seems to be another distinguishing feature, but a stability of this feature is uncertain. Based on known collections, P. pseudoinsidiosus seems to have a more southern distribution than P. insidiosus, but the sample size is too small to judge if this tendency is reflecting a real difference. Further experience with both taxa is needed, and at present these species may be regarded as cryptic and only separable by sequence data. Basidiomata of P. pseudoinsidiosus with a bluish stipe may resemble Pluteus cyanopus Quél. However, by its protologue [45], this species has a black or purplish pruinose pileus, greyish or lilac lamellae and basidiospores about 6 µm. Vellinga [3] interpreted this species based on recent collections with a bluish stipe by having a brown pileus, (narrowly) utriform to pedunculate and ovoid-conical pleurocystidia and narrowly utriform to ovoid cheilocystidia without rostrum. A discussion on the true identity of Pluteus cyanopus falls out of the scope of the present paper, but it will be discussed in another prepared article about the taxa placed in the /cinereofuscus and /phlebophorus clades sensu Menolli et al. [9] and Malysheva et al. [14]. Pluteus phaeocyanopus, Minnis and Sundb., also has a bluish stipe, but has larger basidiospores (6.2-8.4 × 5.7-7.9 µm), (narrowly) lageniform pleurocystidia with pedicel, long neck and obtuse apex with brown intracellular pigment and subglobose to pyriform cheilocystidia with brown intracellular pigment [46]. This species is known from Western North America (San Francisco, CA, USA), and molecular data based on Californian collections show that this species belong in the/cinereofuscus Clade, not related with the P. insidiosus complex (Data not shown).    40 20) in center, light brown towards the margin (RAL 070 60 20) becoming grey-brown with age (RAL 060 60 05); hygrophanous, striate up to half of radius. Surface opaque, velvety or glabrous, weakly to strong venose at the center. Lamellae (L = 40-44/1 = 0-2) moderately crowded, free, slightly ventricose up to four millimeters broad, initially whitish later pink with whitish flocculose edge. Stipe 25-32 × 1.5-2.0 mm, cylindrical usually broadened towards base, pubescent and striate lengthwise, whitish to light grey. Smell and taste not distinctive. Context white. Notes: Pluteus farensis is characterized by having a brown opaque pileus surface, weakly to strongly venose at the center; a whitish to light grey pubescent stipe; cheilocystidia and pleurocystidia mucronate or with short rostrum at the apex, basidiospores mainly globose or subglobose and caulocystidia present only on the upper part of the stipe. Pluteus pseudoinsidiosus is macroscopically similar to P. farensis, but differs as the first has cheilocystidia with longer rostrum at the apex up to 40 µm and sterile lamella edges, although this last feature needs further investigation (see Notes on P. pseudoinsidiosus). Pluteus flavostipitatus is similar to P. farensis but mainly differs for its yellowish stipe, absence of caulocystidia and smaller basidiospore size (4.9-5.7(-6.1) × (4.3-)4.5-5.1(-5.8) µm). Pluteus assimilatus is distinguished from P. farensis by the presence of caulocystidia rather numerous over the entire stipe surface and pleuro-and cheilocystidia without rostrum. Pluteus reisneri is macroscopically similar to P. farensis, but differs in having abundant caulocystidia grouped in clusters on the lower part of stipe, pileipellis with some rostrate elements at the apex and the lack of the pleurocystidia.       Notes: Pluteus flavostipitatus is characterized by rather small basidiomata with wrinkled pileus striate-sulcated at the margin, candle yellow and fibrillose stipe, rostrate pleuroand cheilocystidia, rather small (5.0-6.2 × 4.5-5.7 µm) globose or subglobose basidiospores and the absence of caulocystidia. The most morphologically similar taxon, P. pseudoinsidiosus, differs from P. flavostipitatus in the whitish or silvery grey, rarely bluish stipe, long and prominent needle-like rostra of both pleuro-and cheilocystidia, slightly longer basidiospores (5.0-7.0 × 4.3-5.5 µm) and larger elements of pileipellis (35-58 × 15-31 µm). Differently from P. flavostipitatus, P. insidiosus possesses a larger, dark brown or black-brown pileus, whitish stipe, larger basidiospores and pleurocystidia that are never rostrate. Pluteus farensis is distinguished from P. flavostipitatus by the whitish stipe, the presence of caulocystidia and its different habitat.  Notes: Pluteus flavostipitatus is characterized by rather small basidiomata with wrinkled pileus striate-sulcated at the margin, candle yellow and fibrillose stipe, rostrate pleuro-and cheilocystidia, rather small (5.0-6.2 × 4.5-5.7 µm) globose or subglobose basidiospores and the absence of caulocystidia. The most morphologically similar taxon, P. pseudoinsidiosus, differs from P. flavostipitatus in the whitish or silvery grey, rarely bluish stipe, long and prominent needle-like rostra of both pleuro-and cheilocystidia, slightly longer basidiospores (5.0-7.0 × 4.3-5.5 µm) and larger elements of pileipellis (35-58 × 15-31 µm). Differently from P. flavostipitatus, P. insidiosus possesses a larger, dark brown or black-brown pileus, whitish stipe, larger basidiospores and pleurocystidia that are never rostrate. Pluteus farensis is distinguished from P. flavostipitatus by the whitish stipe, the presence of caulocystidia and its different habitat.
Habitat and distribution: on trunks of broadleaved tree. The holotype was collected in the northwestern region of Russia, and the second collection studied was collected from Estonia. Thus, a presumable distribution area of the species may be limited to the territory of Northern Europe, but for precise knowledge additional finds are needed.
Habitat and distribution: on trunks of broadleaved tree. The holotype was collected in the northwestern region of Russia, and the second collection studied was collected from Estonia. Thus, a presumable distribution area of the species may be limited to the territory of Northern Europe, but for precise knowledge additional finds are needed.   Notes: Pluteus assimilatus shares several morphological features with P. insidiosus, having dark-colored pileus coupled with whitish or light-colored stipe, and broadly ellipsoid or subglobose basidiospores. However, the new species differs from the latter in absence of rostrate cheilocystidia, the presence of numerous caulocystidia, which have not been observed in P. insidiosus, larger cheilocystidia (41.5-71.0 × 16.5-32.5 µm vs 20-54 × Notes: Pluteus assimilatus shares several morphological features with P. insidiosus, having dark-colored pileus coupled with whitish or light-colored stipe, and broadly ellipsoid or subglobose basidiospores. However, the new species differs from the latter in absence of rostrate cheilocystidia, the presence of numerous caulocystidia, which have not been observed in P. insidiosus, larger cheilocystidia (41.5-71.0 × 16.5-32.5 µm vs 20-54 × 6-23 µm) and smaller basidiospores. Due to this characteristic (non-rostrate cystidia), the species is more similar to P. nanus than other members of the P. insidiosus complex, but differs in the smaller size of the basidiospores and the narrower pleurocystidia of a different shape.
Based on the holotype, epitype and several recent collections, P. reisneri is characterized by a medium to dark brown or grey, slightly pruinose pileus, which is usually striate at the margin. In some collections the pileus has been noted as somewhat velvety, but glabrous and neither pruinose nor velvety after rain. The stipe has indistinct dark or rarely whitish floccules, cheilocystidia and caulocystidia are frequently to moderate often rostrate and the pileipellis consist of predominantly sphaeropedunculate, vesiculose to clavate and fusiform elements without apical excrescence. Some collections (BRNM788196, BRNM825833) have also rare elongate elements in the pileipellis, similarly to P. reisneri holotype and epitype. Rare rostrate pileipellis elements around the pileus center are present in both Slovenian and some Czech collections (e.g., BRNM788196, 788198), while only one short appendix was found in the holotype, and also in the epitype collection. In most collections the pileus was indistinctly wrinkled at the center, but the distinctly wrinkled collection (BRNM781263) selected as epitype evokes the P. reisneri protologue. Velenovský in 1921 [16] mentioned the similarity of P. reisneri with P. phlebophorus. The epitype fully match this feature. The protologue of P. reisneri reported that the stipe is pruinose only on the upper part and the species is known only from Rubus Notes: In the protologue [16], the stipe of P. reisneri was described as white, the pileus as grey-brown with a rugulose center and a striate margin, and it was stated to be growing on a Rubus sp. stem. Velenovský [16] mentioned subglobose basidiospores (7.0-8.0 µm long), but our type study found them to be slightly smaller. The pileipellis is formed by mostly sphaeropedunculate, broadly clavate or broadly fusiform elements, but also rare elongate elements up to about 70 µm long or rostrate elements are present. Cheilocystidia are rostrate, and caulocystidia are rostrate in some collections, while pleurocystidia were not found. The holotype is preserved in Velenovsky fluid (formaldehyde and ethanol solution [16,47]), and all attempts to sequence this more than 100 years old holotype have failed. To support an unambiguous identity of P. reisneri, we therefore designate an epitype based on collection from the Czech Republic, which fully corresponds to the original macroscopical description and microscopically match all features observed in the holotype.
Based on the holotype, epitype and several recent collections, P. reisneri is characterized by a medium to dark brown or grey, slightly pruinose pileus, which is usually striate at the margin. In some collections the pileus has been noted as somewhat velvety, but glabrous and neither pruinose nor velvety after rain. The stipe has indistinct dark or rarely whitish floccules, cheilocystidia and caulocystidia are frequently to moderate often rostrate and the pileipellis consist of predominantly sphaeropedunculate, vesiculose to clavate and fusiform elements without apical excrescence. Some collections (BRNM788196, BRNM825833) have also rare elongate elements in the pileipellis, similarly to P. reisneri holotype and epitype. Rare rostrate pileipellis elements around the pileus center are present in both Slovenian and some Czech collections (e.g., BRNM788196, 788198), while only one short appendix was found in the holotype, and also in the epitype collection. In most collections the pileus was indistinctly wrinkled at the center, but the distinctly wrinkled collection (BRNM781263) selected as epitype evokes the P. reisneri protologue. Velenovský in 1921 [16] mentioned the similarity of P. reisneri with P. phlebophorus. The epitype fully match this feature. The protologue of P. reisneri reported that the stipe is pruinose only on the upper part and the species is known only from Rubus sp. stem and in the moss among the grass [16]. The epitype (BRNM781263) was found on mosses decaying trunk of Quercus. No recent collection growing on Rubus sp. stem was found. It is rather distantly related to P. insidiosus and all other taxa treated in this paper, forming a sister clade to all these taxa and the P. thomsonii complex.

Discussion
The European specimens of Pluteus with a brown pileus, a whitish or silvery grey stipe, a pileipellis formed as a hymeniderm of sphaeropedunculate to narrowly clavate elements and some cheilocystidia with rostrum were traditionally identified as Pluteus insidiosus [3,12,48,49]. Without the molecular analysis support, it would be impossible to decide whether the subtle differences observed between individual collections represented one or more species, but with this study, we have revealed at least six species within the P. insidiosus broad sense from Eurasia. Several of these appear to be semicryptic, at least based on the current knowledge, while others are easier to differentiate. Macroscopically, typical basidiomata of P. reisneri clearly differ from all similar species by the combination of a slightly velvety-pruinose pileus and a stipe with delicate dark or rarely whitish floccules. However, glabrous basidiomata were also found, especially after rain. Furthermore, P. farensis may appear with an opaque, velvety or glabrous pileus. Pluteus flavostipitatus is macroscopically recognizable by a candle yellow stipe color, while P. pseudoinsidiosus differs from all similar species only when a greenish-blue stipe is present.
Microscopically, important characteristics usable for species delimitation are the shape and size of cheilocystidia and pleurocystidia, and also the presence and shape of caulocystidia. Rostrate cheilocystidia are a predominant feature for most species in this group, and their shape varies in being narrowly to broadly clavate, narrowly to broadly (sub)utriform, (sub)fusiform, rarely inflated-fusiform, oblong or ellipsoid. Pluteus assimilatus is only one species without rostrate cheilocystidia, while cheilocystidia in the single known collection of P. flavostipitatus presented only short rostra. The presence of a sterile or heterogenous lamellar edge does not appear to be a useful feature in separating taxa recognized here (see notes of P. insidiosus), while the shape and partly also size of pleurocystidia are significant characteristics. Unfortunately, pleurocystidia are very rare in most taxa, which reduces the usefulness of this characteristic for distinguishing these species. Caulocystidia in tufts are common in the entire stipe of P. reisneri and P. assimilatus, mostly only near apex in P. farensis, rare in P. pseudoinsidiosus; and usually lacking in P. insidiosus and P. flavostipitatus. Some rostrate caulocystidia are present in P. reisneri and P. pseudoinsidiosus. More collection of these species may verify more distinct differences between them.
Most of the species described here appear on decayed wood (stumps or trunks) of angiosperms, sometimes on wood covered with mosses, rarely on the wood debris, only P. farensis has terrestrial growth. Pluteus reisneri has been recorded also from mossy ground among grasses and from Rubus sp. stem. The biogeography of the treated species needs further study as, previously, most of these collections would have been identified as Pluteus insidiosus. Based on phylogenetically confirmed findings, the presumable distribution area of P. flavostipitatus is Far Eastern Russia, P. assimilatus may be limited to the territory of Northern Europe (Estonia and Northwestern Russia), P. pseudoinsidiosus is widespread, but rare from Central Europe across southern Europe to Western Asia (the Czech Republic, France, Spain and Iran) and P. farensis is so far known only from Italy.
The hotspot of P. reisneri seems to be Central Europe, especially the Czech Republic, but this species is also known in Slovenia and Turkey. Basidiomata of true P. insidiosus were collected and molecularly confirmed only from the Netherlands. However, data from Estonian soil samples show that P. insidiosus is also present in Estonia. Environmental DNA samples have also detected P. assimilatus, P. farensis and P. pseudoinsidiosus, broadening their known geographic distribution beyond the collection-based records. The distribution of the European species in this complex seems to be broad and overlapping to some degree.
The nuclear ribosomal internal transcribed spacer (nrITS) has been proposed as the universal barcode marker for fungi [50], but currently, a universal specific threshold value has not been recommended for Pluteus in previous studies. It seems likely that the traditional 97% similarity cut-off point is too broad to accurately separate Pluteus species in many instances. We do recommend the use of TEF1-α as a complementary barcode in Pluteus, especially when nrITS variation suggests the presence of more than one species within well-supported nrITS clades. Considering the "Clade I" which includes P. insidiosus, our results indicate that an nrITS patristic distance of 0.0134 (corresponding to nine different nucleotides including gaps in the whole region) is enough to separate two different species. The nrITS1 region is less variable than nrITS2, and patristic differences greater than 0.0082 can be considered as a possible threshold value in nrITS1, whereas this threshold value represents the maximum intraspecific value. The absence of conflicts between the nrITS and TEF1-α phylogenetic analyzes conducted separately confirms that this combination of markers is suitable for the separation of different phylospecies, as already reported in previous studies [51,52]. Conversely, the use of a combined nrITS and TEF1-α dataset in phylogenetic analyses do not accurately resolve the relationship between different species within "Pluteus insidiosus complex" suggesting that other markers will be needed to confirm these relationships. Data Availability Statement: Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov, accessed on 7 January 2021; https://www.mycobank.org, accessed on 5 April 2022.