New data in Porotheleaceae and Cyphellaceae: epitypification of Prunulus scabripes Murrill, the status of Mycopan Redhead, Moncalvo & Vilgalys and a new combination in Pleurella Horak emend.

Mycopan is a genus established for Hydropus scabripes by Redhead, Moncalvo & Vilgalys (in Redhead 2013). They considered the genus to be distinct based on morphology and the phylogenetic analysis by Moncalvo et al. (2002) which included a sequence of Hydropus scabripes (AF042635, DAOM 192847) unrelated to the type species of Hydropus (H. fuliginarius). Subsequent sequences of material identified as Hydropus scabripes are not conspecific with the sequence of DAOM 192847. We consider this sequence (obtained from a mycelium culture) to be misidentified. We investigated the true phylogenetic position of authentic Mycopan including genera previously included in Cyphellaceae and Porotheleaceae. Sixteen collections of M. scabripes from Europe and North America were studied on morphological and molecular basis (nrITS and nrLSU sequences). No sequences were obtained from the holotype of Mycopan scabripes, and we designate an epitype to fix the interpretation of this species and the genus Mycopan. Mycopan is maintained as a good genus nested within Cyphellaceae as sister to the mycenoid genus Atheniella. The misidentified Hydropus scabripes AF042635 (DAOM 192847) represents a different species that is closely related to the holotype (and a new Italian collection) of Hebelomina microspora and the monospecific genus Pleurella described from New Zealand. Consequently, Hebelomina microspora is transferred to the emended genus Pleurella, which is sister to Baeospora within the Cyphellaceae. Additionally, based on these phylogenetic results, an updated taxonomic arrangement of Cyphellaceae and Porotheleaceae is proposed, emphasizing once again the polyphyletic nature of Hydropus and Gerronema.


Introduction
The genus Hydropus Kühner ex Singer, based on Agaricus plexipes b fuliginaria Batsch (1783), traditionally contains pileostipitate saprotrophic species with mycenoid, omphalinoid to collybioid habit, non-or weakly amyloid trama, non-diverticulate cystidia, amyloid or inamyloid spores, and a pileipellis without diverticulate elements, generally at least in part subhymeniform and/or with ventricose/ swollen pileocystidia (Singer 1982(Singer , 1986Hausknecht et al. 1997;Contu and Robich 1998;Bas 1999;Estève-Raventós et al. 2002). It was described originally by Kühner (1938) as an informal group/subsection of the genus Mycena (Pers.) Roussel, within the subgenus Eu-Mycena section Spuriae which encompasses puzzling species with non-or weakly amyloid trama and non-diverticulate cystidia and pileipellis elements. Hydropus was then raised to the genus level by Singer (invalidly 1942Singer (invalidly , validly 1948. It was enlarged by Kühner (1980) to encompass Megacollybia Kotl. & Pouzar (as subgenus). Species of Hydropus are widespread especially in tropical areas (e.g., Singer and Grinling 1967;Singer 1973Singer , 1982Pinheiro et al. 2013;Cooper et al. 2018), but they also occur in the temperate zone (e.g., Kotlaba and Pouzar 1962;Moser 1968;Bas 1983Bas , 1999Bas and Weholt 1984;Robich 1986Robich , 1990Robich , 1992Maas Geesteranus and Hausknecht 1993;Contu and Robich 1998;Esteve-Raventós et al. 2002;Moreau and Courtecuisse 2004;Holec 2008a, b;Laessøe 2008Laessøe , 2012Deceuninck 2012Deceuninck , 2019. There has been no detailed molecular phylogenetic study of the genus, but it is recovered as strongly polyphyletic (Moncalvo et al. 2002;Bodensteiner et al. 2004;Matheny et al. 2006;Eberhardt et al. 2018;Antonín et al. 2019;Varga et al. 2019;Vizzini et al. 2019;Kaygusuz et al. 2020;Matheny et al. 2020;Sánchez-García et al. 2020). Species placed in the genus are spread over numerous independent evolutionary lines (hydropoid fungi) in the Porotheleaceae Murrill and Cyphellaceae Lotsy (Eberhardt et al. 2018), families which are part of the suborder Marasmiineae Aime, Dentinger & Gaya as circumscribed on a phylogenomic basis by Dentinger et al. (2016) (= Marasmioid clade s. Matheny et al. 2006;Binder et al. 2010). The first evolutionary line of hydropoid fungi to be recognized as an independent genus from Hydropus was the monospecific Mycopan Redhead, Moncalvo, Vilgalys (in Redhead 2013). Mycopan is based on Prunulus scabripes Murrill [≡ Hydropus scabripes (Murrill) Singer]. It was established as independent using a reference (nrLSU) sequence present in the phylogenetic analyses by Moncalvo et al. (2000Moncalvo et al. ( , 2002 (AF042635, D A O M 1 9 2 8 4 7 ) w h e r e t h e s e q u e n c e , l a b e l l e d H. scabripes, is sister to Baeospora Singer and unrelated to the type of Hydropus (H. fuliginarius (Batsch) Singer). Prunulus scabripes was originally described based on material from New York state (USA) (Murrill 1916a). It has been found subsequently in other parts of North America and Europe (e.g., Kühner 1938;Smith 1947;Hausknecht et al. 1997;Bas 1999;Laessøe 2008Laessøe , 2012 and North Africa (Morocco, Malençon and Bertault 1975). Only a few sequences attributed to Hydropus/Mycopan scabripes have been deposited in public databases (GenBank) after the work by Moncalvo et al. (2002) and these unexpectedly do not match the AF042635 sequence (see the phylogenetic analysis in Eberhardt et al. 2018). The aim of the present paper is to i) morphologically study and sequence European and North American collections of Hydropus scabripes (holotype included); ii) establish their placement within Cyphellaceae/Porotheleaceae; iii) select a North American newly sequenced collection as an epitype; and iv) provide a modern morphological description of Hydropus scabripes and comment on the discrepancy between the sequences of H. scabripes present in GenBank.

Collections studied
Samples studied are identified with the code of the public fungaria (Thiers, continuously updated) where they are stored: AMB, BRNM, MCVE, NY, PDD and TENN-F. Sample collection data are presented in the Taxonomy part and supplementary materials.

Morphological studies
Macroscopic morphological features were studied in fresh specimens. Colour abbreviations follow Munsell (1988). The following abbreviations are employed: L = number of lamellae reaching the stipe, l = number of lamellulae between each pair of lamellae. Microscopic structures were examined in dried material or fresh material (Pleurella microspora) using different mounting media: water, L4 (Clémençon 1972), Melzer's reagent, ammoniacal Congo red, phloxine, Cresyl blue and Cotton blue. Dried pieces of the samples were rehydrated in water and mounted in L4. Microscopic measurements were carried out with a Nikon Eclipse 80i, Olympus BX-50 and Zeiss Primostar light microscopes, using immersion oil at ×1000. Spore measurements were made by capturing images of a single visual field with multiple spores (taken from lamellar squashes of exsiccate material of mature specimens) which were then measured using the DS-L1 Nikon camera control unit. Spore dimensions do not include the hilar appendix, and are reported as follows: average minus standard deviation of length − average plus standard deviation of length × average minus standard deviation of width − average plus standard deviation of width; Q = average minus standard The approximate spore volume was calculated as that of an ellipsoid (Gross 1972;Meerts 1999). The notation [n/m/p] indicates that measurements were made on "n" randomly selected spores from "m" basidiomes of "p" collections. The width of the basidia was measured at the widest part, and the length was measured from the apex (sterigmata excluded) to the basal septum. Microscopy images were taken using a Nikon DS 5M digital camera connected to the microscope with both brightfield and interferential contrast optics.

DNA extraction, amplification, and sequencing
Total DNA was extracted from dry specimens (Table 1) employing a modified protocol based on Murray and Thompson (1980). PCR amplification (Mullis and Faloona 1987) included 35 cycles with an annealing temperature of 54°C. Primers ITS1F and ITS4 or ITS4B (White et al. 1990;Gardes and Bruns 1993) were employed for the ITS rDNA region, while LR0R and LR5 (Vilgalys and Hester 1990;Cubeta et al. 1991) were used to amplify the LSU/28S rDNA region, EF1-728F, EF1-983F and EF1-1567R (Carbone and Kohn 1999;Rehner and Buckley 2005) for the translation elongation factor 1α (TEF1) gene, and bRPB2-6F2 (reverse of bRPB2-6R2), bRPB2-7.1R2 and bRPB2-7R2 for the RNA polymerase II second largest subunit (RPB2) gene (Liu et al. 1999;Matheny et al. 2007). PCR products were checked in 1% agarose gels, and positive reactions were sequenced with one or both PCR primers. Chromatograms were checked searching for putative reading errors, and these were corrected. The sequences obtained were assembled separately and checked on sight for possible errors in reading with MEGA 6.0 (Tamura et al. 2013), pre-aligned with its MUSCLE application and then manually corrected.

Phylogenetic analyses
BLAST (Altschul et al. 1990) was used to select the most closely related sequences from public databases (INSDC/ GenBank https://www.ncbi.nlm.nih.gov/genbank/, UNITE https://unite.ut.ee/, BOLDSYSTEMS http://www. boldsystems.org/). Available sequences of generic type species were also included. Only ribosomal markers were used because there are few species of the two families represented by deposited sequences of extra ribosomal (protein-coding) genes. Sequence data management was carried out using MESQUITE (Maddison and Maddison 2017). The nrITS and nrLSU alignments were carried out separately.
In the ITS alignment the 18S region was trimmed; it was not partitioned into ITS1, 5.8S and ITS2. Gaps and variable regions were filtered using GBlocks version 0.91b software (Talavera and Castresana 2007) with less stringent selection allowing smaller final blocks and gap positions within the final blocks. After trimming both ends the gaps were treated as missing data (as question marks). The accession numbers of the sequences are reported in Table 1. Two distinct datasets were built. (1) First, an alignment including 28S rDNA (nrLSU) sequences from species of the Cyphellaceae/Porotheleaceae found in previous phylogenetic studies (Moncalvo et al. 2002;Matheny et al. 2006;Eberhardt et al. 2018

Porotheleaceae+Cyphellaceae
The LSU alignment (890 bp), which included 108 sequences, was analysed using Bayesian Inference (BI) and the Maximum Likelihood (ML) methods implemented on the CIPRES Science Gateway v. 3.3 (Miller et al. 2010). The Bayesian analysis was performed using MrBayes 3.2.7a (Zhang et al. 2019) with the parameters of 2 simultaneous runs, 6 chains, temperature set at 0.2, sampling every 1000 generations, until reaching convergence (standard deviation less than 0.01) and PSRF (Potential Scale Reduction Factor) (Gelman and Rubin 1992) equal to 1 (after 2730000 generations). The 25% of sampled trees were discarded as burn-in. RAxML was run (Stamatakis 2014) using the standard search algorithm with 1000 bootstrap cycles. We estimated the best fit substitution model for each single alignment using the Bayesian information criterion (BIC) with jModelTest 2 (Darriba et al. 2012) and therefore selected the GTR+G+I model for all alignments. Flammulina velutipes (Physalacriaceae Corner) was used as the outgroup. The resulting trees were read with the SEAVIEW version 4 (Gouy et al. 2010) and saved in a vector format for printing. Both BI and ML analyses produced the same topology.

Cyphellaceae
The data matrices of ITS (572 bp) and LSU (911 bp), which covered a total of 66 sequences and 56 sequences, respectively, were combined into a single multilocus matrix. This matrix (1483 BP), which covered a total of 78 sequences, was analyzed by using the Bayesian Inference (BI) and the maximum likelihood (ML) criteria as above and using the same parameters (Fig. 2).
As all attempts (also including Illumina MiSeq technology) made to produce molecular data from the Prunulus scabripes holotype failed, a North American collection whose morphological characteristics fit well the protologue, the morphological data of the holotype and the descriptions of most of the authors was selected as epitype. As previously pointed out by Kühner (1938) and Smith (1947) (only on a morphological basis), the North American and European collections are conspecific ( Fig. 2 and Table 2).
A morphologically closely related species, Mycena trichoderma Joss., was described from France (in Kühner 1938) and later also combined in Hydropus . After the original description, this species has been found in several European areas (Josserand 1965;Enderle 1985;Robich 1986Robich , 1990Dämon 1995;Hausknecht et al. 1997;Watling and Turnbull 1998;Hübner 2004;Laessøe 2008Laessøe , 2012Pérez-de-Gregorio and Picón 2011) and in the USA (Michigan, Smith 1947). Hausknecht et al. (1997) described H. trichoderma var. lobauensis from Austria, as a taxon with bisporic basidia and absence of clamp connections, taxon found also in Italy by Robich (2004). The main characters differentiating Hydropus trichoderma from M. scabripes seem to be the greater abundance of larger pileocystidia and the presence of heavily encrusting pigment on the hyphae of the pileipellis and subcutis. However, these incrustations can be rather inconspicuous and scarce.
Our molecular analysis (Fig. 2) Fig. 7). The third subclade consists of a newly sequenced collection of Hydropus paradoxus Moser from Italy (AMB 18772). These subclades are not completely resolved using ribosomal markers, but some differential morphological characters (see below) and TEF1/RPB2 sequences indicate that they are independent entities (Table 2). As "Hydropus scabripes" DAOM 192847 falls within the Pleurella clade, it is a misidentified collection.
The above mentioned Italian Mycena dura collection (incorrectly referred to as Swedish in GenBank) represents a misidentification because the real M. dura Maas Geest. & Hauskn. is a member of Mycena sect. Calodontes (M. pura complex), as stated by Harder et al. (2010Harder et al. ( , 2013 and Olariaga et al. (2015). The close molecular affinity of Mycena dura MCVE 507a with H. scabripes was already highlighted by Cooper (2016a) and Eberhardt et al. (2018), but without providing any comment. Original microscopical observations by HS indicate the collection as conspecific with M. scabripes.

Robich 2004
Hydropus trichoderma var. lobauensis 9-11 × (6-)7.5-9 2 -s p . stipe base (Smith 1947;Lonati 1986;Esteve-Raventós and Ortega 1995;Esteve-Raventós et al. 2002). According to the literature and our observations, M. scabripes is a macro-and microscopically quite variable species depending on the collection examined. The pileus can be grey, dark grey-brown, dark olivaceous grey-brown to nearly blackish (Fig. 3-4a, b); the surface can be glabrous or slightly rugulose to tomentose. There are variants with an almost glabrous, or a white-pruinose or brownish pruinose stipe. Some collections show very rare and short pileocystidia, while in others pileocystidia are rather large and more abundant. The pigment in the elements of the pileipellis is always vacuolar, accompanied or not by scarce or abundant encrusting extracellular pigment. A great range of variation in spore size is observed (Table 3), as already noted by Kühner (1938), Smith (1947), Josserand (1965) and Bas (1999), and this variability is related to the number of basidial sterigmata. As previously highlighted by Smith (1936), Bas (in Maas Geesteranus and Hausknecht 1993) and Bas (1998Bas ( , 1999, the holotype collection turned out to be bisporic. In our observations on the holotype (MM, HS), we found spores measuring 10-12 × 6-7.5 μm (n = 49), a mixture of bi-and tetrasporic basidia and clamp connections, as observed in the selected epitype. Most collections we have studied display clamp connections, irrespective of being tetrasporic, bi/ tetrasporic or mono/bisporic (Table 3). The number of sterigmata varies not only according to the collection studied but also to the portion of lamella examined within the single basidiome. Our collections AMB 18738 (mono-bisporic) and AMB 18854 (bi-tetrasporic) exhibit no clamp connections. A possibility remains also of rare completely bisporic collections without clamp connections (Hausknecht et al. 1997 andRobich 2004 as H. trichoderma var. lobauensis;Enderle 1985;Bon 1995;Bas 1999;Laessøe 2008Laessøe , 2012Deceuninck 2019 and AA VV 2020 as H. scabripes var. scabripes).
Notes-The genus Pleurella was erected by Horak (1971) for Leucopaxillus ardesiacus, a lignicolous pleuropodal species from the North Island of New Zealand, characterized by smooth, amyloid spores [4.5-6(-6.5) × 2-3 μm], presence of cheilocystidia, abundance of extracellular brown encrusting pigment in the pileipellis elements, and clamp connections (Stevenson 1964;Horak 1971). Horak believed that the genus might have an affinity with Melanoleuca Pat. and Leucopaxillus Boursier due to its habit and amyloid spores. Pleurella was synonymized with Hydropus by Singer (1982). The earliest molecular-based inferences about the phylogenetic position of this genus are those of Sánchez-García et al. (2014): they first recognized a close relationship with Dennisiomyces Singer or Porpoloma Singer s. str. based on morphological similarities, but recovered, after a BLASTn search, the nearest alliance between the ITS sequence (JQ694106) publicly available from a collection (PDD 87446) referred to as Pleurella ardesiaca from New Zealand to Baeospora in the Marasmioid clade (Matheny et al. 2006;Binder et al. 2010;= Marasmiineae Dentinger et al. 2016) or to Hypsizygus (Gillet) Singer in the Lyophyllaceae. Cooper (2016a, b) considered Pleurella a member of Cyphellaceae Lotsy together with Cyphella, Cheimonophyllum, Baeospora, + "Mycopan" s. Redhead, without providing molecular evidence, but probably based on previous results which indicated, on a BLASTn basis, an affinity of Pleurella with Baeospora, the latter genus having been shown to belong to the Cyphellaceae by Moncalvo et al. (2000Moncalvo et al. ( , 2002 and Matheny et al. (2006). Eberhardt et al. (2018) recovered a well-supported Cyphellaceae clade consisting of Cyphella digitalis (type species of the genus), Baeospora myosura, Pleurella ardesiaca, Hebeloma microsporum holotype MF039240, and 'Hydropus scabripes' DAOM 192847 (indicated as "Mycopan?"). He et al. (2019) and Kalichman et al. (2020) still considered it as an incertae sedis genus at a family level within the Agaricales (Marasmiineae).
Habitat and distribution: Solitary or gregarious on or associated with well decayed wood or forest litter. So far known only from Italy. Very rare.
Notes-Hebelomina microspora Alessio & Nonis (belonging to the genus Hebelomina which is artificial according to Cittadini et al. 2008;Vizzini et al. 2014;and Eberhardt et al. 2018) was originally established (Alessio and Nonis 1977) to replace Hebelomina microspora Huijsman (Huijsman 1946), invalidly published without a Latin diagnosis (Art. 39.1; Turland et al. 2018). Later, Huijsman (1978) validated it unaware of Alessio & Nonis' proposal, but as unfortunately that species seems to be quite different from the H. microspora described in his paper, he erected, in a note added in proof, Hebelomina neerlandica Huijsman as a nomen novum for Hebelomina microspora. Hebelomina neerlandica was later transferred to Gymnopilus (Cittadini et al. 2008;Eberhardt et al. 2018). Contu (in Cittadini et al. 2008) combined the species of Alessio & Nonis in Hebeloma, without providing any justification and above all without having seen the holotype.
Hebelomina microspora was described by Alessio and Nonis (1977) and Alessio (1981) as characterized by an hemispheric-convex pileus with an obtuse umbo, surface dry, at first whitish-cream, soon ochre-brown and clay to pale orange spotted; emarginate lamellae, whitish cream, then with strong orange hues, ochre-brown when old; a cylindrical stipe, central, concolorous to the pileus, flocculose at apex; a white spore print; strongly amyloid spores, 6-8(-9) × 4-4.5 μm; abundant colourless cheilocystidia; a smell at first of elderberry flowers then rancid floury, taste bitter; and growth on buried and decayed conifer wood (Pinus strobus) in May, in a city park (Turin, Italy).
The species, no longer found after the original description, remained of dubious generic attribution until Eberhardt et al. (2018) examined and sequenced the holotype. They found it in bad condition and without spores, but cheilocystidia and pleurocystidia, the latter being numerous and not reported in the original diagnosis, were detected. In the molecular analysis published in their work, the ITS/partial LSU sequence of the holotype clustered within Cyphellaceae as sister (MLB 100%) to Hydropus scabripes DAOM 192847 whose sequence was considered a reference for the establishment of the genus Mycopan by Redhead (2013). This couple is sister (MLB 92%) to Pleurella ardesiaca PDD87446, GB JQ694106. The clade Mycopan + Hebelomina microspora + Pleurella is sister (MLB 97%) to Baeospora myosura AFTOL-ID 1799.
In our phylogenetic analyses (Figs. 1-2) a well-supported Pleurella clade is recovered, containing Pleurella collections, the holotype and the novel Italian collection of H. microspora (AMB 18917) and the reference collection of M. scabripes.
The novel collection of H. microspora fits quite well the original description (Alessio and Nonis (1977) and its characteristics are such that it can be mistaken for Hydropus scabripes apart from the lamellae with apricot tones, and the strongly amyloid subcylindrical spores. There is no reasonable doubt that the two collections of H. microspora are congeneric with Mycopan scabripes DAOM 192847. The phylogenetic analysis also suggests that H. microspora can be considered to belong to the genus Pleurella. Pleurella ardesiaca and H. microspora share a whole range of characters such as emarginate crowded lamellae, white spore print, ellipsoid to subcylindrical strongly amyloid spores, presence of cheilocystidia and caulocystidia, elements of the pileipellis with abundant extracellular encrusting pigment, a sarcodimitic stipe trama and growth on dead wood. Pleurella ardesiaca differs by having usually pleuropodal basidiomes (stipe excentric or lateral, Fig. 4e), but some collections may show a nearly central stipe (Horak 1971), a trichodermic/ hymenidermic pileipellis, and an absence of pleurocystidia (Horak 1971 and Cooper, https://scd.landcareresearch.co.nz/ specimen/PDD_87446), even though facial cystidia may be rare and scattered in H. microspora (the novel collection) and can easily go unnoticed. Since these differences appear to be of minimal importance, we decided to transfer H. microspora to Pleurella and we propose the emendation of this genus (see above). Cooper (2016b) had previously suggested a possible congenericity between Hydropus scabripes DAOM 192847 (GB AF042635) and Pleurella ardesiaca.
Pending future studies on the tropical species of Baeospora, for the moment we consider Pleurella and Baeospora as two distinct sister genera. In case of synonymization of both taxa by future authors, Baeospora would have priority, on a temporal basis, over Pleurella.

Discussion
The current state of knowledge of the Porotheleaceae and Cyphellaceae and polyphyly of the genus Hydropus Data availability The sequence data generated in this study are deposited in NCBI GenBank.
Code availability Not applicable.

Declarations
Ethics approval Not applicable.

Consent to participate Not applicable.
Consent for publication Not applicable.

Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.