Mycological Progress (2019) 18:713–739 https://doi.org/10.1007/s11557-019-01483-5 ORIGINAL ARTICLE Progress on the phylogeny of the Omphalotaceae: Gymnopus s. str., Marasmiellus s. str., Paragymnopus gen. nov. and Pusillomyces gen. nov. Jadson J. S. Oliveira 1,2 & Ruby Vargas-Isla 2 & Tiara S. Cabral 2,3 & Doriane P. Rodrigues 4 & Noemia K. Ishikawa 1,2 Received: 9 August 2018 / Revised: 22 February 2019 / Accepted: 26 February 2019 # German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Omphalotaceae is the family of widely distributed and morphologically diverse marasmioid and gymnopoid agaric genera. Phylogenetic studies have included the family in Agaricales, grouping many traditionally and recently described genera of saprotrophic or parasitic mushroom-producing fungi. However, some genera in Omphalotaceae have not reached a stable concept that reflects monophyletic groups with identifiable morphological circumscription. This is the case of Gymnopus and Marasmiellus, which have been the target of two opposing views: (1) a more inclusive Gymnopus encompassing Marasmiellus, or (2) a more restricted Gymnopus (s. str.) while Marasmiellus remains a distict genus; both genera still await a more conclusive phylogenetic hypothesis coupled with morphological recognition. Furthermore, some new genera or undefined clades need more study. In the present paper, a phylogenetic study was conducted based on nrITS and nrLSU in single and multilocus analyses including members of the Omphalotaceae, more specifically of the genera belonging to the /letinuloid clade. The resulting trees support the view of a more restricted Gymnopus and a distinct Marasmiellus based on monophyletic and strongly supported clades on which their morphological circumscriptions and taxonomic treatments are proposed herein. The results also provide evidence for the description of two new genera: Paragymnopus and Pusillomyces. Pusillomyces manuripioides sp. nov. (type species of the genus) is described with morphological description, taxonomic and ecological remarks, and illustrations. Keywords Agaricales . Amazon forest . Marasmioid . Gymnopoid . Plant pathogen . Taxonomy Section Editor: Zhu-Liang Yang Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11557-019-01483-5) contains supplementary material, which is available to authorized users. * Jadson J. S. Oliveira oliveira.j.j.s.86@gmail.com 1 Divisão do Curso de Pós-graduação em Botânica – DIBOT, Instituto Nacional de Pesquisas da Amazônia – INPA, Av. André Araújo 2936, Manaus, AM 69067-375, Brazil 2 Coordenação de Biodiversidade – COBIO, Instituto Nacional de Pesquisas da Amazônia – INPA, Av. André Araújo 2936, Manaus, AM 69067-375, Brazil 3 Divisão do Curso de Pós-graduação em Genética, Conservação e Biologia Evolutiva – DIGEN, Instituto Nacional de Pesquisas da Amazônia – INPA, Av. André Araújo 2936, Manaus, AM 69067-375, Brazil 4 Laboratório de Evolução Aplicada, BLM, Divisão de Biotecnologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. General Rodrigo Otávio Jordão Ramos 3000, Manaus, AM 69077-000, Brazil Introduction Several groups in the euagarics have been the target of important changes in their classification as multiple molecular phylogenetic studies have shaped new systematic understanding in the Agaricales (Moncalvo et al. 2000, 2002; Binder and Hibbett 2002; Matheny et al. 2006; Dentinger et al. 2016). One of the groups of this order that has undergone changes, the family Omphalotaceae was initially proposed within the Boletales (sensu Kühner) by Kämmerer et al. (1985) based on the typical sesquiterpenes and the ability to cause white-rot and including genera such as Omphalotus Fayod and Lampteromyces Singer. Additionally, many current members of Omphalotaceae were classified in Tricholomataceae in Singer’s system (Singer 1986) and in Marasmiaceae in Kühner’s system (Kühner 1980). However, based on morphology, Halling (1996) and Antonín et al. (1997) recombined several species or transferred complete sections out of Collybia (Fr.) Staude to Gymnopus (Pers.) Roussel. 714 The family, as currently accepted, was classified in the Agaricales based on phylogenetic studies and Collybia was shown to be polyphyletic (Moncalvo et al. 2000, 2002, Matheny et al. 2006). Collybia dryophila (Bull.) P. Kumm. and C. polyphylla (Peck) Singer ex Halling [combined in Gymnopus in Murrill (1916) and Antonín et al. (1997), respectively], and C. maculata (Alb. & Schwein.) P. Kumm. [combined in Rhodocollybia in Singer (1939)] grouped in the Bclade A^ (Moncalvo et al. 2000) that corresponds to the clade /omphalotaceae in Moncalvo et al. (2002), which is unrelated to the clade /collybia. In Matheny et al. (2006), the clade Omphalotaceae [equivalent to /omphalotaceae in Moncalvo et al. (2002)] grouped within the Marasmioid major clade (IV), but C. tuberosa (type of Collybia) branched in the subclade Clitocybeae [Collybia - Clitocybe group including /collybi a i n M oncalvo e t al . ( 20 02 )] w i t h i n t h e Tricholomatoid major clade (V). Gymnopus branched as a member of the clade Omphalotaceae showing no close relatedness with Collybia s.str. The clade Omphalotaceae grouped as sister to the clade Marasmiaceae with strong support in the same major clade (Matheny et al. 2006) and in the suborder Marasmiineae (Dentinger et al. 2016). However, Gymnopus branched as multiple, nonmonophyletic groups as also did Marasmiellus Murril (Moncalvo et al. 2002; Mata et al. 2004a; Wilson and Desjardin 2005; Hughes et al. 2010; Petersen and Hughes 2017), this latter historically segregated from Marasmius Fr. [see Mata et al. (2004a), Wilson and Desjardin (2005) and Antonín and Noordeloos (2010)]. Based on analysis of nrITS data, Mata et al. (2004a) argued that, by placing Marasmiellus juniperinus Murrill (type species of the genus) within a clade dominated by accepted Gymnopus taxa, either Gymnopus would be monophyletic including Marasmiellus as a synonym, or the limited clade to which M. juniperinus belongs would be a segregated genus (Marasmiellus sensu Wilson and Desjardin (2003) or Collybiopsis Earle) and Gymnopus would be polyphyletic. Mata et al. (2004a) accepted the first scenario, proposing the combination of M. juniperinus in a broad concept of Gymnopus. However, Wilson and Desjardin (2005) argued for keeping Marasmiellus and Gymnopus as distinct genera. Their results based on nrLSU data revealed an unsupported, but discrete clade grouping M. juniperinus and Marasmiellus synodicus (Kunze) Singer (sect. Dealbati), also including many taxa of Gymnopus sect. Vestipedes. They named the clade /marasmiellus, which was not closely related to their clade /gymnopus, this latter including Gymnopus fusipes (Bull.) Gray (type species) along with other taxa of the genus, as well as Setulipes androsaceus (L.) Antonín (type of Setulipes) and Micromphale Gray taxa [among them the type M. foetidum (Sowerby) Singer]. They concluded that until further species belonging to Gymnopus sect. Vestipedes and Levipedes, Marasmiellus (all sections) and Setulipes are added to the Mycol Progress (2019) 18:713–739 analyses, they do not support recognition of a more inclusive Gymnopus [sensu Mata et al. (2004a)]. They suggested that Marasmiellus s. str. could accommodate the members of Gymnopus sect. Vestipedes (i.e., /marasmiellus). Another argument was that by including Marasmiellus within a broad Gymnopus, then Rhodocollybia, Lentinula and Mycetinis Earle also would have to be accepted as synonyms of Gymnopus and then it would be monophyletic (Wilson and Desjardin 2005). Moreover, other Marasmiellus representatives grouped in other multiple unrelated positions. Despite these arguments, the broad concept of Gymnopus—encompassing Marasmiellus, Micromphale, and Mycetinis—was kept by Mata et al. (2006), using the most comprehensive sampling of nrITS sequence data for gymnopoid/marasmielloid taxa to date. This broad Gymnopus was represented by the clade A–N whereas it is possible to observe two major clades: (1) a superclade grouping the clades A–C, and (2) superclade D consisting of subclades E–N. In comparison with Wilson and Desjardin (2005), the first major clade can correspond to /gymnopus and the second (excluding current Mycetinis species) to /marasmiellus. Mata et al. (2006) also observed that their clade D is populated predominantly by members of Gymnopus sect. Vestipedes and corresponded to such section. Micromphale also had problematic phylogenetic relationships (Moncalvo et al. 2002; Mata et al. 2004a, 2006; Wilson and Desjardin 2005; Petersen and Hughes 2016). See more about the taxonomy of Micromphale in Antonín and Noordeloos (2010) and Petersen and Hughes (2016). Petersen and Hughes (2016) focused more on the uncertain phylogenetic position of Micromphale perforans (Hoffm.) Gray, analyzing it along with other taxa that could be considered in Micromphale sect. Perforantia with respect to the traditional taxonomy. They found a distinct and monophyletic clade grouping M. perforans, Gymnopus foliiphilus R.H. Petersen, G. pinophilus R.H. Petersen, G. ponderosae R.H. Petersen, G. sequoiae (Desjardin) R.H. Petersen, and G. sublaccatus R.H. Petersen, as sister (with very weak support) to a clade dominated by Gymnopus spp. corresponding to /gymnopus in Wilson and Desjardin (2005). According to the broad view of Mata et al. (2004a), using nrLSU for the large tree (Fig. 85 in their study), Petersen and Hughes (2016) combined Micromphale sect. Perforantia in Gymnopus sect. Perforantia. Hughes et al. (2010) proposed the new genus Connopus R.H. Petersen to accommodate Gymnopus acervatus (Fr.) Murrill, based on nrITS or nrLSU data, finding it somewhat closely related to Rhodocollybia taxa likewise in Mata et al. (2006). Dutta et al. (2015) also used nrITS or nrLSU data in singlelocus analyses for the phylogenetic placement of their new species, Marasmiellus foliiphilus A.K. Dutta, K. Acharya & Antonín, and records of four Gymnopus spp. based on collections from India. Marasmiellus foliiphilus grouped in the moderately supported clade A (Fig. 6a in their study) which Mycol Progress (2019) 18:713–739 corresponds to the clade /marasmiellus (Wilson and Desjardin 2005). Sandoval-Leiva et al. (2016), based on ITS–5’-28S rDNA, provided a more resolved tree of various marasmioid and gymnopoid genera [Marasmioid clade of Matheny et al. (2006)] and proposed the new and monotypic genus Gymnopanella Sandoval-Leiva, J.V. McDonald & Thorn. The pleurotoid, flabelliform to reniform Gymnopanella nothofagi Sandoval-Leiva, J.V. McDonald, and Thorn was sister to the clade named BGymnopus, including Setulipes^ [corresponding to /gymnopus in Wilson and Desjardin (2005)], but with no statistical support. They also found the clade /marasmiellus, corresponding to /marasmiellus in Wilson and Desjardin (2005), but with good statistical support. They did not discuss this finding though. Petersen and Hughes (2017) conducted an investigation on Mycetinis, also segregated from Marasmius. The genus was resurrected by Wilson and Desjardin (2005) based on a monophyletic group formed by members of the previous Marasmius sect. Alliacei (Kühner 1933; Singer 1976, 1986). This clade was distinct and strongly supported, named Bclade F^ within the Bclade A^ assigned to Omphalotaceae. In the Gymnopus phylogeny with related clades/genera, Petersen and Hughes (2017) also observed two major clades, one with numerous Gymnopus taxa, including G. fusipes, G. androsaceus (L.) Della Magg. & Trassin. (previously Setulipes), and Gymnopus sect. Perforantia, and the second also grouping several Gymnopus species, and additional clades treated as Connopus, Lentinula, Marasmius pallidocephalus Gilliam (probably a monotypic genus), and Rhodocollybia. They also included Gymnopanella as part of this second major clade. These studies approached groups within Omphalotaceae with single locus phylogenetic analyses using nrLSU or nrITS, except for Sandoval-Leiva et al. (2016) and Petersen and Hughes (2016). Sandoval-Leiva et al. (2016) used the combined ITS–5’-28S rDNA of a broad dataset composed of members of Omphalotaceae (broader sampling), Marasmiaceae, Physalacriaceae, and the outgroup formed by Pleurotaceae, Panellus P. Karst., and Entolomataceae strains, recovering a relatively well-resolved tree with many main clades strongly supported. However, their analyses (Bayesian and neighbor-joining) were apparently not partitioned and they only focused on the establishment of Gymnopanella, distinguishing it (phylogenetically and morphologically) from other genera. Petersen and Hughes (2016) used combined nrITS and nrLSU only for Gymnopus sect. Perforantia taxa (Fig. 86 in their study). Their restricted analyses were apparently not partitioned and only dealt with species level comparison within the section. According to MycoBank (Robert et al. 2013), Omphalotaceae has currently ten associated genera: Anthracophyllum Ces., Caripia Kuntze, Gymnopanella, Gymnopus, Lentinula, Marasmiellus, Mycetinis, Neonothopanus R.H. Petersen & Krisai, Omphalotus, and Rhodocollybia (all included in various 715 phylogenetic studies during these approximately past two decades). We do accept the combination of Micromphale in Gymnopus (but see the results of this study), and it would be probably the same for Caripia, awaiting more study. We also defend Mycetinis as a distinct genus, in agreement with Wilson and Desjardin (2005) and Petersen and Hughes (2017). Initially, this study began with the intent to infer the phylogenetic affiliation of an unusual fungal species (very similar to Manuripia Singer) collected in the central Amazonian tropical forest (Amazonas, Brazil), but with distinct micromorphological and ecological characteristics. LSU sequences (also combined with ITS) were used to determine in which marasmioid family this taxon was a member. The results from preliminary analyses not only indicated that this fungus is member of a new genus but also led to a partial revision of the Omphalotaceae, the family of the new fungus. This partial revision addresses the question of which view of Gymnopus would reflect more the natural systematics within the family: (i) the more inclusive Gymnopus sensu Mata et al. (2004a, 2006), or (ii) a more restricted Gymnopus suggested by Wilson and Desjardin (2005). Also, this study allowed the evaluation of the recently proposed genera, sections, and informal clades. Thus, we conducted thorough and broad Bayesian and maximum likelihood analyses to reconstruct phylogenies in Omphalotaceae, with more emphasis on the groups of the clade /lentinuloid (Moncalvo et al. 2002), using combined nrITS and nrLSU (multilocus) in partitioned processes, along with separate single locus analyses as explained in the methodology. The results include the description of the plant pathogen Pusillomyces manuripioides sp. nov. et gen. nov., Paragymnopus gen. nov. and a solution to the case of Gymnopus and Marasmiellus. Phylogenetic trees are provided, and morphological description, taxonomic and ecological comments, and illustrations of the new Amazonian species ascribed to Omphalotaceae. Material and methods Areas sampled T h e h o l o t y p e a n d 11 a d d i t i o n a l s p e c i m e n s o f Pusillomyces manuripioides sp. nov. were collected from the BReserva Biológica de Campina^ (RBC), Km 60 on the road BR-174, between Manaus and Boa Vista districts. The area has 900 ha of typical BCampina^ and BCampinarana^ (Online Resource – Fig. O6a) vegetation types, the former characterized by sparse shrubby and herbaceous vegetation on sandy white soil in an open and sunny savanna-like area while the latter consists of a wood of arboreal to subarboreal vegetation, with slightly open canopy, also on sandy soil. The specimens were found in the Campinarana area, which forms a matrix 716 where islands of Campina are found. The RBC is a protected area under the BInstituto Nacional de Pesquisas da Amazônia^ (INPA) management. Morphological examination The specimens were photographed both when fresh and dried. Macroscopic description was carried out based on fresh and dried material using a stereo microscope. Color coding follows Küppers (2002). For microscopic examination, samples were rehydrated in 70% ethanol, and thin sections were mounted in 3% KOH or in Melzer’s reagent. Basidiospores were described including dimensions as the range of length × the range of width, followed by xrm, the range of the arithmetic means of length × the same for width; xmm, the mean of the arithmetic means of length (± standard deviation (SD)) × the same for width (± SD); Qrm, the range of the means of length/ width; Qmm, the mean of the means of length/width (± SD); n, the number of spores measured; and s, the number of sampled collections. Basidiomata were photographed in the field using a digital camera, and in the laboratory using a Stereoscopic Microscope Leica M205 C with camera Leica MC 190 HD. Microstructures were also photographed using a digital camera coupled to the Leica DM 2500 optical microscope, with an image capture via LEICA EC3 camera and pre-edited in LAS EZ. Line drawings of the microstructures were made with a drawing tube and edited in CorelDRAW X7. The collections were deposited in the Herbarium INPA. DNA extraction, amplification, and sequencing DNA was extracted directly from cultures using a DNeasy Plant Mini Kit (Qiagen). The nrITS and nrLSU regions were amplified using ITS5/ITS4 and LR0R/LR5, respectively (White et al. 1990). The PCR reactions had a final volume of 25 μl and included 1 U Platinum® Taq DNA Polymerase, 1× PCR Buffer and 1 .5mM MgCl2 (Invitrogen), 3 mM of each dNTP (Promega Corporation), 2 μM of each primer, and 1 μl of DNA at 25 ng/μl. The PCR profile for the nrITS and nrLSU amplifications consisted of an initial step of 2 min at 95 °C, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and then a final extension step at 72 °C for 10 min. PCR fragments were visualized in a 1.5% agarose gel stained with GelRED™ (Biotium), under ultraviolet light. The fragments were purified using ExoSAP-IT™ (ThermoFisher Scientific) and then sequenced using BigDye™ Terminator v3.1 Cycle Sequencing (ThermoFisher Scientific). The quality of the electropherograms was analyzed in Geneious R7 (Biomatters Ltd.). Mycol Progress (2019) 18:713–739 Molecular data processing and phylogenetic analyses Corresponding/complementary forward and reverse reads were assembled to obtain clean consensus sequences in Geneious R7, with trimming of extremities. Sequences of nrLSU and nrITS regions were included in datasets according to three approaches: (a) DATASET 1, newly generated nrLSU sequences with those in the GenBank database (NCBI) of closest taxa (BLAST - http://blast.ncbi.nlm.nih.gov/) along with representatives of mostly genera of Omphalotaceae to form the ingroup, also including members of other families of the Marasmioid clade and the Mycenaceae clade, and the outgroup with members of the Tricholomatoid clade (Matheny et al. 2006); (b) DATASET 2, only nrITS data using the newly generated sequences with sequences of closest taxa/ genus revealed via BLAST searches in the GenBank database (NCBI) along with representatives of genera belonging to Omphalotaceae and representatives of Marasmiaceae as the outgroup (Matheny et al. 2006); (c) DATASET 3, newly generated sequences nrITS + nrLSU of the same collection combined data of representatives of genera belonging to Omphalotaceae in the GenBank database (NCBI) as the ingroup and combined sequences of a few representatives of Marasmiaceae as the outgroup. Sequences in GenBank (NCBI) were downloaded (Online Resource - Table O1), their quality checked, and used in the respective dataset. DATASET 1 served as a comprehensive analysis to verify the phylogenetic placement of the new species (and new genus) within the Marasmioid major clade of the Agaricales [or suborder Marasmiineae according to Dentinger et al. (2016)], testing the family where it belongs and whether or not the taxon represents a new genus in Omphalotaceae (family and genus level). DATASET 2 grouped representatives of a very comprehensive breadth of species of genera in Omphalotaceae to test the phylogenetic relationship of the new taxon at Bspecies level,^ since most of the Omphalotaceae species with publicly available sequence data are represented by nrITS. DATASET 3 tested the phylogenetic placement of the new taxon at genus and species level in a tentatively better tree resolution of the Omphalotaceae. All data used in this study are listed in the Online Resource - Table O1. The alignments were produced via MUSCLE (Edgar 2004). Ambiguously aligned regions were excluded from the nucleotide matrices by visual inspection in Geneious R7 (Kearse et al. 2012). The nucleotide substitution models were inferred via jModeltest 2.1.3 (Darriba et al. 2012) or MrModeltest 2.3. (Nylander 2004). All datasets and alignments can be found in TreeBASE 23780. The model selected per dataset was GTR+I+G for all partitions. We conducted MC3 Bayesian analyses (BA) with MrBayes 3.2.1 (Ronquist et al. 2012), using default settings from the model (Nst = 6). For all datasets, BA consisted of two independent runs: (a) 10,000,000 generations, sampling frequency Mycol Progress (2019) 18:713–739 717 every 1000 generations, six independent chains and two swaps, even for partitioned analysis (LSU and ITS concatenated). All burnin was set at 10%. Final trees were based on 50% majority-rule consensus method. Branch lengths went across the 95% highest posterior density trees. For maximum likelihood (ML), the trees (Online Resource Figs. O2, O4–O5) were reconstructed using the GTR+Γ+I model in RAxML 7.0.4 (Stamatakis 2006) with fast-bootstrapping implementing CAT approximations for 1000 pseudoreplicates and a full ML optimization for the final tree. Pairwise comparisons were performed in Geneious R7 using nrITS data when necessary. The phylogenetic trees were visualized and pre-edited in FigTree 1.3.1, and the main edition was conducted in CorelDRAW X7. Results Phylogenetic analyses Three analyses were conducted on three different datasets explained in the BMaterial and methods.^ The analyses on DATASET 1 resulted in the nrLSU trees (50% majority-rule consensus (tree length (TL)) = 23.821670 – Fig. 1, Online Fig. 1 Bayesian 50% majorityrule consesus tree from the single locus (nrLSU) analysis of DATASET 1 [tree length (TL) (mean of the means from two runs), 23.821670; −lnL (mean), − 14,951.21]. Support values at the nodes consist of PP ≥ 0.95 and BS ≥ 70; unsupported nodes under PP 0.5 are collapsed. Thicker stems in black represent highly supported nodes, and those in gray are moderately to weakly supported nodes. Major clades are simplified, representing family level groups as depicted in the figure. Outgroup consists of members of Clitocybe, Collybia, Entoloma, Lepista, Mycena s. str., and Tricholoma Resource Figs. O3, O4; and ML tree (TL) = 3.961914 in Online Resource Fig. O5). With the estimated marginal likelihood arithmetic mean between BA runs in − 14951.21, model parameters are summarized in Table 1. On an alignment of 737 distinct patterns, the ML analysis and applying GAMMA model parameters estimated up to an accuracy of 0.1000000000 log likelihood units, the final ML optimization likelihood was − 14,090.021877, and the parameters for DATASET 1 alignment are in Table 1. The tree ingroup (PP 1.0/BS 65) consisted of five relevant clades: /omphalotaceae (PP 0.95, BS 72), /physalacriaceae (PP 1.0, BS 97), /cyphellaceae (PP 0.99, BS 44), /hydropoid (PP 0.54, BS 18), and /marasmiaceae (PP 1.0, BS 66); the clade /mycenaceae (PP 1.0, BS 99), however, grouped within the outgroup. Pusillomyces manuripioides branched within /omphalotaceae. In the same broad tree, the new species grouped with Gymnopus asetosus Antonín, R. Ryoo & K. H. Ka and G. funalis (Har. Takah.) Antonín, R. Ryoo & K. H. Ka, all these taxa forming a distinct subclade (PP 1.0, BS 100). We also observed these three taxa closely related in the ITS trees (Online Resource Figs. O1, O2) from the analyses on the DATASET 2, forming a strongly supported subclade (PP 1.0, BS 100) in the ingroup (only members of Omphalotaceae). Omphalotaceae 0.98 Pusillomyces manuripioides sp. nov. 1.0 1.0 1.0 Marasmiaceae Physalacriaceae Hydropoid clade Cyphellaceae Outgroup 0.99 1.0 0.2 718 Table 1 Mycol Progress (2019) 18:713–739 Data from ML and BA analyses of DATASET 1 DATASET 1 alignment Taxa Characters 217 1340 Calculations from ML and BA ML (means) Alpha 0.209886 BA (means) 0.268692 Pinvar 0.440729 Substitution rates (A<=>C) (A<=>G) 1.027500 7.738497 0.047838 0.381134 (A<=>T) (C<=>G) 2.201518 0.482952 0.084752 0.026203 (C<=>T) (G<=>T) 11.054926 1.000000 0.423704 0.036369 pi(A) 0.270127 0.261056 pi(C) pi(G) pi(T) 0.191077 0.293142 0.245654 0.179064 0.258431 0.301449 Bases freq. The BA and ML analyses on DATASET 3 rendered wellresolved trees (50% majority-rule consensus tree (TL) = 36.167380—Fig. 2; and ML tree (TL) = 2.927921 in Online Resource Fig. O5) for the phylogenetic reconstruction of generic groups (included in this study) within the family. With the estimated marginal likelihood arithmetic mean between BA runs in − 16,947.29, model parameters are summarized in Table 2. On a partitioned alignment of 356 distinct patterns for nrITS and 395 distinct patterns for nrLSU, the ML analysis and applying GAMMA model parameters estimated up to the accuracy of 0.1000000000 log likelihood units for the two partitions, the final ML optimization likelihood was − 16,425.972776, and the parameters for DATASET 3 alignment are in Table 2. The tree ingroup (PP 1.0/BS 96) revealed 12 distinct generic representatives: /clade A—Marasmiellus s. str. (PP 1.0/BS 57), /clade B—Pusillomyces (PP 1.0/BS 100), /clade C—Connopus (PP 1.0/BS 100), /clade D— Pallidocephalus (PP 1.0/BS 96), /clade E—Rhodocollybia (PP 1.0/BS 66), /clade F—Lentinula (PP 1.0/BS 99), /clade G—Gymnopus s. str. (PP 1.0/BS 88), /clade H—Gymnopus sect. Perforantia (PP 1.0/BS 100), /clade I—Gymnopanella (PP 1.0/BS 77), /clade J—Mycetinis (PP 1.0/BS 100), /clade K—Omphalotus (PP 1.0/BS 100), and the lineage Anthracophyllum archeri (Berk.) Pegler (one strain). The representatives of Omphalotus and Anthracophyllum form a weakly supported clade (PP 0.87 / BS 50). /Clade B and /clade C are sister with weak support (PP 0.96/BS 47). These clades tend to group with /clade D, /clade E, and /clade F, but in an unsupported major clade (PP 0.66/BS -). This major clade (B, C, D, E, and F) is sister to clade A by an unsupported node (PP 0.76/BS 28). /Clade G and /clade H are paraphyletic because of the strain BKY026621 Marasmius sp.1 TFB3940,^ which without support appears as sister to /clade G (PP 0.83/BS 57). Thus, there are two unsupported major clades: (1) clades A, B, C, D, E, and F; (2) clades G and H. /Clade I and /clade J branched individually. An unsupported clade grouping /clade K and lineage L is the most basal in the ingroup. The lineage named as Gymnopus contrarius (Peck) Halling branched in an isolated position basal to the whole family, and may represent a distinct genus too, but more information is needed to reach a conclusion. The tree generated from the analyses on DATASET 2 (broad sampling of nrITS data) as well as the parameters data are shown only in the Online Resource material. Comparing the combined nrITS and nrLSU tree (Fig. 2) with the nrLSU tree (Fig. 1) and the nrITS tree (Online Resource Figs. O1, O2), we observe coherence in the topologies considering that the nrLSU tree and nrITS tree have broader sampling (more taxa included), but limited resolution for the tree at various points. On the other hand, the nrITS + nrLSU tree is better resolved due to the combination of molecular data with high statistical support for all generic representative clades, but various taxa present in the DATASET 1 and 2 are absent in DATASET 3 because of the lack of nrLSU and nrITS data of the correspondent strain. Most clades and subclades found in nrITS + nrLSU tree can also be found in the nrLSU tree and nrITS tree, showing consistency in the topology. Taxonomy Pusillomyces J.S. Oliveira, gen. nov. MycoBank MB 827357. Etymology: Pusillus (latin) = small; myces = mushroom; it refers to the small size of the marasmioid basidiomata. Diagnose: Basidiomata marasmioid, thin, tiny. Pileus hemispheric to convex, or plane, sometimes slightly concave, center plane to depressed, with or without papilla, smooth, or slightly rugulose at the center, and striate-sulcate at the disc and margin, surface finely tomentose, membranous to firmly coriaceous. Hymenophore entirely smooth or with well-developed lamellae, adnate to occasionally adpressed to a false collarium, distant, whitish to pale cream. Stipe filiform, wiry, insititious, rising directly from the substrate or branching from rhizomorphs; chitinous, horny, pliant, hollow; strongly pigmented, mostly brown to dark brown; glabrous or rarely finely pruinose, or even entirely pubescent to hairy. Rhizomorphs present, glabrous to pubescent, rare to abundant. Odor and taste not distinctive. Basidospores Fig. 2 Bayesian 50% majority-rule consensus tree from the multilocus„ (combined nrITS and nrLSU) analysis of DATASET 3 (TL, 35.696274; −lnL, − 14,951.21). Support values at the nodes consist of PP ≥ 0.95 and BS ≥ 70; unsupported nodes under PP 0.5 are collapsed. Thicker stems in black represent highly supported nodes, and those in gray are moderately to weakly supported nodes. Outgroup consists of members of the clade Marasmiaceae (Fig. 1) Mycol Progress (2019) 18:713–739 a 0.4 719 TFB13939 Gymnopus confluens LEBIN1178 Gymnopus confluens TFB14072 Gymnopus confluens TFB14389 Gymnopus confluens TFB14409 Gymnopus confluens 1.0/100 BUR1 Gymnopus confluens WTU394 Gymnopus confluens WTU005 Gymnopus confluens WTU514 Gymnopus confluens TFB7219 Gymnopus confluens TFB14075 Gymnopus confluens 100 MICHPK6820 Gymnopus confluens MICHPK6943 Gymnopus confluens CUH-AM083 Gymnopus confluens TFB13744 Gymnopus confluens TFB14114 Gymnopus confluens TFB14132 Gymnopus confluens TFB13754 Gymnopus confluens 1.0/88 TFB14115 Gymnopus confluens LEBIN1212 Gymnopus confluens LEBIN2294 Gymnopus confluens LEBIN2357 Gymnopus confluens 0.96 ZJ0002XPS01 Gymnopus confluens CUHAM089 Gymnopus menehune 1.0/100 AWW87 Gymnopus menehune AWW15 Gymnopus menehune 0.98/73 CUHAM074 Gymnopus menehune TFB11005 Gymnopus mesoamericanus TFB13890 Gymnopus biformis 1.0/100 TFB14250 Gymnopus biformis TFB14251 Gymnopus biformis 74 TFB13814 Gymnopus biformis TENN68136 Gymnopus disjunctus 1.0/96 TFB13739 Marasmiellus vaillantii TENN69123 Gymnopus eneficola 0.96/100 TENN69128 Gymnopus eneficola TENN69127 Gymnopus eneficola TENN69121 Gymnopus eneficola 1.0/100 TENN69122 Gymnopus eneficola 1.0/100 MICHPK6976 Gymnopus eneficola 1.0/95 MICHPK6975 Gymnopus eneficola 1.0/100 TFB14278 Gymnopus nonnullus TFB14617 Marasmiellus sp. Gymnopus nonnullus var. attenuatus 1.0/99 AWW05 AWW54 Gymnopus melanopus 1.0/98 CUHAM093 Gymnopus melanopus TFB10494 Gymnopus sp. AWW01 Gymnopus brunneigracilis AWW112 Gymnopus gibbosus AWW51 Gymnopus trogioides TFB9121Gymnopus luxurians 1.0/100 TFB14290 Gymnopus pseudoluxurians 1.0/93 Gymnopus luxurians FB10350 Gymnopus luxurians 1.0/100 CUHAM082 Gymnopus polygrammus AWW10 Gymnopus aff. moseri TFB14615 Gymnopus quercophilus TFB14616 Gymnopus quercophilus 1.0/100 SFSU 25220 Gymnopus quercophilus TFB14570 Gymnopus quercophilus TFB13755 Marasmiellus ramealis 1.0/100 WRW05-1170 Gymnopus sp. 1.0/77 TFB14334 Gymnopus utriformis nom. prov. TFB14228 Gymnopus aff. melanopus 1.0/100 TFB9889 Marasmiellus juniperinus TFB10826 Marasmiellus juniperinus 1.0/100 TFB13743 Gymnopus peronatus 1.0 LEBIN1898 Gymnopus peronatus 1.0/100 LEBIN1364 Gymnopus peronatus 1.0 TFB12577 Gymnopus subnudus WRW08462 Gymnopus subnudus Gymnopus dichrous 94 TFB10015 TFB11601 Gymnopus dichrous 1.0/81 TFB10009 Gymnopus aff. dichrous 1.0/95 TFB14111 Gymnopus aff. dichrous TFB14282 Gymnopus micromphaleoides KFRI1321 Gymnopus funalis KFRI1322 Gymnopus funalis 88 BRNM718813 Gymnopus funalis BRNM718747 Gymnopus funalis 1.0/100 KFRI1848 Gymnopus funalis BRNM747542 Gymnopus funalis 0.96/90 1.0/100 KFRI1863 Gymnopus funalis KFRI1870 Gymnopus funalis 1.0 JO674 Pusillomyces manuripioides 100 JO1121 Pusillomyces manuripioides BRNM714994 Gymnopus asetosus 1.0/100 1.0/98 BRNM718733 Gymnopus asetosus BRNM718820 Gymnopus asetosus 1.0/100 BRNM714999 Gymnopus asetosus 0.97/94 BRNM715010 Gymnopus asetosus 0.96 BRNM714965 Gymnopus asetosus 1.0/100 KFRI1886 Gymnopus asetosus KFRI1918 Gymnopus asetosus TFB7498 Connopus acervatus 1.0/100 TFB13532 Connopus acervatus 1.0/100 FB12621 Connopus acervatus TFB13579 Connopus acervatus TFB13581 Connopus acervatus 1.0/100 0.99/70 SAT11 179 Marasmius pallidocephalus TFB13933 Marasmius pallidocephalus TFB5015 Marasmius pallidocephalus 1.0/73 TFB5698 Marasmius pallidocephalus TFB5610 Marasmius pallidocephalus BRNM718676 Gymnopus glabrocystidiatus TFB14606 Micromphale brevipes (= Gymnopus nidus-avis) TFB14607 Micromphale brevipes (= Gymnopus nidus-avis) 77 1.0/96 TFB14498 Marasmius pallidocephalus (= Gymnopus nidus-avis) TFB9087 Micromphale brevipes (= Gymnopus nidus-avis) DPL11763 Micromphale brevipes (= Gymnopus nidus-avis) KFRI1935 Gymnopus glabrocystidiatus 1.0/95 LEBIN2526 Rhodocollybia butyracea f. asema 0.99/92 LEBIN1232 Rhodocollybia butyracea f. asema 1.0/97 TFB14368 Rhodocollybia butyracea TFB14382 Rhodocollybia butyracea TFB13006 Rhodocollybia butyracea 1.0 TFB14131 Rhodocollybia maculata 1.0/100 AFTOL540 Rhodocollybia maculata 1.0/100 TFB14317 Rhodocollybia maculata TFB14253 Rhodocollybia maculata TFB13989 Rhodocollybia maculata 1.0/100 DSH 92143 Lentinula lateritia 1.0/99 OE9 Lentinula edodes 1.0/99 TFB8682-2 Lentinula raphanica TFB10292-6 Lentinula boryana Clade A Marasmiellus s. str. Clade B Pusillomyces Clade C Connopus Clade D Pallidocephalus Clade E Rhodocollybia Clade F Lentinula 0.4 720 Mycol Progress (2019) 18:713–739 b 1.0/83 1.0/95 AWW116 Gymnopus bicolor AWW126 Gymnopus sepiiconicus AWW118 Gymnopus aurantiipes AWW125 Gymnopus indoctoides BCNSCMB4058 Gymnopus inusitatus 1.0 BCNSCMB4057 Gymnopus catalonicus 99 BCNSCMB4065 Gymnopus bisporus BRNM737257 Gymnopus inusitatus var. cystidiatus 1.0/99 TFB13758 Gymnopus ocior 1.0/99 AFTOLID 559 Gymnopus dryophilus TFB13781 Gymnopus aff. dryophilus 89 TFB13782 Gymnopus aff. dryophilus 1.0/98 1.0/74 1.0 TFB14333 Gymnopus spongiosus 100 TFB13975 Gymnopus spongiosus AWW127 Gymnopus vitellinipes 1.0/81 1.0/95 BRNM714849 Gymnopus impudicus 1.0/84 JVG1130531 Gymnopus impudicus 1.0/100 TFB14110 Gymnopus barbipes 1.0/99 TFB14618 Gymnopus foetidus TFB6520 Gymnopus iocephalus 87 VA12117 Gymnopus dysodes JMCR143 Caripia montagnei 1.0/100 TFB13911 Gymnopus foetidus 1.0/100 TFB14340 Gymnopus foetidus 0.99/76 TFB14583 Gymnopus foetidus BRNM747547 Gymnopus cremeostipitatus 1.0/99 TFB14489 Gymnopus neobrevipes TFB14594 Gymnopus neobrevipes 1.0/100 TFB14599 Gymnopus neobrevipes TFB4548 Gymnopus portoricensis 1.0/100 TFB4549 Gymnopus portoricensis TFB4512 Gymnopus portoricensis TFB11631 Gymnopus androsaceus 0.98 TENN F 69268 Gymnopus androsaceus 0.99/86 TFB5021 Gymnopus androsaceus DED8813 Gymnopus adventitius nom. prov. TFB5609 Gymnopus androsaceus 1.0/88 TFB9031 Gymnopus frigidomarginatus nom. prov. TFB4975 Gymnopus novaeangliae nom. prov. 1.0/100 TFB11333 Gymnopus fusipes 1.0/95 TFB14558 Gymnopus fusipes TFB2221 Gymnopus inflatotrama nom. prov. TFB4919 Gymnopus inflatotrama nom. prov. TFB4529 Gymnopus inflatotrama nom. prov. TFB4930 Gymnopus inflatotrama nom. prov. DED5097Gymnopus novomundi nom. prov. TFB3940 Marasmius sp.1 1.0/98 TFB4550 Gymnopus foliiphilus TFB4551 Gymnopus foliiphilus TFB3642 Gymnopus foliiphilus TFB7243 Gymnopus foliiphilus DED5272 Gymnopus foliiphilus TFB4928 Gymnopus foliiphilus TFB8782 Gymnopus foliiphilus 0.98 0.99/98 TFB10364 Gymnopus foliiphilus TFB14291 Gymnopus foliiphilus TFB14422 Gymnopus foliiphilus TFB14048 Gymnopus foliiphilus TFB14063 Gymnopus foliiphilus TFB9166 Gymnopus foliiphilus TFB9167 Gymnopus foliiphilus TFB13875 Gymnopus foliiphilus TFB14332 Gymnopus foliiphilus 0.99/90 TFB13122 Gymnopus perforans subsp. transatlanticus 0.4 TFB13121 Gymnopus perforans subsp. transatlanticus TFB14348 Gymnopus perforans subsp. transatlanticus TFB14395 Gymnopus perforans subsp. transatlanticus TFB13319 Gymnopus perforans subsp. transatlanticus TFB14350 Gymnopus perforans subsp. transatlanticus TFB14613 Gymnopus perforans subsp. transatlanticus TFB14384 Gymnopus perforans subsp. transatlanticus 1.0/78 TFB14592 Gymnopus perforans subsp. transatlanticus TFB10826 Gymnopus perforans TFB4721 Gymnopus perforans 1.0/100 TFB7477 Gymnopus perforans TFB4722 Gymnopus perforans AV100918 Gymnopus perforans subsp. transatlanticus 1.0/90 TFB13319 Gymnopus perforans subsp. transatlanticus UBC25212 Gymnopus sublaccatus 1.0/100 TFB14620 Gymnopus sequoiae 1.0/100 TFB14395 Gymnopus perforans subsp. transatlanticus TFB14377 Gymnopus perforans subsp. transatlanticus TFB13123 Gymnopus perforans subsp. transatlanticus TFB14511 Gymnopus pinophilus TFB13913 Gymnopus pinophilus 1.0/100 TFB14517 Gymnopus pinophilus TFB5256 Gymnopus pinophilus TFB5627 Gymnopus ponderosae TFB14059 Gymnopus pinophilus TFB14097 Gymnopus pinophilus TFB7588 Gymnopus caulocystidiatus nom. prov. 1.0/99 TFB7572 Gymnopus caulocystidiatus nom prov. TFB7148 Gymnopus caulocystidiatus nom. prov. 1.0 1.0/100 TFB7589 Gymnopus caulocystidiatus nom. prov. TFB4033 Gymnopus austrobrevipes nom. prov. 1.0/77 TFB3591 Gymnopus austrobrevipes nom. prov. TFB3585 Gymnopus austrobrevipes nom. prov. SGO 163625 Gymnopanella nothofagi 1.0/100 DAOM 175251 Mycetinis salalis TENN F 55408 Mycetinis copelandii 0.98/70 UPS F012968 Marasmius prasiosmus 1.0/95 TENN F 59615 Mycetinis scorodonius 1.0/93 0.96 1.0/100 TENN F 53474 Mycetinis scorodonius TENN F 69200 Mycetinis opacus 1.0/100 TENN F 55630 Mycetinis alliaceus TENN F 69243 Mycetinis alliaceus 1.0/100 AFTOLID 556 Mycetinis alliaceus 1.0/100 VT455 Omphalotus olivascens AFTOLID 1718 Omphalotus olearius AFTOLID 973 Anthracophyllum archeri PBM 2711 Gymnopus contrarius 1.0/100 ATCC 42449 Campanella subdendrophora MCA1689 Campanella sp. 1.0/98 CMRUB 2041 Moniliophthora perniciosa 1.0/96 MCA2500 Moniliophthora sp. 1.0/100 OKM 25450 Crinipellis zonata 1.0/100 MCA1527 Crinipellis sp. AFTOLID 1505 Marasmius rotula AFTOLID 1559 Marasmius oreades 1.0/99 93 Clade G Gymnopus s. str. Clade H Gymnopus sect. Perforantia (Paragymnopus gen. nov.) Clade I Gymnopanella Clade J Mycetinis Clade K Omphalotus 0.4 Fig. 2 (Continued) Mycol Progress (2019) 18:713–739 Table 2 Data from ML and BA analyses of DATASET 3 721 DATASET 3 alignment Taxa 244 Characters 545 (ITS) + 955 (LSU) Calculations from BA by partition Alpha ITS (means) 0.758406 Pinvar 0.302564 Substitution rates Calculations from ML by partition LSU (means) 0.190530 0.497144 Alpha ITS (means) 0.356165 LSU (means) 0.200742 Pinvar Substitution rates (A<=>C) 0.070389 0.036804 (A<=>C) 1.900609 0.485606 (A<=>G) (A<=>T) 0.390588 0.105534 0.207500 0.085793 (A<=>G) (A<=>T) 11.363035 3.210914 4.003744 1.300596 (C<=>G) 0.043284 0.037125 (C<=>G) 1.243795 0.492606 (C<=>T) 0.339083 0.601554 (C<=>T) 9.601055 6.098430 (G<=>T) Bases freq. 0.051122 0.031224 (G<=>T) Bases freq. 1.000000 1.000000 pi(A) 0.236077 0.278227 pi(A) 0.235424 0.273905 pi(C) pi(G) pi(T) 0.190142 0.204093 0.369687 0.171529 0.283168 0.267077 pi(C) pi(G) pi(T) 0.199045 0.214724 0.350808 0.184005 0.292899 0.249191 obovoid to shortly oblong, ellipsoid, ellipsoid-fusoid, lacrymoid to subclavate, smooth, hyaline, thin-walled, inamyloid. Basidia clavate, 2- to 4-sterigmate. Cheilocystidia not applicable (smooth hymenophore) or when present, in the form of Siccus-type broom cells. Pleurocystidia absent. Lamellar and pileus trama irregular, inamyloid, hyphae smooth or incrusted. Pileipellis nongelatinized, non-hymeniform, a trichoderm of disorganized, packed elements, composed of cylindrical, thin- to slightly thick-walled, diverticulate, smooth or incrusted hyphae, mixed with elements similar to Siccus-type broom cells, or Ramealesstructures. Stipitipellis and Stipe trama dextrinoid. Caulocystidia absent or present, dextrinoid. Clamp connections absent. Chemical reactions: In Melzer’s reagent, only the stipe trama is dextrinoid; otherwise, inamyloid. No staining in alkaline solution (NH4OH or KOH). Ecology: Saprotrophic or parasitic in plant (phytopathogen), epiphytic or in the forest litter, gregarious, on leafy or woody substrate. Distribution: Probably worldwide; to date only recorded from the Neotropic (Tropical, Amazon forest, Brazil) and Paleoarctic regions (Temperate Broadleaf and Mixed Forest, Republic of Korea). Type species: Pusillomyces manuripioides J.S. Oliveira. Pusillomyces asetosus (Antonín, R. Ryoo & K.H. Ka) J.S. Oliveira, comb. nov. (MB 827358) Gymnopus asetosus Antonín, R. Ryoo & K.H. Ka, Mycological Progress 13: 704 (2014) Pusillomyces funalis (Har. Takah.) J.S. Oliveira, comb. nov. (MB 827359) Marasmius funalis Har. Takah., Mycoscience 43 (4): 344 (2002) Gymnopus funalis (Har. Takahashi) Antonín, R. Ryoo & Shin, Mycological Progress 13: 710 (2014) Notes: To see taxonomic and classification comments on the evidence for the combination of G. asetosus and G. funalis, and the establishment of Pusillomyces, refer to Phylogeny and Morphology in the Discussion. A synoptic key comparing Pusillomyces with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). Pusillomyces manuripioides J.S. Oliveira, sp. nov. (Figs. 3, 4, and 5) Mycobank MB 827360 Barcode sequences (GenBank accession numbers): nrITS – MK434210 and nrLSU – MK434211 from the holotype J.J.S. Oliveira & L.S. Bento JO674; and nrITS – MK434212 and nrLSU – MK434213 from the paratype J.J.S. Oliveira & N.K. Ishikawa JO1121. Etymology: The epithet is based on the strong similarity to Manuripia bifida Singer, a monotypic genus. The name Manuripia comes from Manuripi (Bolivia), the holotype locality of M. bifida. Diagnose: Basidiomata manuripioid, tiny. Pileus 0.3– 1.8 mm diam., plano-convex to subinfundibuliform, smooth, brown, chestnut brown to cream. Hymenophore smooth, white to cream. Stipe 1–4 × 0.2–0.3 mm, central, filiform, insititious, glabrous, dark brown, growing mostly from rhizomorphs. Basidiospores 6–9 × 2.8–4.6 μm, usually ellipsoid, inamyloid. Pileus trama inamyloid, irregular, 722 Mycol Progress (2019) 18:713–739 Fig. 3 Pusillomyces manuripioides. a Branch of Eugenia sp. with net of rhizomorphs and infected parts (JO1117). b, c Rhizomorphs bearing basidiomata of P. manuripioides (JO674— holotype). d, e Rhizomorphs strongly attached to dead foliar face, some producing basidiomata (JO674—holotype) hyphae smooth or incrusted, clamp-connections absent. Pileipellis a trichoderm of Rameales-structures. Rhizomorphs like the stipe, some densely covered by conidia. Epiphytic, forming abundant rhizomorphs in hanging living and dead leaves and juvenile living branches. Pileus 0.3–1.8 mm diam., orbicular, plano-convex, plane, to plano-infundibuliform or subinfundibuliform, center with a shallow papilla especially when young, sometimes depressed, or papilla usually receding in old or larger pileus, disc and margin smooth, margin straight to slightly uplifted, edge entire; surface glabrous, dry, dull, papyraceous or subvelutinous; membranous at the margin, a little thicker, tough or coriaceous at the disc towards the center, context thin (< 1 mm), white to whitish cream; with brown to dark brown (N60Y50M30 to N90Y60M40) center or papilla, disc chestnut brown (N50Y60M30) becoming yellowish brown or buff brown (N40Y50M30) towards the margin, or paller (N20Y30–40M20), becoming cream (N 00 Y 10 M 00 ) when very fresh. Hymenophore smooth (lamellae absent), white to pale cream (N00Y10M00), papyraceous, dull, dry. Stipe 1–4 × 0.2– 0.3 mm, central, filiform, very thin, equal, cylindrical, insititious, chitinous, hollow, glabrous, smooth, opaque, dark chestnut brown (N90Y70M30) or very dark brown (N99Y50M30), growing directly from the substrate or more frequently arising Mycol Progress (2019) 18:713–739 723 Fig. 4 Pusillomyces manuripioides (JO674—holotype): illustration of macroscopic structures of basidiomata and rhizomorphs, habit and substrate. Scale bar 1 cm from abundant, very elongate, hair-like rhizomorphs, concolorous with the stipes, part glabrous, part densely pubescent or even hirsute, resembling those found in Manuripia bifida, with dark brown to black pubescence. Basidiospores (Fig. 5a) 6– 9 × (2.2)2.8–4.6(5) μm (xrm = 6.9–7.4 × 2.8–3.6 μm, xmm = 7.3 (± 0.3) × 3.3 (± 0.4) μm, Qrm = 2–3.3, Qmm = 2.6 (± 0.7), n = 30, s = 3), obovoid, rarely shortly oblong, usually ellipsoid to subellipsoid, or amygdaliform, lacrymoid to subclavate, smooth, thin-walled, hyaline, inamyloid. Basidia (Fig. 5b) 20–27.5 × 5– 7 μm, clavate, smooth, hyaline, thin-walled, inamyloid, 2–4 sterigmata. Basidioles (Fig. 5c) 17–26.8 × 3.7–6.4 μm, clavate to broadly clavate, some with tapered apex, smooth, hyaline, thinwalled, inamyloid. Hymenial cystidia absent. Pileus trama inamyloid, irregular, non-gelatinized, packed, but also lacunose in some parts, especially at the central part of the context, forming a gradient, pale brownish near the pileipellis, fading to more hyaline near the hymenial layer, hyphae interwoven, cylindrical, 2–8.2 μm diam., regular in outline, branched, smooth, or more frequently incrusted (irregularly ornamented) towards the pileipellis (Fig. 5e), hyaline or some more opaque (wall thickness), thin- to more thick-walled next to the pileipellis, clamp connections absent. Pileipellis non-hymeniform, non-gelatinized, 724 Mycol Progress (2019) 18:713–739 Fig. 5 Pusillomyces manuripioides (JO674—holotype): microscopic structures. a Basidiospores. b Basidia. c Basidioles. d Diverticulate elements of the pileipellis. e Incrusted hyphae of the pileipellis to the upper layer of the pileus trama. Scale bar a–e 10 μm formed by a layer of disorganized elements or trichoderm, pale chestnut brown in 3% KOH with abundant and dominant Rameales-structures (Fig. 5d), some in transition to irregular Siccus- or Rotalis-type cells (Fig. 4d), usually interconnected with smooth or incrusted hyphae of the pileus trama (Fig. 4e), main body 8.8–29 × 3.3–10.5(− 21) μm, hyphoid, branched, Mycol Progress (2019) 18:713–739 lobed or irregular, but clavate to cylindrical in the Siccus-/Rotalistype cells, hyaline when isolated, smooth, thin-walled, inamyloid; diverticula apical or divergent to the laterals, generally short, 0.7–2.2 × 0.6–1.2 μm, vesiculose, verruciform, to shortly digitiform, sometimes branched, hyaline, solid, with obtuse and rounded apex. Stipe trama mostly inamyloid, or cortical hyphae apparently dextrinoid (at least in the stipe apex) or only orangish brown in Melzer’s reagent due to pigmentation, parallel, not compact, hyphae easily disassociating, then chestnut brown, losing pigmentation towards the inner trama, cylindrical, 3–8.4 μm diam., generally regular in outline, septate, thick-walled, smooth or with granular incrustations, sometimes very prominent looking like diverticula, clamp connections absent; internal hyphae 2.5–7.4 μm diam., with thinner walls, hyaline, usually smooth, sometimes rough, clamp-connections absent. Stipitipellis without elements or vesture on the glabrous stipes and rhizomorphs. Rhizomorphs corresponding to the stipe trama for the glabrous segments in micromorphology, but some pubescent segments densely covered by elongate, cylindrical to fusoid (tapered at the apex) conidia (Fig. 6), 106–237.2 × 7.4–13.2 μm diam., presenting an apparent apical orifice, body with numerous septa, each short segment slightly inflated, thick-walled (including septa), dark brown, very distinct from the hyphae of Fig. 6 Pusillomyces manuripioides (JO674— holotype): Conidiophore and conidia from the cortex of the pubescent segments of the rhizomorphs. Scale bar 10 μm 725 the trama, non-fragmentary, but breaking at septal point when compressed, interconnected with one another at the base via connecting, thinner, irregular, sometimes branched, repent, similar segments, the whole structure forming the conidiophores which are immersed in the trama, but do not appear to have originated from the hyphae, reaction in Melzer’s reagent not distinctive due to the dark pigmentation. Habit and Substrate: Manuripioid (Fig. 3b, c) to gloiocephaloid (those basidiomata growing on the substrate—Fig. 3d, e), tiny, gregarious, epiphytic (about 1.5 m above the ground upward), forming abundant rhizomorphs in hanging living and dead leaves, attaching also on twigs, branches and limbs, specifically or frequently of Eugenia cf. subterminalis DC (Myrtaceae), in Bcampinarana^, sometimes in transition to Bterra-firme^, Amazon Forest. Specimens examined: Brazil, Amazonas, Manaus, Reserva Biológica de Campina, hanging branches with both living and dead leaves of Eugenia cf. subterminalis and fallen trapped leaves from the canopy of multiple dicot tree species, 17. 11. 2016, J.J.S. Oliveira & L.S. Bento JO674, holotypus (INPA 280704!); hanging branches with both living and dead leaves of Eugenia cf. subterminalis and fallen trapped leaves from the canopy of multiple dicot tree species, 28. 11. 2017, J.J.S. Oliveira & G.S. Sarkis JO950 (INPA 280705!); hanging 726 branches with with both living and dead leaves of Eugenia cf. subterminalis and fallen trapped leaves from the canopy of multiple dicot tree species, 15. 2. 2018, J.J.S. Oliveira & G.S. Sarkis JO986 (INPA 280706!); hanging branches with both living and dead leaves of Eugenia cf. subterminalis and fallen trapped leaves from the canopy of multiple dicot tree species, 15. 2. 2018, J.J.S. Oliveira & G.S. Sarkis JO987 (INPA 280707!), 23. 10. 2018, J.J.S. Oliveira & N.K. Ishikawa JO1117 (INPA 282046!), J.J.S. Oliveira & N.K. Ishikawa JO1118 (INPA 282047!); J.J.S. Oliveira & N.K. Ishikawa JO1119 (INPA 282048!), J.J.S. Oliveira & N.K. Ishikawa JO1120 (INPA 282049!), J.J.S. Oliveira & N.K. Ishikawa JO1121 (INPA 282050!), J.J.S. Oliveira & N.K. Ishikawa JO1122 (INPA 282051!), J.J.S. Oliveira & N.K. Ishikawa JO1123 (INPA 282052!), J.J.S. Oliveira & N.K. Ishikawa JO1124 (INPA 282053!). Discussion Phylogeny We based the analysis of DATASET 1 on the Marasmioid Clade depicted in Matheny et al. (2006), including representatives of the Tricholomatoid Clade (Matheny et al. 2006) as the outgroup. In the present analysis, the /mycenaceae subclade grouped within the outgroup, which does not follow the results in Dentinger et al. (2016). The analysis of DATASET 1 confirmed that, among all families and undefined groups within the Marasmioid Clade or suborder Marasmiineae represented by the major clades in the nrLSU tree (Fig. 1), P. manuripioides belongs to Omphalotaceae since it groups with the representatives of the family in the Omphalotaceae clade. This clade is weakly supported if Gymnopus contrarius is considered in the family; but disregarding this species, the clade is strongly supported (see Online Resource material). In the same tree (see again the Online Resource material), it is already possible to observe that P. manuripioides is part of a very distinct lineage and, therefore a representative of a different genus along with Gymnopus asetosus and G. funalis. In the tree reconstructed from the analysis of the combined nrITS and nrLSU data—DATASET 3 (Fig. 2), the ingroup consisted only of representatives of the Omphalotaceae while the ou tg roup is comp osed of rep resen ta tives of the Marasmiaceae. Once again, P. manuripioides branched within the very distinct and unique /clade B (Pusillomyces). Both trees (Figs. 1 and 2) are strong evidence that P. manuripioides, G. asetosus, and G. funalis do represent a new genus in the family. Similar to the analysis of the DATASET 3, we also conducted an analysis including only nrITS data with a very large sampling of members of the Omphalotaceae forming the ingroup, and members of the Marasmiaceae as the outgroup. Based on the Mycol Progress (2019) 18:713–739 trees, it is possible to observe the phylogenetic placement of taxa of the Omphalotaceae in clades that can be correlated to those clades depicted in the nrITS + nrLSU tree (Fig. 2), but such taxa are absent in this multilocus analysis because of the lack of nrLSU data from the same collection. This can restrict and broaden new circumscriptions for genera based on the present phylogeny, strengthening, or redefining boundaries for the groups. Moreover, the resulting nrITS tree (Online Resource Fig. O3) also revealed that P. manuripioides is closely related to G. asetosus and G. funalis, grouping in a strongly supported subclade. This consistently indicates that G. asetosus and G. funalis are congeneric with P. manuripioides, despite all the remarkable morphological divergence between the former two and the latter. We discuss further this relationship in the BMorphology^ section (next part of this discussion). The combined nrITS and nrLSU data tree (Fig. 2) in the present study also brought light to the understanding of the Omphalotaceae family at the genus level. Gymnopus and Marasmiellus are polyphyletic and have been considered artificial groups in previous studies such as Moncalvo et al. (2002), Mata et al. (2004a, 2006), Wilson and Desjardin (2005), Hughes et al. (2010), and Petersen and Hughes (2016, 2017). In our analyses, however, Gymnopus and Marasmiellus appear as monophyletic groups corresponding to /clade G and /clade A, respectively. This agrees with the findings of Wilson and Desjardin (2005), who depicted separate clades assigned for these genera (clade B or /marasmiellus and clade D or /gymnopus in their study) and did not accept Marasmiellus as a synonym of Gymnopus. Similar distinct clades can also be found in Petersen and Hughes (2016, 2017) and Sandoval-Leiva et al. (2016). The clades depicted in the tree are discussed in detail below: /Clade A (Marasmiellus s. str.): This clade is strongly supported and contains Marasmiellus juniperinus, type species of Marasmiellus along with M. ramealis (Bull.) Singer and M. vaillanti (Pers.) Singer, and all members of Gymnopus sect. Vestipedes included in the analysis. This is one of three distinct and unrelated lineages grouping species of Gymnopus and corresponds to the statistically unsupported clade B or /marasmiellus in Wilson and Desjardin (2005). These authors, while discussing the phylogenetic affiliation between M. juniperinus and Gymnopus sect. Vestipedes, argued that they share characteristics such as the production of basidiomes with insititious to subinsititious stipe, acyanophilous, inamyloid, depigmented (and hyaline) basidiospores, and a pileipellis which is a cutis of radially oriented cylindrical hyphae that are non-diverticulate or weakly diverticulate, typically roughened and vestured with annular to zebroid, brownish pigmented incrustations. They further indicated that the pileipellis does not have well-developed Rameales structures, but rather, that it is composed of strongly diverticulate hyphae Mycol Progress (2019) 18:713–739 as in M. ramealis and as in possibly most Marasmiellus spp. In the present paper, we found M. ramealis present in the same clade of M. juniperinus and members of Gymnopus sect. Vestipedes, and thus, the later advocated pattern is not consistent. Also, the need to resurrect Collybiopsis, considering the discussion presented in Wilson and Desjardin (2005), may be dismissed. Thus, Marasmiellus is monophyletic if the genus is restricted based on this clade and a redefinition of the traditional genus is provided in the following section. /Clade B (Pusillomyces): Pusillomyces manuripioides grouped with G. asetosus and G. funalis, forming the distinct and highly supported clade B. This clade represents the new genus Pusillomyces formally described along with the new species in this paper. Morphological and ecological characteristics shared by these taxa are discussed in the next sections. Also, in /Clade B, G. asetosus seems to be rather three different species while G. funalis seems to be two and, then, the involved strains should be revised. 727 obtained from the same geographic region—the US Gulf Coast. Petersen and Hughes (2017) suggest that Marasmius pallidocephalus probably forms a monotypic genus while our results point to the inclusion of at least two additional species: G. glabrocystidiatus and BMicromphale brevipes^ (pro parte). Considering this later, a just published paper (César et al. 2018) has revealed that the strains TFB14606, TFB14607, TFB14498, TFB9087, DPL11763, named as BMicromphale brevipes^ are rather conspecific with the new species Gymnopus nidus-avis César, Bandala & Montoya. We agree with their conclusion, although this very recently proposed new species is not Gymnopus s. str. according to the present study, but also part of /Clade D (Pallidocephalus). This clade deserves to be more carefully studied to formalize the establishment of another genus in Omphalotaceae. A synoptic key comparing members of this clade with the genera of /letinuloid clade is provided in Online Resource (Chart O1). /Clade E (Rhodocollybia): This highly supported clade groups all species of Rhodocollybia present in this analysis, including the type species R. maculata (Alb. & Schwein.) Singer. The genus is monophyletic in this study and agrees with Wilson and Desjardin (2005) and Mata et al. (2006) but differs from Hughes et al. (2010) and Sandoval-Leiva et al. (2016); however, additional taxa of the genus should be included in future analyses. For more information about Rhodocollybia see Mata et al. (2004b). A synoptic key comparing Rhodocollybia with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). /Clade C (Connopus): This highly supported clade is composed of six specimens of Connopus acervatus (Fr.) R.H. Petersen, and recently erected as the monotypic genus Connopus R.H. Petersen in Hughes et al. (2010). Connopus is sister to Pusillomyces gen. nov., but without statistical support. The genus is mainly characterized by producing connate, collybioid or mycenoid basiomata, usually growing in deep polytrichaceous moss juxtaposed to conifer trunks or stumps in temperate to cool forests; hemispheric to convex, hygrophanous, smooth pileus; free to seceding, abundant, close lamellae; central, cylindrical, fistulose, apically glabrous then pruinose stipe, with tomentose base; small, ellipsoid to cylindrical, hyaline, thin-walled, inamyloid basidiospores; lacking pleurocystidia; absent or occasional, slender lecythiform cheilocystidia; conspicually clamped hyphae in the tramae, and a pileipellis consisting of a lax trichoderm, with occasional, weakly banded, erect terminal cells (Hughes et al. 2010). A synoptic key comparing Connopus with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). /Clade F (Lentinula): This is a strongly supported clade encompassing all Lentinula Earle species included in the analysis: L. lateritia (Berk.) Pegler, L. edodes (Berk.) Pegler, L. raphanica (Murrill) Mata & R.H. Petersen and L. boryana (Berk. & Mont.) Pegler. Based on this analysis, the genus is monophyletic. For further information about the genus Lentinula, see Pegler (1983) and Mata et al. (2001). A synoptic key comparing Lentinula with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). /Clade D (Pallidocephalus): This strongly supported clade may correspond to the clade B/austrobrevipes and /pallidocephalus^ in Petersen and Hughes (2016, 2017). However, the clade contains Gymnopus pallidocephalus comb. prov. (officially, Marasmius pallidocephalus Gilliam), Gymnopus glabrocystidiatus Antonín, R. Ryoo & K.H. Ka and strains named as BMicromphale brevipes^ (DPL11763, TFB9087, TFB14606, TFB14607, TFB14498). A second lineage previously determined as Micromphale brevipes (Berk. & Ravenel) Singer appears again in /clade F, recently proposed as Gymnopus neobrevipes Petersen (Petersen and Hughes 2019). Collections of both BMicromphale brevipes^ lineages were all /Clade G (Gymnopus s. str.): This strongly supported clade is composed of Gymnopus fusipes, the type species of the genus and the only member of Gymnopus sect. Gymnopus, and members of Gymnopus sect. Androsacei, sect. Impudicae, and sect. Levipedes. In this clade, Gymnopus s. str. is represented as a monophyletic and less inclusive genus, corresponding to the statistical supported clade D or /gymnopus (excluding Micromphale peforans branch) in Wilson and Desjardin (2005). These authors disagreed with the more inclusive genus suggested in Mata et al. (2004a). Mata et al. (2006) maintained a broad view of Gymnopus corresponding to a major clade grouping the clades A–N in their tree. In the 728 trees provided by Hughes et al. (2010) that focused on the establishment of Connopus, their ITS tree shows Gymnopus as a single monophyletic clade (but not including G. fusipes nor G. androsaceus, and members of Gymnopus sect. Levipedes) while the LSU tree presents multiple unrelated clades bearing members of Gymnopus (including G. fusipes, G. androsaceus, and members of Gymnopus sect. Levipedes). Sandoval-Leiva et al. (2016) provided a tree based on concatenated ITS–5’-28S, but via neighbor-joining method and values of support via Bayesian and BioNJ analyses focused in the establishment of the new genus Gymnopanella and presented monophyletic, strongly supported clades B/marasmiellus^ and BGymnopus, including Setulipes,^ unrelated in the tree, where the former is sister to BRhodocollybia, including Connopus^ and the latter is sister to Gymnopanella. Similar results are found in Petersen and Hughes (2016). In the present study, the concept of less inclusive Gymnopus (s. str.) as defended by Wilson and Desjardin (2005) is strongly supported, and also observed in Sandoval-Leiva et al. (2016) and corresponds to clades A–C in Mata et al. (2006). In the concatenated nrITS + nrLSU tree (Fig. 2), clade G is distinctly resolved and strongly supported, and includes G. fusipes and members of Gymnopus sect. Levipedes, Impudicae and Androsacei. Wilson and Desjardin (2005) discussed that G. fusipes and members of Gymnopus sect. Levipedes share a pileipellis composed of relatively short, broad, branched, non-diverticulate hyphae forming a Dryophila-structure based on Halling (1983) and Antonín and Noordeloos (1997), but the type species of the genus only differs in developing a rooted stipe and pale pinkish or ochraceous spore print. However, based on Wilson and Desjardin (2005), species of Micromphale and those of the old Setulipes are quite distinct from members Gymnopus sect. Levipedes and G. fusipes, because the first two groups produce small, marcescent basidiomata with wiry and insititious stipes, usually accompanied by black wiry rhizomorphs and pileipellis with hyphae which are non-diverticulate and (a) gelatinized and encrusted with a brown pigment (Micromphale) or (b) diverticulateknobby and usually non-gelatinized (Setulipes). Also, in Wilson and Desjardin (2005), Micromphale perforans branched as sister to the clade D and without statistical support. Although being included in /gymnopus in the same study, their results regarding M. perforans are clarified as part of a separate clade corresponding to Gymnopus sect. Perforantia (Singer) R.H. Petersen, resulting from the combination of Micromphale sect. Perforantia Singer in Gymnopus by Petersen and Hughes (2016). Our results also show a similar separate clade (clade H in the present study), but instead, support the establishment of a new genus Paragymnopus. The clade also bears some informal taxa that need to be confirmed. The strains TFB14489, TFB14594, and TFB14599 represent the species Micromphale brevipes (Berk. & Ravenel) Singer (≡ Marasmius brevipes Berk. & Ravenel), according to Mycol Progress (2019) 18:713–739 Petersen and Hughes (2019), species now belonging to Gymnopus. César et al. (2018) proposed Marasmius brevipes to be a synonym of Marasmius westii Murrill. This proposal was rejected by Petersen and Hughes (2019), which provided a new name to the species, Gymnopus neobrevipes R.H. Petersen, since there is already Gymnopus brevipes (Bull.) Gray [now Melanoleuca brevipes (Bull.) Pat.]. /Clade H (Gymnopus sect. Perforantia): This clade is composed of Gymnopus foliiphilus R.H. Petersen, G. sublaccatus R.H. Petersen, G. sequoiae (Desjardin) R.H. Petersen, G. perforans (Hoffm.) Antonín & Noordel., G. pinophilus R.H. Petersen and G. ponderosae R.H. Petersen. The first evidence of this lineage is found in Wilson and Desjardin (2005), but was further explored in Petersen and Hughes (2016) (see discussion in the previous clade). According to Petersen and Hughes (2016), the species in this clade form Gymnopus sect. Perforantia and are morphologically characterized by: (1) pileus and lamellar tramae as well as stipe medullary hyphae and pileipellis embedded in slime matrix; (2) pileipellis of two kinds— (i) a layer of repent, incrusted hyphae, conspicuously clamped, usually with thickened walls (through gelatinization) and embedded in slime matrix, or (ii) well-developed Rameales structure; (3) cheilocystidia generally absent, if present either clavate to utriform or like Siccus-type broom cells; (4) stipe often thread-like (than 1 mm) but usually up to 40 mm long, and often a high ratio of the pileus breadth to stipe length; (5) stipitipellis often with cellular differentiation (excepting G. glabrosipes R.H. Petersen), many times minutely barbed under a 30× lens; 6) always black, branched or unbranched rhizomorphs almost present in all cases, from 2 to 40 × 0.2–0.7 mm; (7) basidiospores not significantly differing in dimension; (8) basidiomata found on dead conifer needles or rotting deciduous leafy debris, more or less host-specific; (9) fusiform pleurocystidia consistently present, with slight differences at the summit; and (10) clamp connections found in all taxa. More morphological characteristics assigned to the section are detailed in Petersen and Hughes (2016). The stipe in this section is mostly insititious but can also be subinsititious or sometimes non-insititious. Petersen and Hughes (2016) recovered a tree based on nrLSU where the clade assigned to Gymnopus sect. Perforantia is sister to /gymnopus, but with low support (ML BS 44). In the tree from our analysis based on concatenated nrITS and nrLSU data, clade H (Gymnopus sect. Perforantia) appears paraphyletic relative to clade G (Gymnopus s. str.), considering that the taxon labeled BKY026621 Marasmius sp.1 TFB3940^ is a distinct lineage sister to clade G (then, clade H is sister to Bclade G + ‘TFB3940 Marasmius sp.1’^ without statistical support). Thus, we propose the establishment of a new genus, Paragymnopus, instead of recognizing this clade Mycol Progress (2019) 18:713–739 729 at a lower rank, Gymnopus sect Perforatia (see clade G above). Mycetinis with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). /Clade I (Gymnopanella): Clade I is formed by Gymnopanella nothofagi Sandoval-Leiva, J.V. McDonald & Thorn, plus strains of informal taxa named Gymnopus caulocystidiatus nom. prov. and G. austrobrevipes nom. prov. in Petersen and Hughes (2016). Unfortunately, a morphological comparison is not possible in order to classify these informal taxa in Gymnopanella, since no anatomical data are provided to date. But according to our tree, we suggest that Gymnopus caulocystidiatus nom. prov. and G. austrobrevipes nom. prov. are better placed in Gymnopanella. This genus is mainly characterized by being saprotrophic, producing campanelloid basidiomata that are flabelliform, convex, have a gelatinous pileus; a reticulate-lamellate hymenophore; and a lateral, short, glabrous stipe. Basidiospores are broad ellipsoid to ovate, hyaline, thin-walled, non-amyloid; subhymenium is ramose; gelatinous, dense trama, composed of subparallel to tangled hyphae, with fine ring-like incrustation; and the pileipellis is a cutis of cylindrical, thick-walled hyphae, seldom branched, coarsely externally incrusted with deposits, some hyphae forming fascicles (Sandoval-Leiva et al. 2016). A synoptic key comparing Gymnopanella with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). /Clade K (Omphalotus): Clade K groups together Omphalotus olearius (DC.) Singer (type species) and O. olivascens H.E. Bigelow, O.K. Mill. & Thiers and represents the genus (type of Omphalotaceae) in the analysis. Omphalotus appears as sister to Anthracophyllum Ces. (/Lineage L), represented by A. acheri (Berk.) Pegler, but this relationship is unsupported. /Clade J (Mycetinis): This strongly supported clade represents the genus Mycetinis Earle, grouping M. alliaceus (Jacq.) Earle (type species of the genus), M. copelandii (Peck) A.W. Wilson & Desjardin, M. opacus (Berk. & M.A. Curtis) A.W. Wilson & Desjardin, M. prasiosmus (Fr.) R.H. Petersen, M. salali s (Desjardin & Redhead) Redhead and M. scorodonius (Fr.) A.W. Wilson & Desjardin. The genus was resurrected in Wilson and Desjardin (2005) based on clade F in the tree shown in their study. Species of this genus were previously included in Marasmius sect. Alliacei. Based on the same study, Mycetinis species share characteristics such as sub- to non-insititious stipe; inamyloid, acyanophilic, white or pale cream spore print, inamyloid trama; and a hymeniform pileipellis made up of smooth, clavate to lobed elements. However, M. opacus may have Rameales-type structures in the pileipellis at the pileus margin when fully mature along with the usual clavate to lobed, often thickwalled cells (Desjardin et al. 1993) and an insititious stipe accompanied by abundant rhizomorphs (Desjardin et al. 1993; Wilson and Desjardin 2005) and was therefore, previously considered as a Marasmiellus species. Interestingly, many taxa in this clade have basidiomata mimicking garlic or cabbage odor and flavor, but the same characteristic can be found in Gymnopus foetidus, G. perforans, G. iocephalus (Berk. & M.A. Curtis) Halling, G. polyphyllus (Peck) Halling and several species of the Androsacei group (Wilson and Desjardin 2005). A synoptic key comparing Morphology Pusillomyces manuripioides is very similar to Manuripia bifida in the macromorphology (Singer 1960, 1976, 1986). In the field, the specimens were immediately associated with the only species of Manuripia on account of the very small basidiomata, an orbicular, smooth pileus with smooth hymenophore, and a filiform, dark-colored, insititious stipe growing directly from abundant, both glabrous or densely pubescent rhizomorphs (Fig. 3a–c). Microscopically (Fig. 5), the new species is also like M. bifida (Singer 1960, 1976) in having ellipsoid, smooth, hyaline, inamyloid basidiospores, with smaller but still compatible dimensions (6– 9 × 2.8–4.6 μm vs 8–9.7 × 3.8–4.8 μm), the absence of cystidia in the hymenium, and by the inamyloid pileus trama while the stipe/rhizomorphs trama can be inamyloid or scarcely dextrinoid. However, P. manuripioides differs strikingly from M. bifida in having a non-hymeniform pileipellis composed of Rameales structures, with some Siccus-type broom cells, and also abundant incrusted hyphae in the upper to the mid-trama of the pileus, gradually disappearing near the hymenial layer. On the other hand, M. bifida has a hymeniform to subhymeniform pileipellis composed of balloon-shaped or vesiculose-clavate Rotalis-type broom cells and no incrusted hyphae in the pileus trama (Singer 1976). The rhizomorphs in P. manuripioides, when pubescent (Fig. 3c), are covered by elongate, dark-brown, multiseptated, smooth conidia on multiple conidiophores (Fig. 6) growing along the cortical trama of the stipe. According to Singer (1976), the rhizomorphs of M. bifida are covered by setoid, thick-walled, fuliginous to fuliginous-chestnut hairs (Singer 1960), clearly different from the elements found in the cortical layer of the rhizomorphs in P. manuripioides. Singer (1976) also described the rhizomorphs in M. bifida as thicker than the stipe whereas this distinction is absent or not clear in P. manuripioides. The two species also differ in the kind of substrate/habitat, strongly suggesting different niches (see the next section). Anatomically, the pileipellis arrangement and elements of P. manuripioides are compatible with species of Gymnopus sect. Androsacei (Mata et al. 2004a; Wilson and Desjardin 2005), previously Marasmius sect. Androsacei. On the other hand, the 730 arrangement and elements of the pileipellis in M. bifida are very similar to the pileipellis of members of Marasmius sect. Marasmius subsect. Marasmius (Singer 1976). In the phylogenetic discussion in the present paper, P. manuripioides is confirmed as a member of Omphalotaceae though this lineage does not branch close to G. androsaceus. If the hypothesis that M. bifida is truly a member of Marasmiaceae, the present study suggests that although P. manuripioides and M. bifida are strongly similar macroscopically and in some microscopic characteristics, the resemblance possibly represents a case of convergence. The inclusion of M. bifida in future analyses is important to have a final conclusion. In the BPhylogeny^ section, P. manuripioides (JO674 and JO1121) represents a distinct, new genus and that G. asetosus and G. funalis (Antonín et al. 2014) are congeneric. Pusillomyces manuripioides strongly differs from these two species in producing an even smaller basidioma with a smooth, discoid, up to 1.8 mm diam. pileus, a completely smooth hymenophore, a short and very thin, wiry stipe mostly arising from abundant, hair-like rhizomorphs or directly from the substrate, and the absence of cheilocystidia. In spite of often having some pubescent rhizomorphs, P. manuripioides is devoid of true caulocystidia, instead, elongate, dark brown, multiseptated conidia and conidiophores penetrating the cortical layer. Otherwise, both stipe and rhizomorphs are glabrous, which is similar to G. asetosus (Antonín et al. 2014). However, these three species share ellipsoid basidiospores, the absence of pleurocystidia, the arrangement and the elements of the pileus trama and the pileipellis, and the stipe trama and stipitipellis (excepting the caulocystidia in G. funalis) (Antonín et al. 2014). Below, a protologue is provided for Paragymnopus gen. nov. according to the clade H (Fig. 2), and Gymnopus (s. str.) and Marasmiellus (s. str.) are redefined based on clade G and clade A (Fig. 2) respectively, with correspondent combinations. Paragymnopus J.S. Oliveira, gen. nov. MycoBank MB 827363. Micromphale sect. Perforantia Singer, Sydowia 2: 32 (1948) Gymnopus sect. Perforantia (Singer) R.H. Petersen, MycoKey 18: 8 (2016) Etymology: It refers to the phylogenetic relationship of this group with Gymnopus s. str. Diagnose: Basidiomata marasmioid, small, thin. Pileus convex to plano-convex, occasionally slightly umbonate, sulcate-striate. Lamellae adnate, adnexed to decurrent, sometimes collariate to pseudocollariate, pallid or off-white. Stipe often up to 40 mm long, ratio of pileus breadth to stipe length often large (see G. pinophilus), central, cylindrical, thin, usually less than 1 mm, insititious, usually vestured (excepting Mycol Progress (2019) 18:713–739 G. glabrosipes), often minutely barbed under the × 10 lens, apex pale brown, then darker, dark sooty brown to nearly black towards the base. Rhizomorphs usually present, black, thin, filiform, branched or unbranched. Basidiospores obovoid, ellipsoid, amygdaliform, hyaline, smooth, thin-walled, non-amyloid. Pleurocystidia consistently present, fusiform, somewhat apically versiform. Cheilocystidia often absent; if present, either clavate to utriform or similar to Siccus-type broom cell. Pileus and lamellar tramae irregular, usually loosely interwoven, with hyphae embedded in a slime matrix. Pileipellis in the form of a gelatinized layer of repent, incrusted hyphae, often thick-walled, conspicuously clamped and embedded in a slime matrix, or as a well-developed Rameales-structure. Stipe medullary with hyphae embedded in slime matrix. Clamp connections present in all tissues. Chemical reactions: In Melzer’s reagent, dextrinoid only in the stipe cortical trama and caulocystidia of some species; otherwise, all tissues inamyloid. No part staining in alkaline solution (NH4OH or KOH). Ecology: Seemingly host-specific, saprophytic, on dead conifer needles or rotting deciduous leafy debris. Type species: Paragymnopus perforans (Hoffm.) J.S. Oliveira (see new combination below). Delimitation: According to Petersen and Hughes (2016), Gymnopus sect. Perforantia (herein as Paragymnopus) may be difficult to separate from Gymnopus sect. Androsacei. Basidiomata of some species in this latter produce a thin slime matrix in the basidiomata, a characteristic traditionally found in Micromphale sect. Perforantia, the genus/section where G. perforans belonged before being combined in Gymnopus sect. Perforantia (Petersen and Hughes 2016). This characteristic is also relevant for all species in Gymnopus sect. Perforantia, therefore, also in Paragymnopus. However, Gymnopus sect. Androsacei has a pileipellis consisting of a trichoderm (hymeniform in primordia) made up by diverticulate hyphae together with Siccus-type broom celllike and setulose endings or well-developed Rameales structure and lacking pleurocystidia while Gymnopus sect. Perforantia subsect. Perforantia (then, Paragymnopus) has a pileipellis formed by a cutis of repent, usually incrusted or varying from diverticulate to non-divericulate hyphae and having pleurocystidia (Antonín and Noordeloos 2010; Petersen and Hughes 2016). This distinction based on elements of the pileipellis is not observed between Gymnopus sect. Androsacei and Gymnopus sect. Perforantia subsect. Pinophili, a problem that was also noted by Petersen and Hughes (2016). A solution for this apparent inconsistency is not provided to date based on morphology since it was surprising to see G. pinophilus and G. ponderosae branching close to Gymnopus sect. Perforantia subsect. Perforantia instead of Gymnopus sect. Androsacei (Petersen and Hughes 2016). Macroscopically, Paragymnopus usually has offwhite to dingy pale gray lamellae, never attached to a Mycol Progress (2019) 18:713–739 pseudocollarium, and almost always a vestured stipe (Petersen and Hughes 2016). On the other hand, the lamellae in Gymnopus sect. Androsacei are often nearly as dark as the pileus, sometimes attached to a pseudocollarium, and the hair-like stipe is usually glabrous and shining (Antonín and Noordeloos 2010; Petersen and Hughes 2016). Finely hairy stipe can be found only in Gymnopus cremeostipitatus Antonín, R. Ryoo & K.H. Ka (Antonín and Noordeloos 2010) if the species is considered within the section based on the concept proposed in the present study (see more in the discussion on Gymnopus sect. Androsacei). Among numerous morphological divergencies, Paragymnopus (Clade H) differs from the other sections of Gymnopus s. str. (Clade G) in having fusiform pleurocystidia and dextrinoid structures (trama or elements). A synoptic key comparing Paragymnopus with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). Selected literature: Antonín and Noordeloos (2010); Petersen and Hughes (2016). Paragymnopus sect. Paragymnopus Gymnopus sect. Perforantia subsect. Perforantia, subsect. auton. Stipe usually vestured; cheilocystidia often absent, when present, clavate to utriform; pileipellis a layer of repent, incrusted or diverticulate hyphae (outgrowths on hyphal segments, not like Rameales structure), conspicuously clamped, usually with thickened walls (through gelatinization) and usually embedded in a slime matrix; fruiting on conifer needles or broad-leafed debris [summarized from Petersen and Hughes (2016) as Gymnopus sect. Perforantia]. Type species: Paragymnopus perforans (Hoffm.) J.S. Oliveira. Paragymnopus foliiphilus (R.H. Petersen) J.S. Oliveira, comb. nov. (MB 828406) Gymnopus foliiphilus R.H. Petersen, MycoKeys 18: 17 (2016) Paragymnopus foliiphilus var. costaricensis (R.H. Petersen & J.L. Mata) J.S. Oliveira, comb. nov. (MB 828407) Gymnopus foliiphilus var. costaricensis R.H. Petersen & J.L. Mata, MycoKeys 18: 28 (2016) Paragymnopus perforans (Hoffm.) J.S. Oliveira, comb. nov. (MB 828408) Agaricus perforans Hoffm., Nomencl. fung.: 215, t.4: 2 (1789) Paragymnopus perforans subsp. transatlanticus (R.H. Petersen) J.S. Oliveira, comb. nov. (MB 828409) 731 Gymnopus perforans subsp. transatlanticus R.H. Petersen, MycoKeys 18: 52 (2016). Paragymnopus sequoiae (Desjardin) J.S. Oliveira, comb. nov. (MB 828410) Micromphale sequoiae Desjardin, Mycologia 77: 894 (1986) Paragymnopus sublaccatus (R.H. Petersen) J.S. Oliveira, comb. nov. (MB 828411) Gymnopus sublaccatus R.H. Petersen, MycoKeys 18: 97 (2016) Paragymnopus sect. Pinophili (R.H. Petersen) J.S. Oliveira, comb. nov. Gymnopus sect. Perforantia subsect. Pinophili R.H. Petersen, MycoKeys 18: 9 (2016) Stipe glabrous-shining; cheilocystidia Siccus-type broom cells; pileipellis composed of well-developed Rameales structure; fruiting on needles of Pinus; discrete clade wellseparated from the clade of members of the previous section [summarized from Petersen and Hughes (2016) as Gymnopus sect. Perforantia subsect. Pinophili]. Type species: Paragymnopus pinophilus (R.H. Petersen) J.S. Oliveira. Paragymnopus pinophilus (R.H. Petersen) J.S. Oliveira, comb. nov. (MB 828412) Gymnopus pinophilus R.H. Petersen, MycoKeys 18: 62 (2016) Paragymnopus ponderosae (R.H. Petersen) J.S. Oliveira, comb. nov. (MB 828413) Gymnopus ponderosae R.H. Petersen, MycoKeys 18: 70 (2016) Gymnopus (Pers.) Roussel, Flore du Calvados et terrains adjacents, composée suivant la méthode de Jussieu: 62 (1806) Basidiomata collybioid, rarely tricholomatoid or marasmioid. Pileus convex, plano-convex to applanate or slightly concave, with or without umbo or papilla, hygrophanous or not, translucently striate or not, dry or slightly viscid, glabrous or innately radially fibrillose. Lamellae free, emarginate or adnate, usually crowded, sometimes fairly distant, regular, segmentiform or ventricose with entire or serrate edge. Stipe central, cylindrical fleshy to filiform, wiry, sometimes broadened towards base, glabrous and polished or fibrillose to finely pubescent (mostly when the basidiomata are fetid or dried or when any tissue reacts to alkaline solution), insititious or more often non-insititious, usually with strigose base, some with whitish rhizoids or sometimes deeply rooting or arising from a sclerotium, tough and firm, solid or fistulose. Rhizomorphs rarely present. Odor indistinct or fetid 732 (rotten cabbage, sewage, etc.) or onion- or garlic-like. Spore print white. Basidiospores ellipsoid to short-oblong (never very elongate or long-clavate), not often subglobose to globose or lacrymoid, thin-walled, hyaline, non-amyloid, with confluent or well-delimitated hilar appendage. Basidia 4-spored, clamped. Cheilocystidia usually present, cylindrical, flexuous, clavate or irregularly coralloid, thin-walled, sometimes as broom cells. Pleurocystidia absent. Lamellar trama regular to subregular. Pileus trama irregular. Pileipellis a cutis or ixocutis of radially arranged cylindrical hyphae, or interwoven, more like a trichoderm or ixotrichoderm, made up of irregular coralloid terminal elements (Dryophila-type structures), sometimes hymeniform in primordial stages only, then soon an irregular trichoderm, composed of irregular, often incrusted, diverticulate hyphal elements, mixed with broom cells and coralloid hyphae. Clamp connections present in all tissues (except for G. bisporiger Antonín & Noordel.). Chemical reactions: No reaction to Melzer’s reagent or Cresyl Blue, but there are rare cases where incrustations of the hyphae are dextrinoid or in Gymnopus sect. Androsacei where at least the stipe trama is dextrinoid; some taxa have also tissue of the basidiome turning green, olivaceous or ochraceous in alkaline solution (NH4OH or KOH). Ecology: Saprotrophic, rarely parasitic; in humus, on wood, rarely on roots of dead, not often on living herbaceous or woody plants (Antonín and Noordeloos 2010). Type species: Gymnopus fusipes (Bull.) Gray. Delimitation: According to the clade G, Gymnopus s. str. is composed of members of Gymnopus sect. Gymnopus, sect. Androsacei, sect. Levipedes (subsections Levipedes and Alkalivirentes) and sect. Impudicae. With only one exception so far, G. barbipes R.H. Petersen & K.W. Hughes that grouped along with members of sect. Impudicae, Gymnopus sect. Vestipedes is segregated and now placed within Marasmiellus s. str. In spite of being very distinct genera based on the phylogenetic trees of the present study, Gymnopus s. str. and Marasmiellus s. str. agree in many features and remain devoid of sharp morphological divergency. As long as members of Gymnopus sect. Vestipedes can be recognized as such using morphological characteristics, dichotomy between this group (sect. Vestipedes) and the other sections of Gymnopus s. str. can be warranted. In other words, this is the limit between Gymnopus s. str. and Marasmiellus s. str. Based on the present study, Gymnopus s. str. contains the four cited sections, and they are found either to be monophyletic (with high support) or tending to monophyletic but unsupported. This result is quite concordant with previous phylogenetic studies (Wilson and Desjardin 2005; Mata et al. 2006; Hughes et al. 2010; Coimbra et al. 2015; Petersen and Hughes 2016, 2017). Wilson and Desjardin (2005) were the first to suggest these possible concepts of distinc genera, despite the fact that their /marasmiellus was unsupported and their /gymnopus included Micromphale perforans (Paragymnopus). Mata et al. (2006), in spite of defending a Mycol Progress (2019) 18:713–739 broad concept of Gymnopus including Marasmiellus, found groupings suggested not only two possible genera, but a possible infrageneric relationship of Gymnopus s. str. into sections. These possible sections were assimilated by Antonín and Noordeloos (2010), but they did not definitively exclude Gymnopus sect. Vestipedes from the genus. Hughes et al. (2010) and Petersen and Hughes (2016, 2017) also found this pattern in their tree, but the concept of two genera (Gymnopus and Marasmiellus) was not established, and only included a new section in the broad Gymnopus (Petersen and Hughes 2016). Coimbra et al. (2015) worked on and developed the concept of Gymnopus sect. Impudicae. The present study not only provides limits between Gymnopus and Marasmiellus, but also strengthens the concepts of at least four possibly natural sections in Gymnopus. The sections are presented below, but the concepts of subsections are not treated in this present paper. Additionally, a synoptic key comparing Gymnopus s. str. with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). Selected literature: Singer (1986), Wilson et al. (2004), Antonín et al. (1997), Antonín and Noordeloos (1997, 2010). Gymnopus sect. Gymnopus Basidiomata fleshy; stipe radicant, forming a distinct pseudorrhiza, deeply sulcate-striate; spore print white to pale ochraceous; cheilocystidia present; pileipellis as a transition between a cutis and a trichoderm, often somewhat gelatinized, composed of irregular, inflated, or often coralloid elements, some resembling Dryophila-type structures; no dextrinoid or cyanophilous structures; parasitic or saprophytic, in bundles at the base of broad-leaved trees, frequently on roots or stumps [summarized from Antonín and Noordeloos (2010)]. Type species: Gymnopus fusipes (Bull.) Gray. Gymnopus sect. Androsacei (Kühner) Antonín & Noordeloos, Czech Mycology 60: 25 (2008) Basidiomata small, marasmioid; pileus dull, dry; lamellae free to adnate, sometimes pseudocollariate; stipe filiform (hair-like), insititious and glabrous (if G. cremeostipitatus belongs to this section, then the stipe can be pallid and finely pubescent or hairy), cheilocystidia in the form of Siccus-type broom cells or coralloid elements; pileipellis hymeniform in primordia only, then non-hymeniform, an irregular trichoderm of often incrusted, diverticulate hyphal elements, irregular in outline, mixed with Siccus-type broom cells and coralloid elements; trama dextrinoid, at least in the stipe apex; saprophytic, on litter of coniferous or broadleaved trees [summarized from Antonín and Noordeloos (2010)]. Type species: Gymnopus androsaceus (L.) Della Magg. & Trassin. Notes: Gymnopus cremeostipitatus Antonín, R. Ryoo & K.H. Ka, G. neobrevipes R.H. Petersen and G. portoricensis R.H. Petersen are additional taxa according to the present circumscription. A small strongly supported clade groups Mycol Progress (2019) 18:713–739 G. androsaceus along with informal taxa named Gymnopus adventitius nom. prov., G. frigidomarginatus nom. prov., and G. novaeangliae nom. prov. Also, perhaps Gymnopus inflatotrama nom. prov. and G. novomundi nom. prov. are close species, but they await description. These informal taxa seem to belong to Gymnopus sect. Androsacei along with the four already described species. However, all these species did not form a clade, so a definitive resolution will wait for future studies. Out of these species, only G. cremeostipitatus produce non-glabrous and pale stipe and may be a taxon of other section, helping to separate Gymnopus sect. Androsacei from Paragymnopus sect. Paragymnopus. Gymnopus sect. Levipedes (Fr.) Halling, Brittonia 48 (4): 487 (1996). Stipe smooth, polished or pubescent; pileipellis mostly as an entangled (never radially oriented) trichoderm, composed of inflated, often lobed elements or coralloid, Dryophila-type structures; trama and elements non-dextrinoid, some species turning green in alkali; saprophytic, usually in coarse humus and forest litter, or on rotten wood [summarized from Antonín and Noordeloos (2010)]. Type species: Gymnopus dryophilus (Bull.) Murrill. Additional species: Gymnopus alkalivirens (Singer) Halling; G. alpinus (Vilgalys & O.K. Mill.) Antonín & Noordel.; G. aquosus (Bull.) Antonín & Noordel.; G. aurantiipes (Corner) A.W. Wilson, Desjardin & E. Horak; G. austrosemihirtipes A.W. Wilson, Desjardin & E. Horak; G. bicolor A.W. Wilson, Desjardin & E. Horak; G. bisporus (J. Carbó & Pérez-De-Greg.) J. Carbó & PérezDe-Greg.; G. catalonicus (Vila & Llimona) Vila & Llimona; G. earleae Murrill; G. erythropus (Pers.) Antonín, Halling & Noordel.; G. exculptus (Fr.) J.L. Mata; G. fagiphilus (Velen.) Antonín, Halling & Noordel.; G. hybridus (Kühner & Romagn.) Antonín & Noordel.; G. inusitatus (Vila & Llimona) Vila & Llimona; G. inusitatus var. cystidiatus Antonín; G. indoctoides A.W. Wilson, Desjardin & E. Horak; G. junquilleus R.H. Petersen & J.L. Mata; G. kauffmanii (Halling) Halling; G. macropus Halling; G. montagnei (Berk.) Redhead; G. nubicola Halling; G. ocior (Pers.) Antonín & Noordel.; G. polyphyllus (Peck) Halling; G. salakensis A.W. Wilson; Desjardin & E. Horak; G. sepiiconicus (Corner) A.W. Wilson, Desjardin & E. Horak; G. spongiosus (Berk. & M.A. Curtis) Halling; G. subsulphureus (Peck) Murrill; G. vitellinipes A.W. Wilson, Desjardin & E. Horak. Gymnopus sect. Impudicae (Antonín & Noordel.) Antonín & Noordel.: 222 (2010) Basidiomata collybioid or marasmioid; smell strong, fetid (rotten cabbage, sewage, etc.) or like onions or garlic; cheilocystidia often inconspicuous; pileipellis composed of diverticulate elements, but never in form of true Dryophila- 733 type structures [summarized from Antonín and Noordeloos (2010)]. Type species: Gymnopus impudicus (Fr.) Antonín, Halling & Noordel. Additional species: G. barbipes R.H. Petersen & K.W. Hughes; G. brassicolens (Romagn.) Antonín & Noordel.; G. brassicolens var. pallidus Antonín & Noordel.; G. dysodes (Halling) Halling; G. foetidus (Sowerby) J.L. Mata & R.H. Petersen; G. impudicus var. graveolens (G. Poirault ex Boud.) Vila & Llimona; G. iocephalus (Berk. & M.A. Curtis) Halling. Marasmiellus Murrill, North American Flora 9 (4): 243 (1915). Marasmius sect. Rameales Kühner, Botaniste 25: 88 (1933). Gymnopus sect. Vestipedes (Fr.) Antonín, Halling & Noordel., emed., Mycotaxon 63: 363 (1997). Basidiomata gymnopoid, collybioid or omphalioid, marasmielloid, to pleurotoid. Pileus orbicular to semicircular, some reniform, applanate to convex, or campanulate, with or without papilla, flattened or wavy-lobate, white, yellow, pink or brown. Lamellae usually well-developed, rarely venose, free, adnate to decurrent. Stipe central, eccentric to almost lateral, sometimes reduced and curved, cylindrical, insititious or subinsititious, rarely with distinct basal mycelium, fistulose to hollow, pale at the apex, often darkening towards base, surface vestured, pruinose to pubescent, pulverulent, fibrillose, minutely flocculose to rather distinctly squamulose, but never glabrous or smooth. Rhizomorphs usually absent, rarely present. Odor indistinct. Spore print white to cream. Basidiospores ellipsoid to oblong, rarely sub-cylindrical, fusiform, lacrymoid or amygdaliform, usually with confluent hilar appendage, smooth, hyaline, thin-walled. Basidioles mostly fusoid. Lamellar edge usually sterile. Cheilocystidia often present, well-differentiated. Pleurocystidia usually absent. Lamellar and Pileus trama regular, subregular to irregular. Pileipellis usually a simple cutis, sometimes in transition to a trichoderm, with weakly to distinctly coralloid or diverticulate terminal elements (Rameales-structures), Dryophila-type structures never present. Caulocystidia present, well-developed, of various shapes. Clamp connections usually present. Chemical reactions: No amyloid, dextrinoid or cyanophilous reactions; usually not metachromatical in Cresyl Blue. Ecology: Usually gregarious, more rarely solitary, saprotrophic, more rarely parasitic on all kinds of living plants; some are host-specific; many species occur in rather exposed habitats, where moisture may vary considerably (costal dunes, xerophytic grassland); some are salt-tolerant (Antonín and Noordeloos 2010). Type species: Marasmiellus juniperinus Murrill. 734 Delimitation: Based on Clade A (Fig. 2), Marasmiellus s. str. is composed of members of Marasmiellus, at least, of sect. Dealbati, sect. Marasmiellus, sect. Rameales, and sect. Stenophylloides [see also Wilson and Desjardin (2005)] and Gymnopus sect. Vestipedes (excepting G. barbipes). Based on Wilson and Desjardin (2005), Marasmiellus sect. Candidi does not belong to this new concept of Marasmiellus s. str. As in Gymnopus s. str., Marasmiellus s. str. is more evidently delimited in cladistic analyses so far. Since the present analysis includes abundant members of Gymnopus sect. Vestipedes, but the sections of Marasmiellus are poorly represented, no infrageneric classifications are tested or newly proposed herein. Based on Wilson and Desjardin (2005) and the present study (Fig. 1), we learn that Marasmiellus sect. Candidi is polyphyletic and its members are not included in the family Omphalotaceae, but possibly belong to Marasmiaceae within multiple lineages. The presence of pleurocystidia is very rare, observed for instance in Marasmiellus phaeomarasmioides G. Moreno, Heykoop, Esteve-Rav. & E. Horak (scarce, filiform, versiform, sometimes with few outgrowths) and M. maasgeesterani Robich & E. Campo (sparse, clavate to sphaeropenduculate) (Antonín and Noordeloos 2010). In the same study, M. trabutii (Maire) Singer is the only one reported as having pileus trama embedded in gelatinous matrix. Unfortunately, these three species have no sequences included in phylogenetic analyses, and these mentioned characteristics are not confirmed in Marasmiellus s. str. A synoptic key comparing Marasmiellus s. str. with the other genera of /letinuloid clade is provided in Online Resource (Chart O1). Retnowati (2018) has just proposed 16 new species of Marasmiellus, two new combinations and eight names are lectotypified in the genus. However, genetic data still need to be provided for phylogenetic evaluation of those taxa. Selected literature: Singer (1986), Wilson et al. (2004), Wilson and Desjardin (2005), Antonín and Noordeloos (1997, 2010). Mycol Progress (2019) 18:713–739 including the correct spelling. Marasmiellus collybioides (Speg.) J.S. Oliveira, comb. nov. (MB 828417) Clitocybe collybioides Speg., Boletín de la Academia Nacional de Ciencias en Córdoba 11 (4): 387 (1889) Marasmiellus confluens (Pers.) J.S. Oliveira, comb. nov. (MB 828475) Agaricus confluens Pers., Annalen der Botanik (Usteri) 15: 8 (1795) Marasmiellus cylindricus (J.L. Mata) J.S. Oliveira, comb. nov. (MB 828477) Gymnopus cylindricus J.L. Mata, Fungal Diversity 16: 118 (2004) Marasmiellus dichrous (Berk. & M.A. Curtis) J.S. Oliveira, comb. nov. (MB 828478) Marasmius dichrous Berk. & M.A. Curtis, Annals and Magazine of Natural History 12: 426 (‘326’) (1853) Marasmiellus disjunctus (R.H. Petersen & K.W. Hughes) J.S. Oliveira, comb. nov. (MB 828479) Gymnopus disjunctus R.H. Petersen & K.W. Hughes, North American Fungi 9: 2 (2014) Marasmiellus eberhardtii (Pat.) J.S. Oliveira, comb. nov. (MB 828728) Laschia eberhardtii Pat., Bulletin de la Société Mycologique de France 25: 8 (1909) Marasmiellus eneficola (R.H. Petersen) J.S. Oliveira, comb. nov. (MB 828480) Gymnopus eneficola R.H. Petersen, Omphalina 5 (5): 5 (2014) Marasmiellus alnicola (J.L. Mata & Halling) J.S. Oliveira, comb. nov. (MB 828414) Gymnopus alnicola J.L. Mata & Halling, Fungal Diversity 16: 115 (2004) Marasmiellus fibrosipes (Berk. & M.A. Curtis) J.S. Oliveira, comb. nov. (MB 828584) Marasmius fibrosipes Berk. & M.A. Curtis, Journal of the Linnean Society. Botany 10: 293 (1869) Marasmiellus biformis (Peck) J.S. Oliveira, comb. nov. (MB 828415) Marasmius biformis Peck, Bulletin of the New York State Museum 67: 25 (1903) Marasmiellus fuscotramus (Mešić, Tkalčec & Chun Y. Deng) J.S. Oliveira, comb. nov. (MB 828481) Gymnopus fuscotramus Mešić, Tkalčec & Chun Y. Deng, Mycotaxon 117: 324 (2011) Marasmiellus brunneogracilis (Corner) J.S. Oliveira, comb. nov. (MB 828416) Marasmius brunneigracilis Corner, Beihefte zur Nova Hedwigia 111: 39 (1996) Notes: The correct spelling of the epithet is brunneogracilis instead of brunneigracilis. The name is herein combined Marasmiellus gibbosus (Corner) J.S. Oliveira, comb. nov. (MB 828482) Marasmius gibbosus Corner, Beihefte zur Nova Hedwigia 111: 55 (1996) Mycol Progress (2019) 18:713–739 Marasmiellus indoctus (Corner) J.S. Oliveira, comb. nov. (MB 828483) Marasmius indoctus Corner, Beihefte zur Nova Hedwigia 111: 60 (1996) Marasmiellus luxurians (Peck) J.S. Oliveira, comb. nov. (MB 828605) Collybia luxurians Peck, Bulletin of the Torrey Botanical Club 24: 141 (1897) Marasmiellus melanopus (A.W. Wilson, Desjardin & E. Horak) J.S. Oliveira, comb. nov. (MB 828484) Gymnopus melanopus A.W. Wilson, Desjardin & E. Horak, Sydowia 56 (1): 181 (2004) Marasmiellus menehune (Desjardin, Halling & Hemmes) J.S. Oliveira, comb. nov. (MB 828587) Gymnopus menehune Desjardin, Halling & Hemmes, Mycologia 91 (1): 173 (1999) Marasmiellus mesoamericanus (J.L. Mata) J.S. Oliveira, comb. nov. (MB 828490) Gymnopus mesoamericanus J.L. Mata, Sydowia 58 (2): 283 (2006) Marasmiellus micromphaloides (R.H. Petersen & K.W. Hughes) J.S. Oliveira, comb. nov. (MB 828491) Gymnopus micromphaloides R.H. Petersen and K.W. Hughes, North American Fungi 9: 6 (2014) Marasmiellus neotropicus (Singer) J.S. Oliveira, comb. nov. (MB 828586) Collybia neotropica Singer, Sydowia 15 (1-6): 54 (1962) Marasmiellus nonnullus (Corner) J.S. Oliveira, comb. nov. (MB 828510) Marasmius nonnullus Corner, Beihefte zur Nova Hedwigia 111: 76 (1996) Marasmiellus nonnullus var. attenuatus (Corner) J.S. Oliveira, comb. nov. (MB 828511) Marasmius nonnullus var. attenuatus Corner, Beihefte zur Nova Hedwigia 111: 77 (1996) Marasmiellus parvulus (J.L. Mata, R.H. Petersen & K.W. Hughes) J.S. Oliveira, comb. nov. (MB 828492) Gymnopus parvulus J.L. Mata, R.H. Petersen & K.W. Hughes, Sydowia 58 (2): 285 (2006) Marasmiellus peronatus (Bolton) J.S. Oliveira, comb. nov. (MB 828512) 735 Agaricus peronatus Bolton, An History of Fungusses, Growing about Halifax 2: t. 58 (1788) Marasmiellus polygrammus (Mont.) J.S. Oliveira, comb. nov. (MB 828585) Marasmius polygrammus Mont., Annales des Sciences Naturelles Botanique 1: 118 (1854) Marasmiellus pseudoluxurians (R.H. Petersen and K.W. Hughes) J.S. Oliveira, comb. nov. (MB 828493) Gymnopus pseudoluxurians R.H. Petersen and K.W. Hughes, North American Fungi 9: 7 (2014) Marasmiellus pseudomphalioides (Dennis) J.S. Oliveira, comb. nov. (MB 828589) Collybia pseudomphalodes Dennis, Kew Bulletin 15 (1): 74 (1961) Notes: Acconding to Dennis (1961), the epithet is due to the resemblance to Collybia omphalodes (Berk.) Dennis (≡ Marasmius omphalodes Berk.). Berkeley (1856) justified the choice of the name by considering the species between Marasmius and Omphalia. The correct spelling is Marasmius omphalioides, and therefore, Collybia pseudomphalioides Dennis. The is combined herein with the correct spelling. Marasmiellus quercophilus (Pouzar) J.S. Oliveira, comb. nov. (MB 828588) Marasmius quercophilus Pouzar, Ceská Mykologie 36 (1): 1 (1982) Marasmiellus stevensonii (E. Horak) J.S. Oliveira, comb. nov. (MB 828726) Collybia stevensoniae E. Horak, New Zealand Journal of Botany 9 (3): 450 (1971) [nom. nov. to replace the illegitimate ≡ Crinipellis readii G. Stev., Kew Bulletin 19 (1): 43 (1964)] Marasmiellus subcyathiformis (Murrill) J.S. Oliveira, comb. nov. (MB 828602) Marasmius subcyathiformis Murrill, North American Flora 9 (4): 269 (1915) Marasmiellus subnudus (Ellis ex Peck) J.S. Oliveira, comb. nov. (MB 828513) Marasmius subnudus Ellis ex Peck, Annual Report of the New York State Museum 51: 287. (1898 [as 1897]) Marasmiellus subpruinosus (Murrill) J.S. Oliveira, comb. nov. (MB 828603) Marasmius subpruinosus Murrill, North American Flora 9 (4): 266 (1915) 736 Mycol Progress (2019) 18:713–739 Fig. 7 Infection and necrosis on living leaves until death by Pusillomyces manuripioides (JO1117). a Rhizomorph attachment to the foliar face. b Early stage of the infection. c Necrosis of the foliar tissue. d Death of the initially infected area. e Spot of necrosis in living leaf and decaying of dead leaf. f Death of the entire leaf Marasmiellus indonesiensis A.W. Wilson, Desjardin & E. Horak ex J.S. Oliveira, sp. nov. (MB 828712) Etymology: refers to Indonesia, where the holotype was collected. Holotype: Indonesia, Bali, Lake Tamblingan, solitary on leaf litter, 15. 01. 2000, Wilson 39, holotypus (SFSU). Diagnosis: The effectively published description in A.W. Wilson, Desjardin & E. Horak, Sydowia 56 (1): 193 (2004), as Gymnopus tamblinganensis nom. prov., according to the Art. 38.1, 38.11 and 38.13 of the Code. Notes: The name Marasmiellus tamblinganensis is already occupied [Marasmiellus tamblinganensis Retn., Gardens Bulletin Singapore 70 (1): 227 (2018)]. Marasmiellus termiticola (Corner) J.S. Oliveira, comb. nov. (MB 828509) Marasmius termiticola Corner, Beihefte zur Nova Hedwigia 111: 101 (1996) Marasmiellus trogioides (A.W. Wilson, Desjardin & E. Horak) J.S. Oliveira, comb. nov. (MB 828494) Gymnopus trogioides A.W. Wilson, Desjardin & E. Horak, Sydowia 56 (1): 195 (2004) Marasmiellus villosipes (Cleland) J.S. Oliveira, comb. nov. (MB 828606) Marasmius villosipes Cleland, Toadstools and mushrooms and other larger fungi of South Australia 1: 166 (1934) Additional species: Marasmiellus ramealis (Bull.) Singer; Marasmiellus stenophyllus (Mont.) Singer; Marasmiellus synodicus (Kunze) Singer; Marasmiellus vaillantii (Pers.) Singer. Ecology of Pusillomyces manuripioides Pusillomyces manuripioides is epiphyte, forming a rhizomorph net entangled with hanging living and dead (or dying) leaves, twigs, branches and limbs about 1.5 m and upwards off the ground, seemingly strictly or at least more frequently of Eugenia spp. (shrubby to subarboreal trees of the subforest) (Fig. 3a, Online Resource Fig. O6a–b). Manuripia bifida, however, was found on dicotyledonous dead fallen woody sticks on the forest floor (Singer 1976). This is a very important distinction in the habit and habitat, Mycol Progress (2019) 18:713–739 suggesting different niches. Being epiphytic, P. manuripioides demonstrates particular adaptations, pattern of reproduction and distribution, and different nutrition mode. It acts as a facultative parasite causing necrosis in living leaves on where the rhizomorphs are attached (Fig. 7), being a kind of Horse Hair Blight disease with true infection of living plant tissues. Field observations suggest that there is host specificity with Eugenia spp. for the parasitic mode, whereas there is no substrate specificity for the saprotrophic mode. To properly understand the ecology of the species, firstly, we observed that the main structural body unit of the hypothetical individuals is represented by an interconnected net of rhizomorphs from where most of the basidiomata grow. Even those basidiomata that seem to grow directly from the leaves were actually arising from rhizomorphs strongly adhered to the foliar face. This adherence of the rhizomorphs is essential since it may serve both for attachment and parasitic nutrition. Therefore, the production of rhizomorphs is the main strategy of the species instead of being an accessory or secondary growth mode. That is, we shall always find the species growing as this interconnected net of rhizomorphs where we possibly can find the tiny basidiomata. Secondly, the habit of the new species strongly indicates that it can act as both a biotrophic on living leaves and saprotrophic on dead leaves resulting from the parasitic activity. Moreover, dead leaves of various tree species that fall from the canopy can be also captured in the rhizomorphs net, serving as food for the fungus. The infection initiates with the adherence established when a free part of rhizomorph touches the foliar face (Figs. 3, 4, and 7a). Then, generative hyphae grow from the inner part of the rhizomorph out to the substrate (Fig. 7a, b), invading the cortical layer of the leaf. These abundant generative hyphae form a visible cream-colored mycelial tomentum along both sides of the rhizomorph. On living substrate, the rhizomorph of P. manuripioides provokes necrosis on previously healthy leaves, starting as a dirty brown difuse spot around the mycelium tomentum of the rhizomorphs (Fig. 7b). Then, the necrosis progresses damaging the immediate area of the leaf causing it to become pale brown with dark brown border (Fig. 7c). The damage gets more severe, killing that area of the leaf. The killed area becomes pale yellow or brownish yellow with some conspicuous and abundant inconspicuous dark brown dots (Fig. 7d, e). It is possible to see a large dark brown spot around the infecting rhizomorph. The necrosis expands to the entire leaf, causing death of the whole structure (Fig. 7f). Once the leaf is dead, the infecting rhizomorph switches to the sapotrophic mode, decomposing the dead structure. The rhizomorphs can also be found attached along living limbs, branches and twigs bearing infected leaves. These parts do not seem to be damaged and may be used only to conduct the rhizomorphs to the leaves, but this need confirmation. More evidences of the parasitic activity of P. manuripioides such as the morphology of the infection apparatus and the 737 manner in which the fungus invades the plant, the life cycle, the pathogenicity, the host specificity and a thorough observation on the ecology of the species will be the subject of another paper. Conclusions Significant progress is reached on the phylogeny of Omphalotaceae, retaining the widely used genera, Gymnopus and Marasmiellus. These genera have been the subject of many efforts firstly (and historically) in morphology and more recently in molecular phylogeny studies to find definitive concepts in the taxonomy and systematics of these groups. The present study offers clear evidences for Gymnopus s. str. consisting of Gymnopus sections Androsacei, Gymnopus, Impudicae and Levipedes while Marasmiellus s. str. is composed of Gymnopus sect. Vestipedes and members of Marasmiellus sections Dealbati, Marasmiellus, Rameales, and Stenophylloides (so far). Based on Wilson and Desjardin (2005), Marasmiellus sect. Candidi is excluded from Marasmiellus s. str. Therefore, we defend the concept and propose the acceptance of a less inclusive Gymnopus; and on the other hand, Marasmiellus forming a distinct genus as suggested by Wilson and Desjardin (2005). Two new genera are now added to Omphalotaceae: Paragymnopus and Pusillomyces. Paragymnopus contains species of Gymnopus sect. Perforantia (Petersen and Hughes 2016). Pusillomyces is a new genus composed of P. manuripioides and the now combined Gymnopus asetosus and G. funalis. All genera recognized in this study follow Vellinga et al. (2015). Pusillomyces manuripioides is a phytopathogenic fungus found with an epiphytic habit more frequently on Eugenia spp. in BCampinarana,^ Amazon forest, but specific studies of its pathogenicity ought to be made to understand its biology in depth. Full taxonomic treatment was provided to fulfill nomenclatural requirements for the new taxa. Acknowledgments The authors thank the INPA Herbarium for contributing to this publication, R.H.S.F. Cruz for doing the illustrations, Dr. Tarciso de Sousa Filgueiras for reviewing the names in Latin, Dr. Genevieve Gates (Honorary Associate in Mycology and Forest Ecology, Tasmanian Institute of Agriculture) for reviewing the English writing and the anonymous reviewers for relevant contribution to this paper. The authors also thank the financial and logistical support from the Fundação de Amparo à Pesquisa do Estado do Amazonas – FAPEAM, the Centro de Estudos Integrados da Biodiversidade Am azônica (INCT- CENBAM), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Biodiversity Research Program (PPBio), and the Japan Science and Technology Agency /Japan International Cooperation Agency - Science and Technology Research Partnership for Sustainable Development (JST/ JICA-SATREPS). Study registered in SISGen (Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado) number A44535D. Funding This study was supported by BCoordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES^ with a 738 scholarship from BPrograma Nacional de Pós-doutorado – PNPD^ granted to J.J.S. de Oliveira, post-doctoral fellow of DIBOT, and other to T.S. Cabral, post-doctoral fellow of DIGEN, INPA. For R. 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