Fungal diversity in the tropics: Entoloma spp. in Panama

Entoloma (Agaricales, Basidiomycota) is a species-rich genus with approximately 2000 species known worldwide. In Central America, however, information about the species of this genus is sparse, despite the generally high biodiversity in this region. Recently, 124 specimens of Entoloma were collected in Panama, Chiriquí Province. In the present publication, the morphology of 20 species represented by more than one specimen is described and depicted with photographs, line drawings, and scanning electron micrographs. Molecular phylograms based on ITS or concatenated ITS and partial nc LSU rDNA sequences are provided. The taxonomic status of these species is evaluated and 17 species of Entoloma are described as new to science. Only one species could be assigned to an already known species, viz. Entoloma belouvense. Nolanea albertinae, described from Brazil, appeared similar and is combined in E. belouvense on varietal level. The identifications of two further species are uncertain. At least 30 other species, including potentially new species, cannot formally be described due to insufficient material. A preliminary key to the species of the genus Entoloma in Panama is provided. The spatial shape of the polyhedroid basidiospores of Entoloma spp. is discussed based on literature and the micrographs generated for the present study. Our re-evaluations indicate that the type of polyhedroid basidiospore and the structure of its base are not reliable as diagnostic characters for the delimitation of subgenera in Entoloma.


The genus Entoloma in Central America
The genus Entoloma (Agaricales, Basidiomycota) includes species with almost exclusively polyhedroid, faceted basidiospores, which are angled in outline and pinkish as seen with a light microscope. It comprises mainly species with agaricoid basidiocarps; few species develop secotioid or gasteroid basidiocarps (Co-David et al. 2009;Kinoshita et al. 2012;Vidal et al. 2016). Currently, approximately 2000 species of Entoloma are known to science (Noordeloos et al. 2018). Two monographic treatments of the genus exist for two regions of North America (Hesler 1967;Largent 1994). Such monographs are not available for South America, but some preliminary and regionally comprehensive treatments have been published (Horak 1978(Horak , 1982Coimbra et al. 2013;Karstedt and Capelari 2013). Coimbra (2014) published a checklist including 271 species of Entolomataceae recorded for Central and South America. However, almost nothing is known about Entoloma species in Panama. Only one species, Entoloma cylindrocapitatum (T.J. Baroni & Ovrebo) Noordel. & Co-David, has been reported until now (Ovrebo and Baroni 2007;Hofmann and Piepenbring 2021). Few Entoloma species are reported from the other Central American countries, Belize, Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua: six species are reported Section editor: Zhu-Liang Yang

Fieldwork
Specimens of Entoloma spp. were collected by the authors and collaborators from 2014 to 2018, mainly in montane forests at 1600-2400 m asl. dominated by Quercus spp., sometimes by Alnus acuminata. Some collections were made in a forest dominated by Oreomunnea mexicana at 1200 m asl., a forest dominated by Calophyllum longifolium Willd. at 40 m asl. on Isla Parida, and close to a path in disturbed secondary vegetation at 120 m asl. in Los Algarrobos. Most of the specimens were photographed in the field. Further photos were taken in the laboratory and ephemeral characters as colour, smell, and taste were registered. Colour terms and codes were determined using Kornerup and Wanscher (1967). Coordinates of the locations were recorded using a Garmin GPSmap 62 (Garmin Deutschland, Garching, Germany). The basidiocarps were dried at 40-45 °C on an electric food dehydrator and frozen for several days before storage in herbaria. Mainly specimens of species represented by at least two collections were studied in detail.

Light microscopy
Micromorphological characters of the basidiocarps were analysed by light microscopy on dried material. The size of the basidiospores, hymenial structures, and features of the pileipellis were investigated in hand-cut sections mounted in tap water or in 5-10% KOH. To measure the long cells of the hymenophoral trama and stipe surface, sections were soaked in KOH for 5 min, rinsed with deionised water, and stained with Congo Red. At least 20 basidiospores were measured from lamellae squash preparations for each collection. For some specimens, the presence or absence of clamp connections at the base of basidia was additionally examined using phase contrast. Spore sizes and Q-values are given in 5th percentile-mean-95th percentile. Spore sizes are rounded to the nearest 0.5 µm, Q-values to the nearest 0.05. Other values of measurements were less strictly rounded off to avoid pseudo-exact indication of sizes.

Scanning electron microscopy (SEM)
Fragments of basidiocarps were prepared for SEM following a protocol including the use of dioctyl sodium sulfosuccinate (DSS), based on Koch et al. (2021) and Erbar (1995) with several modifications. Small pieces of lamellae were soaked in a solution of 5% (w/v) DSS in 4:1 water:ethanol for 24 h. The samples were then rinsed three times in 4:1 water:ethanol and two times in 0.1 M sodium cacodylate buffer and thereafter fixed in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer for 20-24 h. After fixation, the samples were rinsed two times in the cadodylate buffer and then dehydrated in a graded ethanol series of 30, 50, 70, 90, 100, and 100% for 10-20 min each. The dehydrated samples were stored in 100% ethanol until critical point drying using liquid CO 2 . Dried samples were sputtered with gold and studied in a Hitachi (S 4500) scanning electron microscope. The description of basidiospore structures follows Pegler and Young (1979), except that polyhedron terminology is used instead of their spore types. The earlier term "dièdre basal" (Kühner and Boursier 1929) is used instead of "dihedral base" to avoid confusion with mathematical terms.

DNA extraction and PCR
Pieces of lamellae of 3-10 mm 2 were taken from dry basidiocarps and ground in a MM301 Mixer Mill (Retsch GmbH, Haan, Germany). DNA was extracted from the resulting powder using the peqGOLD fungal DNA mini kit (VWR, Darmstadt, Germany) or the innuPREP Plant DNA Kit (analytikjena, Jena, Germany) according to the instruction manuals. The ITS region was amplified by PCR in a peqSTAR gradient thermal cycler (PEQLAB, Erlangen, Germany) using the VWR Taq DNA polymerase (VWR, Darmstadt, Germany). To obtain the ITS sequences, the forward primer ITS1 or ITS1F and the reverse primer ITS4 or ITS4B (White et al. 1990; Gardes and Bruns 1993) were used. Further ITS sequences were obtained as described by Papp and Dima (2018). To obtain the D1/D2 region of the nc LSU rDNA, the primers LR0R together with LR5 (Vilgalys and Hester 1990) or NL1 together with NL4 (O'Donnell 1992) were used. Both rDNA regions were amplified using the same PCR protocol: denaturation at 98 °C for 4 min followed by 35 cycles of 95 °C for 45 s, 53 °C for 30 s, and 72 °C for 60 s, with a final elongation step at 72 °C for 5 min. A partial sequence of the second largest subunit of RNA polymerase II (RPB2) was obtained using the primers rpb2-6F and rpb2-7.1R (Matheny 2005) with a touchdown PCR protocol: denaturation at 95 °C for 4 min followed by 14 cycles of 94 °C for 45 s, 56 °C (-0.5 °C/cyc) for 60 s, and 72 °C for 60 s, thereafter 40 cycles of 94 °C for 30 s, 53 °C for 40 s, and 72 °C for 60 s, with a final elongation step at 72 °C for 10 min. Success of amplification was checked by gel electrophoresis using a 1% (w/v) agarose gel. Successfully amplified products were sent to Microsynth Seqlab (Göttingen, Germany) for purification and Sanger sequencing.

Phylogenetic analyses
Sequences were edited with Geneious 2019.2.1 (Biomatters Ltd., Auckland, New Zealand) and aligned with Mafft (Katoh and Standley 2013) using the E-INS-i algorithm. The ends of the resulting alignments as well as unreliable terminal parts of sequences were manually pruned in AliView (Larsson 2014). For the phylogenetic analysis of sequences of subg. Cyanula, aligned ITS and LSU sequences were concatenated and a piece of 61 nucleotides at the 5ʹ end of the LSU, which was not available for many sequences, excluded from the final alignment. Maximum likelihood trees were built using RAxML 8.2.11 (Stamatakis 2014). The GTRGAMMA model was used for alignments of less than 50 sequences, the GTR CAT model with 45 per site rate categories for 50 sequences, and more, along with a bootstrap analysis (Felsenstein 1985) with 500 repetitions. Resultant phylogenetic trees were visualised using FigTree (Rambaut 2014). All sequences generated are deposited in GenBank (Table 1).

Results
A total of 124 specimens of Entoloma spp. were collected. These specimens represent more than 50 species, with at least 22 species being represented by more than one specimen. Twenty of these species were studied in detail and are described below. Only one species could be assigned to an already described species and two species were identified with considerable uncertainty. Seventeen species are unknown to science and are described as new species below. The most frequently represented subclade was the subgenus Cyanula, with 35 specimens belonging to 18 species. Specimens of the subgenera Alboleptonia, Cubospora Entoloma, and Nolanea were frequently found as well (Table 2). In 2017 and 2018, most specimens were collected in montane forests with abundant to dominating trees of Quercus spp. The highest number of Entoloma specimens for a single location, i.e. 22 specimens found during three visits, was collected in the PILA, in a montane forest dominated by Alnus acuminata, with some Quercus spp. and at least one individual of Podocarpus sp. On the contrary, only two specimens, both of subg. Cyanula, were collected in lowland habitats, one in a forest close to mangrove forest on Island Parida and the other one at a path in disturbed secondary vegetation (Table 3). This path in Los Algarrobos was the most often visited location in these years. The specimens collected in the years before 2017 were mainly collected during a local inventory project and are thus less informative for comparisons of diversity of habitats.

Shape of basidiospores
Polyhedroid basidiospores of 17 species of Entoloma from Panama were analysed by SEM. The basidiospores displayed a considerable degree of variability, increasing with the number of facets. Apart from the cuboid basidiospores, the number of angles of the respective facets was often variable and thus not determined. The adaxial facet is always a single facet and consistent in its position above the apiculus. The spatial formation of facets can abaxially be variable. The basal facet can either be next to the abaxial facet or separated from it by lateral facets (Fig. 2e). When two abaxial facets are formed, they can be next to each other or separated by lateral facets (Figs. 2i,26d). Shape and a consistent base type of the basidiospores of two species of subg. Entoloma (E. nuboocculatum and E. mediorobustum, described below) could not be determined because of considerable variability of these characters. Five species have hendecahedroid spores, three of subg. Cyanula and two of subg.
Nolanea. Two species each have heptahedroid, octahedroid, or decahedroid basidiospores, respectively, while one species each presents cuboid, enneahedroid, dodecahedroid, or tetrahedroid basidiospores. Reduced forms frequently occur, mainly concerning reduction or lack of the apical facet and/ or reduction of the pair of adaxial facets to a single adaxial facet. Basidiospore shapes in subgenera are not consistent. The largest variability was observed in subg. Alboleptonia, including one species with mainly octahedroid, one species with enneahedroid, and another species with tetradecahedroid basidiospores. A simple base is formed in four species, two in subg. Entoloma and two in subg. Nolanea, while basidiospores with a dièdre basal are present in all subgenera, exclusively so in Alboleptonia, Cubospora, and Cyanula. A base built by three facets was found in the newly described species E. mediorobustum (Fig. 2l).

Taxonomy
The traditional infrageneric classification of Entoloma is based on morphological characters, viz., habit, lamellae attachment, pileipellis structure, and basidiospore shape (Romagnesi 1974;Romagnesi and Gilles 1979). This classification has been continuously revised and emended (Noordeloos 1981(Noordeloos , 1992(Noordeloos , 2004Largent 1994). Co-David et al. (2009) demonstrated that a high number of subgroups in this classification were not monophyletic based on molecular phylogenetic results. With the incorporation of DNA sequence data, several subgenera have since been revised, viz., Pouzarella by He et al. (2013), Leptonia by Morozova et al. (2014a), Entoloma (as "Rhodopolia") by Kokkonen (2015), and Claudopus by He et al. (2019). The section Cyanula was elevated to the rank of subgenus by Noordeloos and Gates (2012) and two new subgenera, Cubospora and Cuboeccilia, were described by Karstedt et al. (2019). He et al. (2019) recognised 10 major clades, largely referring to the studies just cited. Baroni et al. (2011) described the genus Entocybe for a few aberrant species of the large, basal clade of Entoloma. This new genus, however, causes the basal clade to be paraphyletic and was, therefore, not recognised by He et al. (2019).

Subgenus Entoloma Fig. 3
As in most other subgenera, species of subgenus Entoloma are hard to identify on macroscopical characters in the field, as they are pretty uniform in colour and stature. Romagnesi (1953, 1954) first considered the structure and pigmentation of the covering layer of the pileus as potential tools to distinguish species, besides size and shape of basidiospores. Noordeloos (1981Noordeloos ( , 1992Noordeloos ( , 2004) expanded the concept, resulting in an overview of sect. Rhodopolia in Europe      with species distinguished by a combination of macro-and microscopical characters. Since an ectomycorrhizal lifestyle was discovered for species of subg. Entoloma (Antibus et al. 1981;Loree et al. 1989;Agerer 1997), ecological characters are also considered to delimit species. Kokkonen (2015) used ITS sequence data as a new tool to tackle concepts of species in subg. Entoloma in Northern Europe. New species were defined based on a combination of morphological and ecological characteristics combined with an ITS phylogeny. As a result, nine species were described as new to science and many species were synonymised. Ongoing studies in Europe (Brandrud et al. 2018;Noordeloos et al. 2018) show that there are still many questions as how to interpret classical species, as well as how to deal with apparently cryptic speciation in this clade. Montañez et al. (2016)    Description: Basidiocarps tricholomatoid. Pileus 3.0-5.0 cm diameter, convex when young, becoming plane to depressed in old basidiocarps, usually with an obtuse umbo, margin initially deflexed, then straight, blackish brown to dark yellowish brown (5F8) in the centre, dark brown to light brown (6D7, 6E7, 6F7, 6F8), olive brown (4F8), yellowish brown (5E7, 5E8) to greyish yellow (4C6, 4C7) towards the margin, pileal surface glabrous, with a waxy appearance, rugulose, slightly translucently striate at the margin up to a quarter of the radius in fresh basidiocarps. Lamellae adnate to sinuate, segmentiform to ventricose, rather distant, relatively thick and broad, initially whitish with greyish to cream tinge, later greyish pink, with smooth, concolorous edges. Stipe 3.5-4.0 × 0.6-0.9 cm, cylindrical, solid in young basidiocarps, becoming hollow, basically yellowish brown (5E5, 5E6, 5E7) to olive brown (4E6, 4E7, 4E8, 4F6, 4F7), overlaid by whitish fibrils, paler, almost white, towards base, whitish fibrillose-flocculose at the apex when young. Basal mycelium white. Odour and taste farinaceous to farinaceous rancid.
Lamellae sinuate, segmentiform to ventricose, crowded, initially almost white to pale greyish, later pink, with slightly blunt serrate, concolorous edges and moderately to strongly transvenose sides. Stipe 8.0-12.0 × 0.8-1.0 cm, cylindrical to somewhat tapering towards base, solid, greyish yellow (4B4, 4B5, 4C3, 4C4, 4C5, 4C6, 4C7) under a whitish fibrillose to Notes: Basidiocarps of E. pruinosum are characterised by a tricholomatoid habit, a brown surface of the pileus with a distinct pruina and relatively small basidiospores. This combination of characters is rather unique. No other known species in subg. Entoloma has such a strongly developed pruina on the pileus. The small, isodiametrical spores are more or less like those found in the /prunuloides clade, and the European E. prunuloides may have a slight, but not so strong pruina on the pileus. Entoloma prunuloides, however, like some other similar species, is phylogenetically very distant from subg. Entoloma (Morgado et al. 2013). Entoloma pruinosum belongs to a clade of species known from Central and North America. In the ITS phylogeny (Fig. 5), it is close to E. brunneocinereum Hesler, with a p-distance of 2.7% in the ITS1. This species has, however, somewhat smaller basidiospores (7.0-8.0 × 6.0-7.0 µm) and intracellular pigment (Noordeloos 1988). Based on type studies, Kokkonen (2015) demonstrated that E. lividomurinum Hesler is conspecific with E. brunneocinereum. Entoloma griseopruinatum Cheype & Noordel., a European species with a pruinose pileus, has larger basidiospores (9.0-11.0 × 7.0-8.0 µm), more greyish colours in stipe and pileus, intracellular pigment in the pileipellis, and is phylogenetically distant (Fig. 5).
Subgenus Nolanea (Fr.) Noordel (Fig. 12) Traditionally, species of the subg. Nolanea are characterised by basidiocarps with a mycenoid habit, a rather smooth pileus surface, and relatively long cells in the hymenophoral trama (Noordeloos 1980). A preliminary study on this subgenus in Europe was published by Vila et al. (2013). Karstedt et al. (2020) described five new species from Brazil, using Nolanea at generic rank. Their molecular phylogeny based on three loci supports Nolanea as a monophyletic clade. The results of a large, worldwide study on this subgenus are currently being prepared for publication (Reschke et al., in prep.).
Holotype WU27132: Several basidiocarps in good condition. Basidiospores heterodiametrical, (Noordeloos and Hausknecht 2007), no clamp connections were observed at the base of basidia. Mycobank: MB840620. Basidiocarps mycenoid. Pileus 1.5-2.0 cm diameter, broadly conical to expanded with pronounced papilla and straight to deflexed, somewhat uneven margin, greyish yellow-golden, brown to dark brown (4C6, 6E5, 6E6, 6F5, 6F6) with dark brown (6F6, 7F5) centre, pileal surface glabrous, somewhat innately fibrillose, translucently striate almost to the centre, hygrophanous and then with sericeous appearance. Lamellae adnexed to free, ventricose, mediumspaced to rather distant, greyish white when young, becoming brownish pink, with smooth and concolorous edges. Stipe Notes: Nolanea albertinae was described from Brazil by Karstedt et al. (2020). Before, it had been treated as E. belouvense by Karstedt et al. (2019). In the phylogeny (Fig. 14), the ITS sequence of the holotype of E. belouvense var. belouvense is nested among the sequences of specimens of E. belouvense var. albertinae. However, the differences in spore size, resulting in higher Q-values in E. belouvense var. belouvense, are confirmed here. Apart from this, no differing characters were observed. Nolanea albertinae is treated here as a variety of E. belouvense because only one specimen of the type variety is available and ITS sequences may not be suitable to resolve such close relationships. More specimens from the type region, La Réunion, are needed to evaluate if this treatment is justified. Similar to the type variety, no clamp connections were observed in the specimens from Panama. Etymology: crème (French) = cream (here: colour), striatus (Latin) = striate; refers to the morphology of the pileus.
Habitat: Basidiocarps solitarily to gregarious on soil, between mosses and litter, in Alnus acuminata-dominated montane forest and Quercus-dominated montane forest between 2300 and 2400 m asl. in Chiriquí, Panama.
Additional Notes: Entoloma cremeostriatum is characterised by a convex-umbilicate, pale, striate pileus, small, heterodiametrical basidiospores, and clamp connections at the base of basidia. With a p-distance of 5.5% between the ITS sequences, it is relatively close to E. readiae G. Stev., described from New Zealand (Stevenson 1962). This species has basidiocarps which are like those of E. cremeostriatum in shape. Moreover, the two species share finely incrusting pigments in the hyphae of the pileipellis and clamp connections at the base of the basidia. The basidiospores of E. 1 3 readiae are larger than those of E. cremeostriatum according to the original description (9-10 × 7-8 µm). Our measurements of two authentic specimens (Table 1) of E. readiae, however, resulted in somewhat smaller basidiospore sizes, 7.5-8.4-9.0 × 6.5-7.4-8.0 µm, Q = 1.00-1.14-1.25 (n = 46 of 2 specimens). However, these are still somewhat larger and have smaller Q-values than those of E. cremeostriatum. In addition, basidiocarps of E. readiae are brown and have a strongly farinaceous odour, while those of E. cremeostriatum are generally paler and have a slightly nitrous odour. Two sequences annotated as E. pulchellum (Hongo) Hongo are relatively close to those of E. cremeostriatum with p-distances of 5.2% between the ITS sequences. Entoloma pulchellum is described as having a convex-umbilicate, yellow to flesh-coloured pileus with brown tones, often a minutely squamulose pileus surface, especially in the centre, a striate pileus margin, heterodiametrical basidiospores of 10.0-12.5 × 7.0-9.0 µm, clavate to capitate cheilocystidia of 25-37 × 13.0-14.5 µm, and no clamp connections at the base of basidia (Hongo 1957). These characters, especially the squamulose pileus surface together with the absence of clamp connections and presence of distinct cheilocystidia, indicate a species of subg. Cyanula, probably close to E. formosum (Fr.) Noordel. Thus, the specimens corresponding to the ITS sequences (KR673495, KC257436) annotated as E. pulchellum in GenBank apparently are misidentified. Reschke & Noordel.,sp. nov.,Figs. 17 and 18 Mycobank number: MB840631. Etymology: f lavus (Latin) = yellow, konikos (Greek) = conical; refers to the colour and shape of the pileus.
Odour indistinct, taste not tested. .2-10.5 × 6.0-6.6-7.5 µm, Q = 1.15-1.40-1.65 (n = 65 spores of 2 specimens), predominantly heptahedroid, composed of an adaxial facet (D), a pair of apico-adaxial facets (AD), an apical facet (A), a pair of lateral facets (L), and a basal facet (Ba) forming a simple base, reduced forms hexahedroid or more rarely pentahedroid (prismatic), with single AD-facet and/or reduced to absent A-facet, heterodiametrical to irregularly cruciform, with 4-5 pronounced angles in outline, weakly pigmented yellowish pink, somewhat thick-walled. Basidia Notes: Entoloma transitionisporum belongs to the clade of E. conferendum and its allies. It is characterised by a conical to convex umbilicate, brown, translucently striate pileus, a silvery fibrillose stipe, absence of clamps, and the shape of the basidiospores, which can be interpreted as a transitional state between heterodiametrical and cruciform. Entoloma belouvense is closely related based on ITS sequence data (Fig. 14). Its basidiocarps are on average smaller and paler than those of E. transitionisporum and their pilei have a contrasting darker centre, which was not observed in pilei of the latter species. In addition, the basidiospores of E. belouvense are somewhat longer and without transitional states to a cruciform shape. Basidiocarps of E. conferendum can have a similar habit, but E. conferendum is easily distinguished from E. transitionisporum by exclusively cruciform basidiospores. Entoloma luteifuscum K.N.A. Raj & Manim., described from India, differs by yellowish tones of the basidiocarps and slightly larger basidiospores, which are exclusively heterodiametrical (Raj et al. 2014). Entoloma dissimile (Singer) Horak, described from Nothofagus-forest in Argentinia, may be similar to E. transitionisporum. However, the pilei of E. dissimile are darker, basidiospores are slightly broader, and pigment is incrusting in the pileipellis (Horak 1978). No similar species were found in Largent (1994), Horak (1982), andHesler (1967).

Subgenus Alboleptonia (Largent & R.G. Benedict) Noordel
The subgenus Alboleptonia in the traditional sense (Largent and Benedict 1970;Noordeloos 1987Noordeloos , 2004 is an assemblage of white or whitish species, often with a differentiated pileipellis. It appears to be polyphyletic, considering recent phylogenies (He et al. 2015c;Largent et al. 2016). Here, we focus on the core group of Alboleptonia, i.e., species around the type species E. sericellum (Fr.) P. Kumm. This species is widespread in Europe and appears to be a complex of several species, both in and outside of Europe. Entoloma cuboidoalbum Noordel. & Hauskn. was described from Austria (Noordeloos and Hausknecht 2009), but there are more species waiting to be described in this group, as evident by the results of the phylogenetic analysis (Fig. 23). In the present study, three new species from Panama are Etymology: named after its similarity to Entoloma sericellum and its occurrence in the Parque Internacional La Amistad, honouring the principle of friendship in nature conservation.

) Noordel
Species of the subgenus Cyanula form a well-supported clade in the ITS/LSU phylogeny (Fig. 30). They develop basidiocarps with often bright colours, a differentiated pileipellis with a trichodermal structure, intracellular pigment, and clampless hyphae. The lamellar edge of many species is formed by tramal hyphae that grow through the hymenium forming a sterile band along the edge, with dense clusters of more or less clavate or fusiform terminal cells. These terminal cells often contain blue or brown intracellular pigment, causing a coloured border along the edge, the "serrulatum-type" lamellar edge (Noordeloos 2004). Species of this clade occur worldwide, from the arctic tundra to the tropical rainforest. In Europe, they are characteristic of oligotrophic grasslands, habitats which are generally threatened in Europe (Janssen et al. 2016). The subgenus Cyanula is the most species-rich subgenus within Entoloma, with approximately 500-600 species already described (Hesler 1967;Horak 1980Horak , 2008Largent 1994;Noordeloos 2004;Noordeloos and Gates 2012;Crous et al. 2021;Noordeloos et al. 2021;Dima et al. 2021). Species delimitation is often rather difficult. Morphological look-alikes occurring on several continents are often phylogenetically distant and must therefore be considered as semi-cryptic. Reschke & Noordel.,sp. nov.,Figs. 31 and 32 Mycobank number: MB840624.
Etymology: arcanus (Latin) = arcane; refers to the dark appearance of the basidiocarps and its isolated position in the phylogenetic tree.
Subgenus Cubospora Karstedt et al. (Fig. 43) The subgenus Cubospora was recently erected by Karstedt et al. (2019) for species with a convex to conical pileus, cuboid basidiospores, abundant clamp connections, and often abundant oily contents. They were formerly placed in subgenus Inocephalus Noordel. Most species of the subgenus Cubospora have a tropical to subtropical distribution (Horak 1976(Horak , 1977Romagnesi and Gilles 1979)  Etymology: aurantium (Latin) = gold, virescent (Latin) = becoming green; refers to colour and staining of the basidiocarps.

Discussion
The investigation of species of Entoloma from Panama revealed 17 species new to science; one species could be identified with certainty, and two further species are presented with uncertain identifications. Further specimens representing more than 30 additional species were not thoroughly studied due to limited material. Further fieldwork and investigation of specimens will increase the number of species known for Panama and will lead to many further species new to science.
Most species recorded for Panama belong to the subgenera Cyanula, Entoloma, Alboleptonia, and Nolanea and thereby show a pattern similar to Entoloma species diversity in temperate zones. Species of the section Calliderma as well as the subgenera Cubospora and Inocephalus are unknown from European temperate to boreal regions, but they are present in North America (Hesler 1967;Horak 1976;Largent 1994;Karstedt et al. 2019). This result is not surprising because most collections were made in montane locations around 2000 m asl. with temperatures similar to subtropical or temperate climate. However, such comparisons based on infrageneric groups of Entoloma are generally difficult to make at this moment, as the infrageneric classification of Entoloma is changing and the position of a large number of species needs to be reassessed. Two specimens representing two species of the subgenus Cyanula were found in lowland habitats at 40 and 120 m asl., respectively. Thereby, this subgenus is not only the most species rich but also the one with the most widely distributed species in the present study. Species of this subgenus have been reported northwards up to arctic habitats (Noordeloos 1984) and southwards to temperate regions in the southern hemisphere (Horak 2008;Noordeloos and Gates 2012).
The results of fieldwork in 2017 and 2018 show that primary forests in the mountains of Chiriquí harbour a higher diversity and abundance of species of Entoloma than disturbed lowland vegetation. This difference may be explained by different elevations or the degrees of disturbance. In Europe, the diversity and abundance of certain grassland fungi, like Entoloma spp., is used to evaluate the value of locations for nature conservation (McHugh et al. 2001;Newton et al. 2003;Griffith et al. 2013). Horak (1978) reported that in South American virgin forests, Entoloma can outnumber all other agaric genera in species richness at certain times of the year. Diversity of Entoloma spp. may be a promising marker for habitats and locations valuable for nature conservation; however, studies that would examine the possibility of a relationship between disturbance and diversity are needed.

Shape of basidiospores
Polyhedroid basidiospores are a unique and striking feature of Entoloma spp. The usefulness of their shape and base type for infrageneric classification has been disputed. Pegler and Young (1979) reported a "precise arrangement and order of development" of the facets for a given species. Kühner and Boursier (1929), however, stated that the variability of basidiospore shape rises together with the complexity of the spores. The present study confirmed the latter because a certain variability of the arrangement at the abaxial side was observed as well as there being two species, for which a general basidiospore type could not be determined. In addition to that, Co-David et al. (2009) and Baroni et al. (2011) presented basidiospores with irregular and incomplete facets. Therefore, a fixed number of facets can most likely not be determined for the basidiospores of every species. A possibility to describe such spores may be the use of ranges of numbers of facets, like x-hedroid to (x + a)-hedroid. Romagnesi (1941) as well as Pegler and Young (1979) suggested that "simple" basidiospore shapes, like prismatic and cuboid ones, are more primitive than those with many facets. This is contradicted by the fact that molecular phylogenies consistently demonstrate that species with prismatic and cuboid basidiospores are located in several clades, but not in the basal one (Co-David et al. 2009;Karstedt et al. 2019). Considering the relatively large fraction of species with irregular and bumpy basidiospores in the basal clade (Co-David et al. 2009;Baroni et al. 2011), it is more likely that the basidiospores of the ancestral species of Entoloma had a rather rhodocyboid morphology, as proposed by Mazzer (1976). Cuboid and prismatic basidiospores are most likely derived from "complex" basidiospores through reduction of the number of facets. Entoloma conferendum is nested between species with heterodiametrical hepta-to hendecahedroid basidiospores in subg. Nolanea. Thus, its prismatic basidiospores are most likely derived from these basidiospores, similar to the frequently reduced basidiospores in E. transitionisporum. Regarding the small difference in ITS sequences between E. paraconferendum and E. conferendum, such reduction-driven evolution can obviously happen relatively rapidly. The cuboid basidiospore with dièdre basal is shared by all members of the subgenera Cubospora and Cuboeccilia (Karstedt et al. 2019). It remains unknown if this shape evolved more than once. Both subgenera, Cyanula and Nolanea, share species with hendecahedroid basidiospores with dièdre basal. Whether this spore type has evolved once or several times is unclear. Diverse basidiospore shapes in species of the subgenus Alboleptonia show that the shape of basidiospores in Entoloma spp. does not reliably reflect systematic relationships. The basidiospore type should thus only carefully be used for infrageneric classification.
In both the subgenera, Entoloma and Nolanea, basidiospores with different base types occur. This is in accordance with the observations of Pegler and Young (1979). Thus, the base type cannot be used as a major character to define subgenera in Entoloma. However, it is possible that this character is consistent in some subgenera or may be useful in subclades at lower rank. More species have to be analysed to assess the phylogenetic value of this character.