First contribution to the genus Gymnopilus (Agaricales, Strophariaceae) in Paraguay

Abstract Gymnopilus is characterized by its ferruginous-yellow basidiomata and lamellae, ferruginous spore print, ellipsoidal basidiospores with warty and rough ornamentation, and lacking a germinative pore. Here, novel data on the Gymnopilus species of Paraguay is presented, macro and microscopic morphological characteristics, distribution, and ecology are described, and a taxonomic discussion is provided. Gymnopilus imperialis is recorded in the Alto Paraná Department, G. lepidotus in the Central Department, G. luteofolius in the Cordillera Department, G. peliolepis in the Paraguarí Department, and G. purpureosquamulosus in the Central Department and Boquerón, all as new records for Paraguay. Photographs of the fresh basidiomata and some microscopic structures such as basidia and basidiospores are attached.


Introduction
The genus Gymnopilus P. Karst. includes more than 200 saprobic species, mainly lignicolous, which is why they represent an important component of the wood mycobiota around the world (Guzmán-Dávalos 2003;Holec 2005;Kirk et al. 2008). Based on the pigments it contains and the non-mycorrhizal habit, Kühner (1984) classified Gymnopilus together with Galerina Earle in the Strophariaceae family. Later, Singer (1986) placed it within Cortinariaceae due to the ornamentation and lack of germinal pore of the basidiospores. Currently, according to the results of phylogenetic analysis based on DNA sequences, Gymnopilus is not related to any of the two families mentioned above, since it forms an independent clade called 2 de 13 Rodriguésia 72: e00752019. 2021 "Gymnopilae" by Matheny et al. (2006) and is currently included in the family Hymenogastraceae (Kirk et al. 2008).
Gymnopilus is characterized by its yellow, ferruginous or purple basidiomata, yellow to ferruginous lamellae, central to eccentric stipe, with a partial cortinoid or fibrillose veil, generally fugacious, or as a membranous ring, bitter to farinaceous flesh, ferruginous spore print, basidiospores ellipsoidal with warty to rugose ornamentation, without germinative pore and in most species with dextrinoid walls, presence of subcapitulated cheilocystidia to capitated and hyphae with clamp connections (Horak 1989;Guzmán-Dávalos 2003;Holec 2005). An important ecological character is the lignicolous habit, being able to grow in woods in different degrees of decomposition (Guzmán-Dávalos 2003;Holec 2005) and apparently without having a preference for the substrate (Singer 1951). However, it has been observed that some tropical species are associated with Angiosperms and others with palms, while in temperate zones they are associated with conifers.

Materials and Methods
Samples were collected in the Departments Alto Paraná, Boquerón, Central, Cordillera and Paraguarí-Paraguay, and analyzed in the Análisis de Recursos Vegetales Laboratory, Mycology lab, Facultad de Ciencias Exactas y Naturales (FACEN), of the Universidad Nacional de Asunción, Paraguay. The macroscopic descriptions were based on fresh material, according to the guidelines proposed by Wright & Albertó (2002) and Lodge et al. (2004). The microscopic characteristics were described from the material mounted in 5% KOH and observed in an optical microscope; in addition, 1% floxin, Congo ammoniac red, Melzer reagent and cresyl blue were used, the last one for the metachromatic reaction. The following notations were followed when performing the measurements of the basidiospores: Q = ratio between the length and width of the spores, indicated as a range of variation; n = number of measured spores and N = number of basidiomata (Niveiro et al. 2012). The specimens studied were deposited in the herbarium of the Facutad de Ciencias Exactas y Naturales of the Universidad Nacional de Asunción, Paraguay (FACEN).

Results and Discussion
Five species of Gymnopilus were recorded: G. imperialis in the Alto Paraná Department, G. lepidotus in the Central Department, G. luteofolius in the Cordillera Department, G. peliopelis in the Paraguarí Department and G. purpureosquamulosus in the Central and Boquerón Departments, all as new records for Paraguay.
It was found in grassland near plantations of Pinus elliotti, during autumn.  The type specimen is from Brazil, also recorded from Argentina, Costa Rica and Jamaica (Spegazzini 1889;Hesler 1969;Guzmán-Dávalos et al. 2003). In Brazil it has been cited for the states of São Paulo and Paraná (Spegazzini 1889;Pegler 1997;Capelari et al. 2015;BFG 2018). This species is grouped within the spectabilisimperialis clade as the basal species, which also includes G. junonius (Fr.) P.D. Orton, G. pampeanus (Speg.) Singer and G. spectabilis (Fr.) Singer, this clade includes robust basidiomata, with thick membranous ring and fibrillose to slightly squamulosus, dark ferruginous colour pileus . The species of this clade are very similar to each other, and in many cases have been considered as synonyms or varieties of the same taxon .
Gymnopilus pampeanus is a related species, and is differentiated from G. imperialis by its cylindrical stipe and adnexed to sinuate-adnate lamellae in the first one, besides G. pampeanus appears to be closely associated with Eucalyptus forests (Pegler 1983(Pegler , 1997. Another closely related species is G. rugulosus R.Valenz, Guzmán & J. Castillo. This species is known for the tropical and subtropical regions of the northern hemisphere, shares characters such as robust basidiomata with tuberculated basidiospores; however, it differs from G. imperialis by the presence of lageniform, ventricular, non-capitated or subcapitulated pleurocystidia of 21-38 × 5-10 μm (Guzmán-Dávalos & Ovrebo 2001;Cardona et al. 2005).
Gymnopilus imperialis was originally described for the south of Brazil (Spegazzini, 1889), and considering the main morphological characters like robust basidiomata, pileus greater than 10 cm in diameter, claviform stipe widened towards the base, membranous ring, basidiospores subglobose to ellipsoidal, tuberculated, and absence of pleurocystidia to be consistent with those observed in the Paraguayan specimen, the material analyzed from Paraguay is identified as G. imperialis. Hesler, Mycologia Memoirs 3: 40 (1969).

Gymnopilus lepidotus
Fig. 2 Basidiomata cespitose. Pileus 3,5-10 cm diam., hemispheric to convex, margin entire when young, plane-concave with revolute margin at maturity; wet to dry surface, ferruginous chestnut with vinous tones, becoming clear towards the margin, covered with chestnut scales, erect, abundant in the center, scattered towards the margin. Lamellae subdecurrent, crowded, up to 0.4 cm wide, reddish brown in fresh, ferruginous in dry, margin entire, concolorous, lamelullae of two lengths. Context thin, up to 0.1 cm thick, ferruginous yellow in dry samples, darkens to reddish brown with contact in fresh samples. Smell and taste not tested. Stipe 2-7 cm × 0,4-1 cm, central, cylindrical, widens slightly towards the base, up to 1 cm in diameter, hollow, beige to light brown, fibrillose, striated, reddish to the touch. Partial veil not observed. Basal mycelium whitish to cream, abundant. A reddish pigment dissolves from the pileus when mounted in KOH. Spore print yellow ferruginous to orange ferruginous.
It was found on decomposing trunk, in urban area.
The type is from Florida, United States America (Hesler 1969;Guzmán-Dávalos 2003). In addition it was recorded from Argentina for Misiones Province (Wright & Wright 2005;Lechner et al. 2006;Wright et al. 2008;Grassi et al. 2016 Gymnopilus lepidotus is characterized by the brownish-colored pileus with small erect or adherent scales, pleurocystidia and basidioles with granular, golden content, usually very abundant, cheilocystidia and pleurocystidia not capitated to subcapitated and radial pileus trama (Guzmán-Dávalos 1996, 2003. The size of the pileus is a character that has been discussed: Hesler (1969) and Wright et al. (2008) described the pileus as small, 4-8 mm in diam.; later Guzmán-Dávalos (1996, 2003, after reviewing the samples from Florida, stated that the measurements obtained by Hesler (1969) maybe mistaken because, even when dry, the samples reviewed by Hesler presented a bigger pileus, from 6-18(-26) mm in diam. In addition, some samples from Mexico reached up to 70 mm in diam. As for the pleurocystidia, Hesler (1969) mentioned that they are hyaline, rare and inconspicuous; abundant clavate basidioles with brown content. Guzmán-Dávalos (1996) interprets these basidioles as pleurocystidia of the pseudocystidia type, which coincides with the samples described by Wright et al. (2008), who mentioned subpyriform, clavate to subutriform pleurocystidia, 20-22 × 5-6 μm, with ferruginous chestnut content.

Gymnopilus luteofolius
It was found in rotting trunk, in urban area. It's distributed in Argentina and some states of the United States of America (Singer 1953b;Hesler 1969). This species is characterized by a dark reddish to reddish brown colour on the surface of the pileus when young, turning pinkish red or yellowish red to yellowish; red to vinous context that shifts to yellowish, and the clavate to ventricose caulocystidia disposed in clusters, in addition the species has fusoid to ventricose pleurocystidia and ventricose cheilocystidia with capitate to non-capitate apex (Hesler 1969). The studied samples do not present dextrinoid basidiospores, which differs from the observations of Hesler (1969). Singer (1969) noted that the dextrinoid reaction could be lost in herbarium samples and it is very difficult to observe. Because the fresh material was not studied for the Paraguayan material, it is inconclusive whether or not the basidiospores are dextrinoid.
It was found on decaying trunk of the palm native to south America, Acrocomia aculeata, during summer.
An important character to consider of G. aculeatus (Bres. & Roum.) Singer is the habit. Gymnopilus aculeatus has been found on dead trunks of palms and orchids (Singer 1951). The same is described for G. palmicola, which is always related to palms. On the other hand, G. purpureosquamulosus grows both on decaying wood (Høiland 1998;Guzmán-Dávalos et al. 2008) and on living palms (Guzmán-Dávalos et al. 2008), and G. peliolepis is restricted to decaying wood (Singer 1951). However, more studies are necessary to determine the substrate specificity of these species. Høil., Mycotaxon 69: 82 (1998).

Gymnopilus purpureosquamulosus
Fig. 5 Basidiomata cespitose. Pileus of 2-9 cm diam., hemispherical, convex to plano-convex at maturity, margin entire to crenate when young, smooth to wavy; wet to dry surface, bright yellow when young, ferruginous chestnut when mature, covered with erect, triangular, permanent scales, evenly distributed or grouped in the center and diminishing towards the margins, in dry samples the margin scales become appressed, reddish, ferruginous chestnut to violet when fresh, purple when dry. Lamellae close, ventricose, up to 0.5 cm wide, sinuated to shortly decurrent, concolour to the surface of the pileus in fresh, orange ferruginous when dry, margin entire, wavy, concolor, lamelullae of three lengths. Context yellowish up to 0.4 cm thick. Smell inappreciable, taste not bitter. Stipe 2-7 × 0.4-1 cm, central, cylindrical, widens slightly towards the base, hollow, flexuous, fibrillose, whitish to yellowish when fresh, stains to reddish brown to the touch. Partial veil leaves fibrillose remnants of ferruginous tonalities in the upper part of the stipe, fleeting. Basal mycelium whitish, abundant. Spore print ferruginous.
It was found in decaying wood or trunks, urban area, found during spring and summer.
The species have pantropical distribution. Zimbabwe (Høiland 1998), Nigeria, Italy, Switerland, Panama (Guzmán-Dávalos et al. 2003, India (Acharya et al. 2017) and Brazil ). Gymnopilus purpureosquamulosus is characterized by having the pileus covered with pointed and erect scales in the center, adpressed towards the margin, reddish, lilac, reddish brown when fresh, and purple when dry, ellipsoidal to oblong basidiospores, with subacute apex, the cheilocystidia of varied forms, lageniform, utriform, cylindrical to fusiform, with claviform, rounded to globose apex. (Høiland 1998;Guzmán-Dávalos et al. 2008;Acharya et al. 2017). Høiland (1998) indicates that G. peliolepis (Speg.) is similar to G. purpureosquamulosus; however, it differs in that the former has smaller basidiospores and less capitated cheilocystidia. Another morphologically similar species is G. dilepis, although it has smaller basidiospores than those found in G. purpureosquamulosus (Høiland 1998). In the description of the type specimen, the scales are adpressed in the center, while Guzmán-Dávalos et al. (2008) mention that the scales are erect in the center and appressed towards the margin.
There is some confusion between G. purpureosquamulosus and G. chrysopellus Singer & Digilio (1951). Gymnopilus chrysopellus was described by Berkeley & Curtis (1869) as Agaricus (Flammula) chrysopellus from Cuba. These authors describe "Pileo umbilicato adpresse tomentoso, quandoque depresso margine subsulcato…". Subsequently, Murrill (1913) proposes the combination of this species to Gymnopilus, adding the characters of the spores. However, neither of the two mentioned authors described the coloration of the pileus and the scales. Singer & Digilio (1951) describe in detail G. chrysopellus based on material from Argentina, Brazil, Peru, Martinique, Guyana, Cuba and USA, noting that it is a common species throughout tropical and subtropical America. These authors provide a description with characters similar to those described by Høiland (1998) for G. purpureosquamulosus. Singer & Digilio (1951) describe the pileus surface as having a "yellow background, with erect scales, concolorous with the surface, gradually becoming deep orange to ferrugineus." Moreover, Hesler (1969) described G. chrysopellus with a golden yellow surface, tomentose, sometime with appressed fibrils. Høiland (1998) when describing a G. purpureosquamulosus does not compare it with the Singer & Digilio descriptions of G. chrysopellus. Considering this, we could currently restrict G. chrysopellus to the specimens with the pileus surface fibrillose, golden yellow, without orange ferruginous scales, leaving the latter as a distinctive character for G. purpureosquamulosus. To know the identity of the specimens studied by Singer & Digilio (1951), is necessary to re-study them, in order to know with certainty the distribution of these two conflictive species. The morphological differences between the material studied in Paraguay and that recorded in the bibliography of the type specimen and additional materials of G. purpureosquamulosus are: (a) erect scales in the center agreeing with the description of Guzmán et al. (2008), but differing from the adpressed scales described by Høiland (1998) for the type specimen, and (b) hyphae of the pileipellis with pigment embedded in bands cited by Høiland (1998) and Guzmán et al. (2008); however, Acharya et al. (2017) did not describe this character for Indian materials. Based on the characters presented by the cited authors, we conclude that the material studied corresponds to G. purpureosquamulosus, cited for the first time in Paraguay. Spegazzini (1919) recorded in Paraguay two species as Flammula picrea (Pers.) P. Kumm. and Flammula sapinea (Fr.) P. Kumm. Later, Flecha & Niveiro (2019) suggested that this two records were synonyms of Gymnopilus based on the data available in Mycobank and Index Fungorum Databases; however, they did not analysed the samples from Spegazzini to confirm that they belong to the genus, hence we can not confirm the presence of Gymnopilus in the previous records made by Spegazzini in Paraguay. Flecha & Niveiro (2019) also mention the species Gymnopilus earlei Murrill recorded by Gullón (2011), which represents a photographic record since there is not a physical record of the sample, therefor this is also an unconfirmed record. After a long hiatus in the study of Funga in general, and of Gymnopilus in particular for Paraguay, this study represent the first contribution towards the description of Gymnopilus in the country, nevertheless a broader sampling and the inclusion of molecular data are necessary to complement the knowledge of Gymnopilus in Paraguay and in the region.