Hygrophorus citrinofuscus: epitypification of a rare waxcap species from Central Europe and its transfer to the genus Neohygrocybe

Abstract

Hygrophorus citrinofuscus is a striking and rarely reported grassland species originally described by J. Favre from the Swiss Alps. In absence of sequence data for the type specimen, a recent collection from Austria, which is well documented based on morphology and sequence data, is designated as the epitype of H. citrinofuscus to stabilise the species concept. Further collections from Austria and the Czech Republic are given. Morphologically similar species are discussed. Hygrophorus citrinofuscus is morphologically and phylogenetically well delimited and macroscopically characterised by yellow-brown pileus colours, a fibrillose pileus surface, pale lamellae and a yellow stipe, making it readily recognisable in the field. Morphology of the lamellar trama as well as phylogenetic analyses of a combined matrix of ITS, LSU, SSU and RPB2 sequence data clearly place this species in the genus Neohygrocybe, and a transfer is proposed.

Citation: Friebes G, Fuljer F, Boertmann D, Voglmayr H, Kautmanová I (2025). Hygrophorus citrinofuscus: epitypification of a rare waxcap species from Central Europe and its transfer to the genus Neohygrocybe. Fungal Systematics and Evolution 15: 121–132. doi: 10.3114/fuse.2025.15.05

INTRODUCTION

The genus Neohygrocybe (Hygrophoraceae, Agaricomycetes, Basidiomycota) is characterised by basidiomata with dull colours and often rimose, non-glutinous pilei, unchanging or reddening flesh and a subregular to regular lamellar trama comprised of relatively short elements (< 400 µm in length) (Herink 1959, Lodge et al. 2013). European Neohygrocybe species are indicators for waxcap grasslands with high conservational value (Rotheroe 1999, McHugh et al. 2001, Adamčík & Kautmanová 2005, Fuljer et al. 2022b). However, in most areas outside of Europe, e.g. North America, Africa and Australasia, waxcaps of the genera Cuphophyllus, Gliophorus, Hygrocybe s. str., Neohygrocybe and Porpolomopsis typically occur in forested areas (Hesler & Smith 1963, Griffith et al. 2004, Boertmann 2010, Griffith et al. 2013).

Broader concepts of the genus Hygrocybe (e.g. Candusso 1997, Boertmann 2010) used to include Neohygrocybe. However, Lodge et al. (2013) demonstrated that N. ovina, N. subovina and N. ingrata form a distinct phylogenetic clade which is to be regarded as its own genus, and they also tentatively included N. nitrata. Four Neohygrocybe species have been added to the genus since the study by Lodge et al. (2013), i.e. N. fumosa from tropical rainforests in Brazil (Cardoso et al. 2023), N. griseonigra from subtropical China (Wang et al. 2018), N. ovinoides from a tropical rainforest in Puerto Rico (Cantrell & Lodge 2004, Cardoso et al. 2023) and N. pseudoingrata from unimproved grasslands in Slovakia and the Czech Republic (Fuljer et al. 2022a).

In the years 2021 and 2022 a waxcap species with striking yellow-brown colours was collected in Austria and the Czech Republic. Initial research revealed similarities with H. citrinofuscus, described by Favre (1960) from the Swiss Alps and later combined in the genus Hygrocybe by Bon (1976). A morphological examination of the holotype of H. citrinofuscus confirmed these similarities. At the same time, the morphology of the lamellar trama strongly suggested a placement of H. citrinofuscus in Neohygrocybe, which was confirmed by phylogenetic analyses of a multigene matrix including sequences obtained from recent collections from Austria and the Czech Republic. Since PCR from a DNA extract of the type was not successful, an Austrian collection is designated as the epitype of H. citrinofuscus.

MATERIAL AND METHODS

Collections and morphological analyses

The collections were studied both in fresh and dried conditions. Macro-morphological features were determined based on fresh material. Then, whole basidiomata were dried in a dehydrator for further micro-morphological examination. Colours were determined using the Pantone colour chart (Pantone Color Finder 2024). Micro-morphological features were studied from dried basidiomata in KOH (3 %) with an ammonial Congo Red solution. The epitype was studied using the Olympus cellSens software with an Olympus B51 microscope and an Olympus DP28 camera. The Czech collection was studied using the AmScope imaging software with a Kapa Mic D117 light microscope and a ToupCam EP camera. In total, 192 basidiospores and 72 basidia were measured and examined: 72 basidiospores and 30 basidia from the epitype (GJO 137205), 50 basidiospores and 22 basidia from the holotype (G-K 13946) and 70 basidiospores and 20 basidia from collection HR B018636. The spore quotient (Q) refers to the length/width ratio of all measured spores. Qav refers to the average value of all obtained Q values. The epitype collection is deposited in the herbarium of the Universalmuseum Joanneum (GJO) under the accession number GJO 137205. The holotype was loaned from the Conservatoire et Jardin botaniques de la Ville de Genève (G), where it is deposited under the accession number G-K 13946. Fungarium acronyms follow Thiers (2024).

DNA extraction, amplification, sequencing

Total genomic DNA of the epitype collection was extracted from dried tissue using the Dneasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, although with prolonged incubation time of up to 3 h after addition of the RNA-lytic enzyme. Amplification reactions were performed using a C1000 Touch™ Thermal Cycler. The target region of the internal transcribed spacer regions of ribosomal DNA (ITS) was amplified using primers ITS5 and ITS4 (White et al. 1990). The large subunit of ribosomal DNA (LSU) was amplified using primers LR0R (5’-ACCCGCTGAACTTAAGC-3’) and LR5 (5’-TCCTGAGGGAAACTTCG-3’; Vilgalys & Hester 1990). The PCR amplifications were conducted in 25 μL total volume using a GoTaq Flexi PCR kit (Promega), the reaction mixture containing 20–25 ng total DNA template, 1 μL of both primers (10 μM), 5 μL of buffer (5×), 2.5 μL of dNTP (2 mM), 2 μL of MgCl₂ (25 mM), 0.2 μL GoTaq Flexi polymerase and the final volume was added with ultrapure water. The PCR reaction for ITS and LSU regions was set up as follows: 3 min initial denaturation at 95 °C, 32 cycles (95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min + increasing time 2 s per cycle), 10 min final elongation at 72 °C. The PCR products were checked on 2 % agarose gel. PCR products were purified using a Thermosensitive Alkaline Phosphatase (FastAP) and Exonuclease 1 (Exo 1) (Thermo Fisher Scientific Inc., USA) according to manufacturer’s instructions. The PCR products were sequenced in a commercial laboratory (Eurofins Genomics GmbH, Cologne, Germany).

Genomic DNA was extracted from dried basidiomata of collection HR B018636 using the CTAB method (Doyle & Doyle 1987). Sequence data were generated for the ITS region using primers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990). The PCR was performed with EliZyme FAST Taq MIX Red (Elisabeth Pharmacon), following a standard protocol with 37 cycles and annealing temperature of 54 °C. The PCR products were purified by precipitation with polyethylene glycol (10 % PEG 6000 and 1.25 M NaCl in the precipitation mixture) and sequenced in both directions using the Sanger method (Macrogen Europe, The Netherlands).

Phylogenetic analysis

The newly generated sequences were aligned to a representative multilocus matrix (ITS, LSU, SSU, RPB2) of Hygrophoraceae subfam. Hygrocyboideae, selecting three species of Chromosera as the outgroup according to Lodge et al. (2013). Sequences were primarily selected from Lodge et al. (2013) and complemented with additional GenBank sequences. The GenBank accession numbers of sequences used in the phylogenetic analyses are given in Table 1.

Table 1.

Details of collections and sequences analysed in this study, including taxon name, country of origin, collection identifiers, references and GenBank accession numbers.

Taxon	Origin	Collection	GenBank accession number				References
			ITS	LSU	SSU	RPB2
Chromosera citrinopallida	Greenland	Boertmann 06/2	KF291072	KF291073	KF291074	—	Lodge et al. (2013)
Chromosera cyanophylla	USA (Wyoming)	PBM-1577	DQ486688	—	DQ435813	KF381509	Matheny et al. (2006), Lodge et al. (2013)
Chromosera lilacina	Greenland1	T. Borgen-86.294	KF291054	—	—	—	Lodge et al. (2013)
Gliophorus graminicolor	Australia (Tasmania)	TJB-10048	KF381520	KF381545	KF381531	KF407936	Lodge et al. (2013)
Gliophorus psittacinus	Denmark	Boertmann 02/10	KF291075	KF291076	KF291077	KF291078	Lodge et al. (2013)
Humidicutis auratocephala	USA (Massachusetts)	JCS071105E	—	DQ457672	DQ440646	DQ472720	Matheny et al. (2006)
Humidicutis dictiocephala	Ecuador	QCAM6000	KY689661	KY780120	—	—	Crous et al. (2017)
Humidicutis marginata var. marginata	USA (Tennessee)	TFB12230	KF291144	—	—	—	Lodge et al. (2013)
Humidicutis marginata var. olivacea	USA (Tennessee)	AK-108	—	KF291145	—	—	Lodge et al. (2013)
Humidicutis pura	USA (North Carolina)	PBM4191a	MT237512	MT228851	—	—	Matheny et al., unpublished
Hygrocybe andersonii	USA (Florida)	DMFL05-6	KF291170	KF291171	—	—	Lodge et al. (2013)
Hygrocybe caespitosa	USA (West Virginia)	DMWV-03-737	KF291104	KF291105	—	KF291107	Lodge et al. (2013)
Hygrocybe cantharellus	USA (New Hampshire)	PBM-2554	—	DQ457675	DQ444857	—	Matheny et al. (2006)
Hygrocybe chlorophana	Denmark	Boertmann 2002/9	EU435148	EU435148	KF381536	KF381513	Lodge & Ovrebo (2008), Lodge et al. (2013)
Hygrocybe coccinea	Denmark	Boertmann 02/8	EU435146	EU435146	KF291113	KF291114	Lodge & Ovrebo (2008), Lodge et al. (2013)
Hygrocybe constrictospora	Denmark	Boertmann 07/38	KF291115	KF291116	—	—	Lodge et al. (2013)
Hygrocybe glutinipes var. rubra	USA (North Carolina)	DJL05NC9	EU435149	EU435149	KF291125	—	Lodge & Ovrebo (2008), Lodge et al. (2013)
Hygrocybe helobia	Russia	Kovalenko 99-9-1	KF291182	KF291183	KF291184	—	Lodge et al. (2013)
Hygrocybe lepida	Denmark	Boertmann 02/2	KF306333	KF306334	—	—	Lodge et al. (2013)
Hygrocybe melleofusca	Puerto Rico	DJL-PR-EV-8/24/2005	KF291154	KF291155	KF291156	—	Lodge et al. (2013)
Hygrocybe miniata	Russia	AK-110	KF291179	KF291180	KF291181	—	Lodge et al. (2013)
Hygrocybe miniata f. longipes	USA (Massachusetts)	PBM-2749	DQ490630	DQ457677	DQ444859	DQ472724	Matheny et al. (2006)
Hygrocybe nigrescens var. brevispora	Puerto Rico	SAC-0005	KF291130	KF291131	KF291132	—	Lodge et al. (2013)
Hygrocybe noninquinans	Puerto Rico	DJL-PR-1	KF291127	KF291129	KF291126	KF291128	Lodge et al. (2013)
Hygrocybe occidentalis var. scarletina	Puerto Rico	Cancerel PR 02	EU435151	EU435151	KF291187	—	Lodge & Ovrebo (2008), Lodge et al. (2013)
Hygrocybe punicea	Scotland	DJL-SCOT-B2	KF291133	KF291134	KF291135	—	Lodge et al. (2013)
Hygrocybe purpureofolia	USA (North Carolina)	DJL04NC1	KF291192	KF291193	KF291191	—	Lodge et al. (2013)
Hygrocybe reidii	Wales	DJL-ENG-15	KF291158	KF291159	KF291160	—	Lodge et al. (2013)
Hygrocybe rosea	Puerto Rico	DJL-PR-4	KF291197	KF291198	—	—	Lodge et al. (2013)
Neohygrocybe citrinofusca	Austria	GJO 137205	PQ283526	PQ282241	—	—	Present study
	Czech Republic	HR B018636	PQ283525	—	—	—	Present study
Neohygrocybe fumosa	Brazil (Mato Grosso)	JS600 FLOR67460	ON616413	—	—	—	Cardoso et al. (2023)
Neohygrocybe griseonigra	China (Guangdong)	GDGM 44492	MG779451	MG786565	—	—	Wang et al. (2018)
Neohygrocybe ingrata	USA (North Carolina)	DJL05TN62	KF381525	KF381558	KF381539	KF381516	Lodge et al. (2013)
	Wales	Griffith	KF291225	KF291226	—	—	Lodge et al. (2013)
	USA (New York)	TJB-9606	—	KF291223	—	KF291224	Lodge et al. (2013)
	Russia	AK-8	—	KF291227	—	—	Lodge et al. (2013)
Neohygrocybe ovina	Wales	Griffith	KF291233	KF291234	KF291235	KF291236	Lodge et al. (2013)
	Wales	GEDC0877	KF291228	KF291229	—	—	Lodge et al. (2013)
Neohygrocybe pseudoingrata	Slovakia	SNMH344	MZ479356	MZ479363	—	—	Fuljer et al. (2022)
	Slovakia	SNMH345	MZ479355	—	—	—	Fuljer et al. (2022)
Neohygrocybe subovina	USA (North Carolina)	DJL05TN61	KF291136	KF291137	—	—	Lodge et al. (2013)
	USA (Tennessee)	DJL04TN16	KF291140	KF291141	—	—	Lodge et al. (2013)
	USA (West Virginia)	WRWV04-752	—	KF291142	KF291139	KF291138	Lodge et al. (2013)
Porpolomopsis calyptriformis	England	E Brown-3	KF291242	KF291243	KF291244	KF291245	Lodge et al. (2013)
Porpolomopsis lewellinae	Thailand (Chiang Mai)	TJB-10034	KF291238	KF291239	KF291240	KF291241	Lodge et al. (2013)

¹ According to the entry in GenBank the country of origin is the United Kingdom.

Sequence alignments were produced with the server version of MAFFT v. 7.490 (http://mafft.cbrc.jp/alignment/server/; Katoh et al. 2019) using the default settings for SSU and RPB2, while the Q-INS-i iterative refinement method was implemented for the ITS and LSU. The resulting ITS, LSU, SSU and RPB2 alignments were checked and manually refined using BioEdit v. 7.2.6 (Hall 1999) and combined for subsequent phylogenetic analyses. After exclusion of long gaps, the final combined data matrix contained 5 247 characters (1 143 nucleotides of ITS, 1 499 nucleotides of LSU, 1 784 nucleotides of SSU and 821 nucleotides of RPB2).

Maximum likelihood (ML) analyses were performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI v. 2.0 (Edler et al. 2021), using the ML + rapid bootstrap setting and the GTRGAMMA + I substitution model with 1 000 bootstrap replicates. The matrix was partitioned for the different gene regions, with the ITS, LSU and SSU regions treated as a single partition following Aime et al. (2006). For evaluation and discussion of bootstrap support, values below 70 % were considered low, between 70 and 90 % medium/moderate, above 90 % high and 100 % maximum.

RESULTS

Phylogeny

The combined multilocus matrix used for phylogenetic analyses comprised 5 247 characters, of which 1 332 were parsimony informative (542 from ITS, 297 from LSU, 140 from SSU and 353 from RPB2). The best ML tree (-lnL = 34490.191492) obtained by RAxML is shown in Fig. 1. Overall topologies were similar to the multilocus tree of Lodge et al. (2013). The genera Chromosera, Hygrocybe and Porpolomopsis received maximum bootstrap support, Neohygrocybe high (98 %), and Gliophorus and Humidicutis medium (77 and 76 %, respectively) support. Within Neohygrocybe, all species with more than a single accession received maximum support. While N. griseonigra, N. ovina and H. subovina formed a highly supported (99 %) clade, with a highly supported (97 %) sister group relationship between N. ovina and N. subovina, backbone support of the remaining branches within the genus was insignificant to moderate. Neohygrocybe citrinofusca was placed in a moderately (66 %) supported clade together with N. fumosa, N. ingrata, N. nitrata and N. pseudoingrata, and sister group relationship of N. citrinofusca to N. fumosa received medium (81 %) support.

Fig. 1.

Phylogram of the ML tree (-lnL = 34490.191492) revealed by RAxML from an analysis of the combined ITS-LSU-SSU-RPB2 matrix of selected Hygrophoraceae subfam. Hygrocyboideae, to show the phylogenetic position of Neohygrocybe citrinofusca. Collection identifiers are given following the taxon names. ML bootstrap support above 50 % is given above or below the branches.

Taxonomy

Neohygrocybe citrinofusca (J. Favre) Friebes, Boertm., Fuljer, Kautmanová & Voglmayr, comb. nov. MycoBank MB 855630. Figs 2–5.

Fig. 2.

Basidiomata of Neohygrocybe citrinofusca from various collections. A. GJO 137205, epitype (photo M. & G. Friebes). B. GJO 137208 (photo H. Pidlich-Aigner). C. GJO 137209 (photo H. Prelicz). D–G. HR B018636 (D, F. P. Nouzovský. E. J. Herčík. G. H. Horová).

Fig. 5.

Neohygrocybe citrinofusca (HR B018636). A. Basidiospores. B. Inflated, sterile elements in the hymenium, stained with ammonial Congo Red solution. Scale bars = 10 µm.

Basionym: Hygrophorus citrinofuscus J. Favre, Ergebn. wiss. Unters. schweiz. NatnParks 6: 586. 1960.

Synonym: Hygrocybe citrinofusca (J. Favre) Bon, Doc. Mycol. 7 (no. 25): 5. 1976.

Typus: Austria, Styria, Leoben, St. Michael in Obersteiermark, Brunn, “Hartner Leitn”, 47.3450556N, 14.9894444E, alt. 672 m, cow-grazed grassland, 11 Sep. 2022, G. Friebes (epitype GJO 137205, designated here, MycoBank MBT 10022028), ITS GenBank PQ283526, LSU GenBank PQ282241. Switzerland, Grisons, Vulpera, above the Hotel Waldhaus Vulpera, alt. 1 350 m, in a clearing in grass, 9 Sep. 1957, J. Favre (holotype G-K 13946 in G).

Habitat and distribution: Known from Austria, the Czech Republic and Switzerland. It grows in nutrient-poor, semi-natural grassland at an alt. of 291 m to 1 350 m from July to September.

Description of GJO 137205 (epitype): Pileus 5–28 mm diam, at first hemispherical, later irregularly convex to applanate, occasionally soon with a compressed to umbilicate centre, rarely with a broad umbo, margin often irregular, wavy, crenate; surface smooth to very finely scaly, shiny, radially fibrillose and occasionally splitting radially, dry, context very thin; margin usually not striate to inconspicuously striate for 1/3 of the cap diam; margin Oil Yellow to Super Lemon (Pantone 15-0743 to Pantone 14-0754), centre Bright Gold to Demitasse (Pantone 16-0947 to Pantone 19-0712). Stipe 15–40 × 2–4 mm, cylindrical, terete to somewhat compressed or furrowed, rarely straight, often slightly bent in the middle or the lower third; surface smooth, some parts faintly white pruinose, often slightly wavy, dry, with white basal mycelium, Oil Yellow to Spruce Yellow (Pantone 15-0743 to Pantone 17-1040). Lamellae adnexed to adnexed with a decurrent tooth, with an entire edge, distant, 18–26 complete with 1–3 tiers of lamellulae, pale, Whitecap Gray to Rich Gold (Pantone 12-0304 to Pantone 16-0836). Context with the same colour as the pileus or the stipe, although a bit paler; without a specific smell or taste. Basidia 4-spored (rarely 2- or 1-spored), narrowly clavate to clavate, awl-shaped, with clamps, (33.9–)35.1–47.8(–50.9) × (8.4–)8.6–12.6(–13.1) µm, av. = 42.0 × 10.7 µm (n = 30); occasionally sterile, inflated elements (basidia?) up to 16 µm wide are observable. Basidiospores smooth, ellipsoid to oblong, thin-walled, hyaline, (5.7–)7.0–12.0(–12.4) × (3.9–)4.2–7.1(–7.4) µm, av. = 9.9 × 5.7 µm, Q = (1.4–)1.5–2.0(–2.1), Qav = 1.7 (n = 72), inamyloid. Cystidia absent, yet occasionally somewhat protruding terminal elements of the lamellar trama can be found inside the hymenium. Pileipellis generally a cutis of 2.8–10.5 µm wide, straight to slightly sinuous hyphae with intracellular, brownish pigment; in slightly scaly parts of the pileus resembling a trichoderm; clamps present, although sometimes difficult to observe. Stipitipellis a cutis of parallel, hyaline to slightly yellowish, 2.3–11.5 µm wide hyphae, clamps frequent. Lamellar trama subregular to regular, comprised of mostly parallel, cylindrical cells up to 250 µm long and 3.5–33 µm wide, not tapering, very compact and complete individual hyphae difficult to discern, clamps present at least on some septa.

Additional materials examined: Austria, Styria, Feldbach, Mühldorf, military cemetery, 46.946710N, 15.896124E, alt. 291 m, nutrient-poor grassland, 2 Sep. 2010, H. Prelicz & B. Wieser (GJO 137209; as Hygrocybe spadicea); Styria, Graz (Stadt), Graz-St. Veit, Hoschweg, 47.1143333N, 15.4088889E, alt. 385 m, in a garden meadow next to Abies, 2 Aug. 2014, H. Pidlich-Aigner (GJO 137206; as Hygrocybe spadicea); ibid., in a garden meadow underneath an apple tree (Malus domestica), 27 Jul. 2016, H. Pidlich-Aigner (GJO 137207; as Hygrocybe spadicea); ibid., in a garden meadow underneath an apple tree (Malus domestica), 8 Sep. 2017, H. Pidlich-Aigner (GJO 137208; as Hygrocybe spadicea). Czech Republic, Heřmanův Městec, camping site Konopáč, 49.9370725N, 15.6461342E, alt. 333 m., mowed semi-natural lawn, 4 Sep. 2021, H. Horová (HR B018636).

DISCUSSION

The Austrian collection GJO 137205 was not initially assignable to any species of Hygrocybe s. l. described in popular monographic treatments (e.g. Candusso 1997, Boertmann 2010). It was the brief account of Hygrocybe citrinofusca in Bon (1990) which brought the first author’s attention to this species originally described by Favre (1960) from the Swiss Alps. A thorough comparison with Favre’s description revealed great similarities, and ultimately additional collections from Austria and the Czech Republic were able to be linked to this name. The holotype of Hygrophorus citrinofuscus was also examined microscopically, which confirmed the similarities (Table 2). Since PCR from a DNA extract of the holotype failed consistently, the collection GJO 137205 is designated as the epitype of Hygrophorus citrinofuscus in this study to stabilise the species concept.

Phylogenetic analyses of a combined multilocus matrix clearly place Hygrocybe citrinofusca in the genus Neohygrocybe (Fig. 1), into which it is therefore combined here. It is revealed as the closest relative of N. fumosa, although this relationship is only moderately supported, and additional sequences are necessary for a better resolution, in particular for N. fumosa for which only an ITS sequence is available.

Favre (1960) reports the lamellar trama cells of the holotype with a length of 400–500 µm, which is longer than the maximum of 250 µm observed by the authors in the other collections studied. However, the lamellar trama cells were extremely compact and often collapsed in the examined collections, thus making it challenging to obtain exact measurements. The possibility that longer cells were present as well cannot be excluded.

The basidiospore size of N. citrinofusca varies to a certain degree. Favre (1960) gives a range of 8–10 × 4.5–5.5 µm from 4-spored basidia and 10–11 × 6 µm from 2-spored basidia. Examination of the holotype revealed a size of (6.9–)7.3–9.5(–10.8) × 4.8–6.0(–6.4) µm (Table 2). The basidiospores of the Czech collection (HR B018636) measured (7.4–)7.7–11.1(–12.6) × (4.7–)4.9–6.7(–7.2) µm, and the measurements of the epitype are (5.7–)7.0–12.0(–12.4) × (3.9–)4.2–7.1(–7.4) µm. The specimen GJO 137208 has measurements of 6.1–9.2 × 5.0–6.4 µm based on spores obtained from a spore deposit by H. Pidlich-Aigner.

Table 2.

A comparison between the holotype and the epitype of N. citrinofusca. Information and measurements in square brackets were obtained by the authors during the examination of the holotype.

	Description of the holotype (based on Favre 1960)	Description of the epitype (GJO 137205)
Pileus: diameter	Up to 30 mm	5–28 mm
Pileus: shape	Irregularly umbonate, at first hemispherical, centrally compressed	At first hemispherical, occasionally soon with a compressed centre, later irregularly convex to applanate, rarely with a broad umbo, margin often irregular
Pileus: surface	Dry, matt, finely fibrillose	Dry, shiny, smooth to very finely scaly, radially fibrillose and occasionally splitting radially
Pileus: colour	Younger specimens with dark brown caps, with a citrine-brown edge, later paler and weakly citrine and brownish	Margin Oil Yellow to Super Lemon, centre Bright Gold to Demitasse
Lamellae: size	16–25, up to 7 mm wide	18–26, not measured
Lamellae: shape	With a finely wavy edge, with a decurrent tooth	With an entire edge, adnexed to adnexed with a decurrent tooth
Lamellae: colour	White	Pale, Whitecap Gray to Rich Gold
Stipe: size	Up to 36 × 5.5 mm	15–40 × 2–4 mm
Stipe: shape	Subequal or more often attenuated, solid, ultimately hollow, occasionally compressed	Cylindrical, terete to somewhat compressed or furrowed, often slightly bent in the middle or the lower third
Stipe: surface	Moist, glabrous, finely fibrillose, with fine silky sheen	Dry, glabrous, some parts faintly white pruinose
Stipe: colour	Weakly brown-citrine when young, later brownish citrine, with white base	Oil Yellow to Spruce Yellow, with white basal mycelium
Context	Pale citrine in the pileus and the top of the stipe, brownish citrine or brownish in the rest of the stipe	With the same colour as the pileus or the stipe, though a bit paler
Smell and taste of the context	Lacking	Lacking
Basidia: number of sterigmata	4 (rarely 2)	4 (rarely 2 or 1)
	[4 (rarely 2)]
Basidia: size	35–41 × 8–9 µm	(33.9–)35.1–47.8(–50.9) × (8.4–)8.6–12.6(–13.1) µm
	[(34.7–)35.2–47.4(–48.2) × 7.3–10.2(–10.9) µm]
Basidiospores: shape	Ellipsoid to cylindrical ellipsoid	Ellipsoid to oblong
	[Ellipsoid]
Basidiospores: colour/surface	Hyaline, smooth, inamyloid	Hyaline, smooth, inamyloid
	[Hyaline, smooth]
Basidiospores: size	8–10 × 4.5–5.5 µm (measured from 4-spored basidia), 10–11 × 6 µm (measured from 2-spored basidia)	(5.7–)7.0–12.0(–12.4) × (3.9–)4.2–7.1(–7.4) µm, on average 9.9 × 5.7 µm
	[(6.9–)7.3–9.5(–10.8) × 4.8–6.0(–6.4) µm, on average 8.2 × 5.4 µm]
Basidiospores: Q	—	(1.4–)1.5–2.0(–2.1)
	[(1.3–)1.34–1.76(–1.84)]
Basidiospores: Qav	—	1.7
	[1.53]
Lamellae: trama	Regular	Subregular to regular
Lamellae: shape of trama cells	Cylindrical	Cylindrical, parallel
Lamellae: size of trama cells	Up to 18 µm wide and 400–500 µm long	3.5–33 µm wide, up to 250 µm long
Cystidia	Pseudocystidia present, observable as terminal hyphae which cross the hymenium, 25 to 40 µm	True cystidia absent, yet occasionally somewhat protruding terminal elements of the lamellar trama can be found inside the hymenium
Pileipellis	Hyphae in a radial arrangement, with brownish content and colourless walls	Cutis, hyphae straight to slightly sinuous, with brownish pigment, in slightly scaly parts resembling a trichoderm
Pileipellis: cell size	2–7 µm in the thinnest cells, otherwise up to 19 µm wide	2.8–10.5 µm wide

The collection HR B018636 from the Czech Republic matches the epitype macro- and micro-morphologically. The sole difference is the shorter basidiospore length in HR B018636, i.e. on average 9.1 × 5.7 µm, Qav = 1.59, vs on average 9.9 × 5.7 µm, Qav = 1.7, in the epitype. This may be due to a higher ratio of 2- and 1-spored basidia in the epitype. The lamellar trama of HR B018636 is subregular and consists of cylindrical, vermiform cells, which are occasionally narrowed at the septa, and measure (15–)21–216(–220) × (4.1–)4.2–18.1(–25.3) µm, av. 99 × 9.9 µm.

Neohygrocybe citrinofusca is a striking species which can hardly be confused with other grassland fungi in the field. It is surprising and most likely a testament to its rarity that very few mentions of this species exist in literature. It was reported from France by Cormont & Deschamps (2005; as Hygrocybe citrinofusca), although no further information is provided by those authors. Romero de la Osa (2008) describes and illustrates Hygrocybe citrinofusca from Spain. However, the photo and description provided in that work clearly differ from N. citrinofusca based on the conical, umbonate cap and free lamellae, and instead may represent a colour variation of H. spadicea.

Indeed, H. spadicea arguably bears the greatest resemblance to N. citrinofusca among European taxa, given the similar colour schemes of their basidiomata. Boertmann (2002) suggests that N. citrinofusca may be a synonym of H. spadicea, and Bon (1990) suspects that N. citrinofusca may be a form of H. spadicea with an obtuse cap. The habitus of H. spadicea, however, is consistently different from N. citrinofusca based on the material studied. The former has a mainly conical cap and ascending lamellae, and the latter is characterised by an often applanate to umbilicate cap and adnexed to slightly decurrent lamellae. Additionally, H. spadicea has lamellar trama cells which are occasionally longer than 1 000 µm (Boertman 2010), whereas they are only up to 250 µm long in N. citrinofusca based on the authors’ own observations (and attaining 500 µm according to Favre 1960). The fact that the collections GJO 137206, 137207, 137208 and 137209 were initially identified as H. spadicea confirms these superficial similarities. Phylogenetically, both species are remote, as H. spadicea is clearly placed within Hygrocybe s. str. (Lodge et al. 2013).

Candusso (1997) argues that N. citrinofusca more resembles H. citrinovirens than H. spadicea based on the coloured illustration in Favre (1960; pl. 1, fig. 4). However, H. citrinovirens is distinguishable from N. citrinofusca by its conical cap, free to adnexed lamellae and a lack of brown colours (Boertman 2010). Phylogenetically, H. citrinovirens belongs to Hygrocybe sect. Microsporae (Lodge et al. 2013).

Fig. 4.

Neohygrocybe citrinofusca (GJO 137205, epitype). A. Basidiospores. B–I. Basidia and basidioles. J. Lamellar trama. K. Pileipellis. A–J stained with ammonial Congo Red solution. Scale bars: A–I = 10 µm; J, K = 50 µm.

Hygrocybe caespitosa, known from eastern North America, and H. macrosiparia, described from Ecuador, share dry basidiomata with similar habits and colour schemes with N. citrinofusca, although they differ morphologically in their distinctly scaly caps (Murrill 1914, Hesler & Smith 1963, Crous et al. 2017). Hygrocybe melleofusca, observed in Puerto Rico and the Lesser Antilles, is another species with yellow-brown basidiomata and broadly adnexed to decurrent lamellae. It was initially placed in Hygrocybe sect. Neohygrocybe, yet it similarly differs in that it occasionally has a squamose cap, in addition to a context staining yellow when cut (Lodge & Pegler 1990). Phylogenetic analyses place both H. caespitosa and H. melleofusca in Hygrocybe subsect. Squamulosae (Lodge et al. 2013).

Neohygrocybe lawsonensis, described from Australia, has broadly adnexed lamellae with yellowish to greenish tints (Young & Wood 1997), and is the only other Neohygrocybe species with yellow colours. However, it is different from N. citrinofusca in various aspects, e.g. the brownish cap and stipe, a red discolouration of bruised lamellae and smaller basidiospores (Young & Wood 1997).

The phylogenetically most closely related species to N. citrinofusca, i.e. N. fumosa, N. ingrata, N. nitrata and N. pseudoingrata (Fig. 1), differ in the nitrous smell and the lack of yellow colours (Boertmann 2010, Fuljer et al. 2022a, Cardoso et al. 2023).

Several more species belonging to Neohygrocybe, not all of which have yet been combined into said genus, have been described from areas outside of Europe. They all deviate from N. citrinofusca in pileus shape, colour, spore size, reactions of context, etc. (e.g. Dennis 1953, Heinemann 1966, Pegler 1983, Horak 1990, Desjardin & Hemmes 1997, Cantrell & Lodge 2004, Young 2005).

The genera Camarophyllopsis and Hodophilus also contain species which are superficially similar to N. citrinofusca, particularly C. schulzeri and the group of Ho. micaceus, although they are readily distinguishable by much smaller basidiospores, and the genus Hodophilus additionally differs in that is has a hymeniform pileipellis (Birkebak et al. 2016, Adamčík et al. 2018).

Based on the reports of N. citrinofusca confirmed by the authors, i.e. five collections from three locations in Austria and one collection from the Czech Republic, in addition to the holotype from Switzerland, this species does not seem to be restricted to any particular type of grassland, geology or elevation. The epitype was found in a pasture grazed by cows rich in grassland fungi, GJO 137206, 137207 and 137208 were collected in a garden where it has been observed for many years (H. Pidlich-Aigner, pers. comm.) and GJO 137209 as well as HR B018636 were equally collected from mowed lawns. The elevation ranges from an altitude of 291 m (GJO 137209) to 1 350 m (holotype). The site of the epitype was revisited on five occasions in 2022, 2023 and 2024; however, no further basidiomata were observed.

It is remarkable that all observations were made between the end of July and the first half of September. This indicates that N. citrinofusca is among the earliest-fruiting grassland species and is therefore likely to be overlooked by mycologists.

Fig. 3.

Details of basidiomata of Neohygrocybe citrinofusca (HR B018636; A–C, E. P. Nouzovský. D, G. H. Horová. F, H. J. Herčík). A–C. Smooth to fibrillose or minutely squamulose pileus surface. D–F. Pale, adnexed lamellae. G, H. Smooth stipe.