Neohygrocybe Pseudoingrata, a New Grassland Species from Slovakia and the Czech Republic

Abstract

Neohygrocybe pseudoingrat a, a new waxcap species known from Slovakia and the Czech Republic, is characterised by its pale greyish coloured and often robust basidiomata (or sporocarps), nitrous smell, context without colour changes, hollow, contorted and compressed stipe and smooth or slightly fibrillose pileus surface. Based on morphology and DNA analysis of ITS and LSU sequences of the collected specimens, N. pseudoingrata belongs to Neohygrocybe sect. Neohygrocybe together with N. ovina , N. nitrata and N. ingrata . Collections of N. pseudoingrata form a well-supported clade in phylogenetic trees.

Full text

Fungal Systemacs and Evoluon is licensed under a Creave Commons Aribuon-NonCommercial-NoDerivaves 4.0 Internaonal License
© 2022 Westerdijk Fungal Biodiversity Instute 11
Editor-in-Chief
Prof. dr P.W. Crous,Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.
E-mail:p.crous@westerdijkinstitute.nl
Fungal Systemacs and Evoluon
doi.org/10.3114/fuse.2022.09.02
VOLUME 9
JUNE 2022
PAGES 11–17
INTRODUCTION
The genus Hygrocybe as delimited in Boertman (2010) has
been split into a number of genera (Chromosera, Cuphophyllus,
Gliophorus, Gloioxanthomyces, Hygrocybe, Neohygrocybe and
Porpolomopsis) by Lodge et al. (2013). Neohygrocybe diers
from most Humidicus, Porpolomopsis and Gliophorus species
in lacking bright pigments. Lodge et al. (2013) described
Neohygrocybe as having swollen, and stued spes that become
hollow; pileus 2–6 cm, hemispherical, becoming umbonate,
smooth to scaly, margin becoming ssured, brick colour to
fuscous cinereous; lamellae few, sublunate, uncinate, broad,
venose, white at rst, becoming cinereous.
Members of the genus Hygrocybe s. l. (Hygrocybe,
Neohygrocybe, Gliophorus, Porpolomopsis) and Cuphophyllus
fall into disnct clades but they usually occur together and
are oen treated as one group for conservaon purposes
(e.g., Boertmann 2010). Most of these genera occur in
“unimproved”, mowed or grazed grasslands in Europe, where
they gure as good indicators of conservaon value of semi-
natural and natural grasslands (Adamčík & Kautmanová 2005,
Boertmann 2010, Fuljer et al. 2020). These grasslands are
usually characterised by very low levels of dissolved nitrate
and phosphate (Ejrnæs & Brunn 1995). Hygrocybe s. l. species,
together with a Clavariaceae, Entoloma and Geoglossaceae,
form a so called “CHEG” group, by reason of sharing ecological
similaries (Rotheroe 2001). However, waxcaps can also
produce basidiomata in habitats such as peat bogs, sand dunes
and woodlands (Cantrell & Lodge 2000, Grith et al. 2004,
Boertmann 2010) and in North America and the tropics they
are mainly found in forests (e.g. Hesler & Smith 1963, Pegler &
Fiard 1978, Læssøe & Boertmann 2008).
The ecological role of waxcaps is sll unclear, despite intensive
research in this eld. Grith et al. (2004) referred to the fact
that some of the waxcaps can occur in the grasslands together
with mosses and this connecon was also noced by Boertmann
(2010). However, their biology remains a mystery since isotopic
signatures indicate that they are neither mycorrhizal nor
saprotrophic (Seitzman et al. 2011, Halbwachs et al. 2013).
Recent studies revealed that some of the waxcaps can be
associated with plant roots and they probably have a biotrophic
lifestyle with plants (Halbwachs et al. 2013, 2018). Tello et al.
(2013) proved that at least one species, Hygrocybe virginea, is
a maternally transmied endophyc fungus associated with
Plantago lanceolata.
In this report we describe a taxon new to science found in
central European grasslands. It is also likely to have a wider
distribuon.
MATERIAL AND METHODS
Collecons and morphological analyses
Waxcaps were collected in Slovakia and the Czech Republic
during 2014–2020, from July to October, at 23 localies by F.
Fuljer, M. Zajac and M. Mička. Most of the collecons were from
the Javorníky Mts. (northwestern part of Slovakia) and the rest
were from Biele Karpaty, Jablunkovské medzihorie, Kysucká
vrchovina, Turzovská vrchovina (Slovakia) and Českotřebovská
Neohygrocybe pseudoingrata, a new grassland species from Slovakia and the Czech Republic
F. Fuljer1, M. Zajac2, D. Boertmann3, D. Szabóová4, I. Kautmanová4*
1Department of Botany, Faculty of Natural Sciences, Comenius University, Révová 39, 811 02 Braslava, Slovakia
2Administraon of Protected Landscape Area Kysuce, U Tomali č. 1511, 022 01 Čadca, Slovakia
3Department of Ecoscience, Aarhus University, Frederiksborgvej 399, DK-4000 Roskilde, Denmark
4Slovak Naonal Museum - Natural History Museum, Vajanského nábrežie 2, P.O. Box 13, 810 06 Braslava, Slovakia
*Corresponding author: ivona.kautmanova@snm.sk
Abstract: Neohygrocybe pseudoingrata, a new waxcap species known from Slovakia and the Czech Republic, is
characterised by its pale greyish coloured and oen robust basidiomata (or sporocarps), nitrous smell, context without
colour changes, hollow, contorted and compressed spe and smooth or slightly brillose pileus surface. Based on
morphology and DNA analysis of ITS and LSU sequences of the collected specimens, N. pseudoingrata belongs to
Neohygrocybe sect. Neohygrocybe together with N. ovina, N. nitrata and N. ingrata. Collecons of N. pseudoingrata
form a well-supported clade in phylogenec trees.
Key words:
Agaricomycetes
grasslands
Hygrophoraceae
meadows
new taxon
Waxcaps
Citaon: Fuljer F, Zajac M, Boertmann D, Szabóová D, Kautmanová I (2022). Neohygrocybe pseudoingrata, a new grassland species from Slovakia and
the Czech Republic. Fungal Systemacs and Evoluon 9: 11–17. doi: 10.3114/fuse.2022.09.02
Received: 27 September 2021; Accepted: 12 November 2021; Effectively published online: 19 January 2022
Corresponding editor: P.W. Crous

© 2022 Westerdijk Fungal Biodiversity Instute
Fuljer et al.
Editor-in-Chief
Prof. dr P.W. Crous,Westerdijk Fungal BiodiversityInstitute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.
E-mail:p.crous@westerdijkinstitute.nl
12
vrchovina (Czech Republic) mountains. Soil type has been
determined based on the geological map of Slovakia (hp://apl.
geology.sk/gm50js).
Descripons of macro-morphological features were based
on fresh material. Colours were coded according to the Pantone
colour chart (Pantone Colour Finder 2021). Twenty basidiomata
were studied and measured from the holotype collecon.
The micromorphology of the studied specimens was
invesgated by F. Fuljer, D. Boertmann and I. Kautmanová using
a Kapa Mic D117 with integrated camera, a Leica SM-Lux, a DIC
microscope Nikon Eclipse Ni-U and microphotography were
captured by a Nikon DS-Ri2 camera. NIS-Elements Basic Research
and MiCam v. 2.4 imaging soware were used to measure and
examine microscopic features. Tissues, spores and other micro-
morphological structures were examined fresh or rehydrated in
H2O or in Congo Red ammonia soluon. Altogether 575 spores
from 14 basidiomata were studied and measured; spores were
measured mainly from spore deposits in H2O. Fiy basidia,
50 sterigmata and 50 basidioles from ve basidiomata were
invesgated from the rehydrated material in ammonial Congo
Red soluon. Other microscopic structures, such as gill trama,
pileipellis and spipellis, were observed in three basidiomata
from the holotype. Q value refers to the division of length and
width of microscopic structures. Qav refers the average value
of Q and av. refers the average length and width of microscopic
features.
Type material was deposited in the herbarium of the Slovak
Naonal Museum-Natural History Museum, Braslava (BRA).
Nomenclature follows Lodge et al. (2013) and Index Fungorum
(indexfungorum.org).
DNA extracon, amplicaon, sequencing
Total genomic DNA was extracted from dried ssue using
DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according
to the manufacturer’s protocol, but with prolonged
incubaon me of up to 3 h aer addion of the RNA-lyc
enzyme. PCR was performed using a BioRad C1000 Touch™
Thermal Cycler. Target region of the internal transcribed
spacer regions of ribosomal DNA (ITS) was amplied using
primers ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4
(5’-TCCTCCGCTTATTGATATGC-3’; White et al. 1990). The large
ribosomal subunit of ribosomal DNA (LSU) was amplied
using primers LR0R (5’-ACCCGCTGAACTTAAGC-3’) and LR5
(5’-TCCTGAGGGAAACTTCG-3’; Vilgalys & Hester 1990). The
amplicaon reacons were conducted in 25 μL total volume
using a GoTaq Flexi PCR kit (Promega), the reacon mixture
containing 20–25 ng total DNA template, 1 μL of both primers
(10 μM), 5 μL of Buer (5×), 2.5 μL of dNTP (2 mM), 2 μL of
MgCl2 (25 mM), 0.2 μL GoTaq Flexi polymerase (5 U) and the
nal volume was added with ultra pure water. The amplicaon
reacon for ITS and LSU regions was set up as follows: 3 min
inial denaturaon at 95 °C, 32 cycles (95 °C for 30 s, 55 °C for
30 s, and 72 °C for 1 min + increasing me 2 s per cycle), 10 min
nal elongaon at 72 °C. The PCR products were analysed on 2 %
agarose gel. PCR products were puried using a Thermosensive
Alkaline Phosphatase (FastAP) and Exonuclease 1 (Exo 1)
(Thermo Fisher Scienc Inc., USA) according to manufacturer’s
instrucons. The paral gene was sequenced in a commercial
laboratory (Eurons Genomics GmbH, Cologne, Germany).
Sequences were visualised, edited and aligned in MEGA-X (Kumar
et al. 2018). Sequence similarity searches were performed using
GenBank BLASTn (hp://www.ncbi.nlm.nih.gov/BLAST/) and
BOLD Idencaon System (hps://www.boldsystems.org/).
Phylogenec analysis
DNA sequences of Neohygrocybe species and selected outgroup
of Cuphophyllus fornicatus were downloaded from NCBI on
21 Jan. 2021. All sequences retrieved in this study were sent
to BOLD database and transferred to GenBank and accession
numbers are listed in Table 1. Evoluonary analyses were
conducted in MEGA X (Kumar et al. 2018) by using the Maximum
Likelihood method and Tamura-Nei model (Tamura & Nei 1993).
The tree with the highest log likelihood (-3667.62) is shown
(Fig. 1). The percentage of trees in which the associated taxa
clustered together is shown next to the branches. Inial tree(s)
for the heurisc search were obtained automacally by applying
Neighbor-Joining and BioNJ algorithms to a matrix of pair wise
distances esmated using the Maximum Composite Likelihood
(MCL) approach, and then selecng the topology with superior
log likelihood value. The tree is drawn to scale, with branch
lengths measured in the number of substuons per site. This
analysis involved 26 ITS sequences. There were a total of 782
posions in the nal dataset. In the tree, Neohygrocybe species
were posioned on a separate branch close to the clade of N.
nitrata, which is consistent with the results from macro- and
microcharacters observaons.
RESULTS
Taxonomy
Neohygrocybe pseudoingrata Fuljer, Zajac, Boertm. &
Kautmanova, sp. nov. MycoBank MB 842316. Figs 2, 3.
Etymology: Name refers to Neohygrocybe ingrata, a species
with similar morphology.
Typus: Slovakia, Javorníky Mts., Melocík, Veľké Rovné, ca. 300 m
E from the main road, N49°20’28.14” E18°30’37.65”, alt. 798 m,
cow grazed and mowed grassland, 21 Jul. 2020, F. Fuljer (holotype
BRA CR33023, ITS GenBank MZ479356, LSU GenBank MZ479363, ITS
BOLD NEOHY001-21).
Habitat & Distribuon: Known from Slovakia and the Czech
Republic, probably more widespread but possibly misidened
as N. ingrata or N. nitrata. Growing gregarious and very oen
caespitose and may also sporulate in half rings, somemes
solitary or scaered. It has been recorded in dierent vegetaon
types, but always in unimproved semi-natural mesic meadows
and pastures, from July to October, on acidic, neutral and
calcareous soils.
Pileus 20–80 mm, at rst hemispherical, later convex to
applanate, irregular, oen irregularly contorted, somemes
umbonate, or centrally compressed and with spling margin;
surface smooth, or radially brillose, dry, when old very oen
uneven, bu brown, pale brownish, greyish brown, dark
brownish grey (Pantone 463C to Pantone 466C). Spe 35–100 ×
8–32 mm, fusiform, clavate; irregularly furrowed, compressed,
oen contorted and tawn; hollow; surface smooth, dry, white
with slightly greyish or brownish nges (Pantone 4246C to

© 2022 Westerdijk Fungal Biodiversity Instute
Neohygrocybe pseudoingrata sp. nov.
Editor-in-Chief
Prof. dr P.W. Crous,Westerdijk Fungal BiodiversityInstitute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.
E-mail:p.crous@westerdijkinstitute.nl
13
Pantone 4247C). Lamellae adnexed, oen very broad and
thick, ventricose, brile, white with brownish or greyish hue,
much paler than pileus, slightly paler than spe (Pantone
P 1-9 C, Pantone 7527C) , somemes with paler edges. Context
not reddening (without any colour changes), white, white
with brownish hue, especially in cap (in spe Pantone 7527C,
in pileus Pantone 4645C, Pantone 4655C or Pantone 4665C);
rather fragile, brillose. Smell unpleasant, signicantly nitrous.
Taste neutral, somemes farinaceous. Spore deposit white.
Basidiospores broadly ellipsoid, ellipsoid to ellipsoid-oblong,
thin-walled, smooth, hyaline, non-amyloid, somemes with one
big vacuole, (6.5–)7.2–10.2(–11.8) × (4.4–)4.7–6.4(–7.5) µm, av.
= 8.4 × 5.5 µm, Q = (1.1–)1.3–1.8(–2.1), Qav. = 1.56 (575 spores
from 14 basidiomata measured from the type collecons).
Basidia (33.5–)35–51(–55) × (5.5–)6.8–9.5(–11.3) µm, av. =
42 × 8 µm (50 basidia from ve basidiomata measured from
the holotype), predominantly 4-spored, narrowly clavate to
clavate, sterigmata (2.5–)2.7–6.6(–6.9) µm (50 sterigmata from
ve basidiomata measured from the holotype), awl-shaped.
Basidioles (30.5–)33–46(–49) × (5.4–)5.9–8.7(–10.1) µm (50
basidioles from ve basidiomata measured from the holotype),
clavate to broadly clavate. Cysdia absent. Pileipellis a cus
with cells 28–146 × 3.5–15 µm. Spipellis a cus with some
free hyphal ends (resembling a thrichoderm) with cells 25–160
× 3.9–17 µm, cells below pileipellis with brownish content. Gill
trama subregular with cells 30–155 × 4–26.5 µm (some up to
400 µm), ± cylindrical, vermiform and somemes with slightly
inated ends, long slender cells in centre and shorter cells to the
sides. Clamps abundant in all ssues.
Addional materials examined: Czech Republic, Českotřebovská
vrchovina Mts., Česká Třebová, alt. 475 m, mesic mowed meadow,
27 Jul. 2020, M. Mička (BRA CR34358). Slovakia, Javorníky Mts.,
Tomborov Salaš, Pšurnovice (Bytča), N49°14’2.85” E18°31’59.94”,
alt. 384 m, cow grazed and mowed meadow, 4 Oct. 2014, F.
Fuljer (BRA CR34502); Melocík, Veľké Rovné, N49°20’33.42”
E18°30’31.18”, alt. 791 m, small overgrown meadow hidden in the
forest, 29 Aug. 2019, F. Fuljer (BRA CR34375); Dučkov, Vysoká nad
Kysucou, N49°21’38.56” E18°31’51.30”, alt. 722 m, mesic mowed
meadow, 31 Aug. 2018, F. Fuljer (BRA CR34374); Škápová, Petrovice,
N49°14’54.65” E18°31’47.48”, alt. 458 m, mesic mowed meadow,
1 September 2019, F. Fuljer (BRA CR34370); under the Holý vrch,
Hvozdnica, N49°12’46.52” E18°27’0.67”, alt. 547 m, mesic mowed
meadow on calcareous soils, 19 Sep. 2019, F. Fuljer (BRA CR34377);
Škápová, Petrovice, N49°15’2.01” E18°31’52.58”, alt. 426 m, mesic
mowed meadow, 2 Oct. 2019, F. Fuljer & M. Zajac (BRA CR34376);
Table 1. Collecons studied and analysed in this study by molecular methods with collecon numbers, country of origin, GenBank and BOLD
accession numbers (some collecons of N. pseudoingrata were not sequenced, for all collecons check Addional materials examined).
Species Herbarium number Origin ITS GenBank Accession No. ITS BOLD Accession No.
N. ingrata BRA CR34493 Slovakia MZ479336 NEOHY 008-21
BRA CR34490 Slovakia MZ479339 NEOHY 019-21
BRA CR34489 Slovakia MZ479337 NEOHY 025-21
BRA CR34488 Slovakia MZ479338 NEOHY 026-21
N. nitrata BRA CR34492 Czechia MZ479340 NEOHY 009-21
N. ovina BRA CR34491 Slovakia MZ479341 NEOHY 010-21
BRA CR34487 Slovakia MZ479342 NEOHY 027-21
N. pseudoingrata sp. nov. BRA CR33023 holotype Slovakia MZ479356 NEOHY 001-21
BRA CR34363 Slovakia MZ479355 NEOHY 002-21
BRA CR34377 Slovakia MZ479354 NEOHY 003-21
BRA CR34369 Slovakia MZ479353 NEOHY 004-21
BRA CR34368 Slovakia MZ479352 NEOHY 005-21
BRA CR34367 Slovakia MZ479351 NEOHY 006-21
BRA CR34364 Slovakia MZ479350 NEOHY 007-21
BRA CR34374 Slovakia MZ479349 NEOHY 011-21
BRA CR34373 Slovakia MZ479348 NEOHY 012-21
BRA CR34362 Slovakia MZ479347 NEOHY 013-21
BRA CR34371 Slovakia MZ479346 NEOHY 014-21
BRA CR34511 Slovakia MZ479345 NEOHY 015-21
BRA CR34382 Slovakia MZ479344 NEOHY 016-21
BRA CR34365 Slovakia MZ479343 NEOHY 017-21
BRA CR34372 Slovakia MZ479362 NEOHY 018-21
BRA CR34502 Slovakia MZ479361 NEOHY 020-21
BRA CR34378 Slovakia MZ479360 NEOHY 021-21
BRA CR34384 Slovakia MZ479359 NEOHY 022-21
BRA CR34383 Slovakia MZ479358 NEOHY 023-21
BRA CR34370 Slovakia MZ479357 NEOHY 024-21

© 2022 Westerdijk Fungal Biodiversity Instute
Fuljer et al.
Editor-in-Chief
Prof. dr P.W. Crous,Westerdijk Fungal BiodiversityInstitute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.
E-mail:p.crous@westerdijkinstitute.nl
14
under the Medvedie hill, Petrovice, N49°15’46.82” E18°31’1.80”, alt.
422 m, mesic overgrown meadow, 2 Oct. 2019, F. Fuljer & M. Zajac
(BRA CR34372); Benková, Petrovice, N49°16’4.53” E18°30’52.90”, alt.
451 m, mesic mowed meadow, 2 Oct. 2019, F. Fuljer & M. Zajac (BRA
CR34371); Medvedie, Petrovice, N49°15’53.80” E18°30’57.08”, alt.
444 m, overgrown part of mesic meadow, 27 Oct. 2019, F. Fuljer (BRA
CR34370); Baránkovci, Šavnik, N49°16’50.71” E18°25’12.50”, alt. 692
m, cow grazed pasture, 8 Jul. 2020, F. Fuljer (BRA CR34363); Benková,
Petrovice, N49°16’2.26” E18°30’47.03”, alt. 477 m, mesic mowed
meadow, 13 Jul. 2020, F. Fuljer (BRA CR34369); Setechov, Petrovice,
N49°16’7.00” E18°29’46.72”, alt. 560 m, overgrown meadow, 16
Jul. 2020, F. Fuljer (BRA CR34368); Vrchrieka, Vysoká nad Kysucou,
N49°21’41.23” E18°33’3.74”, alt. 790 m, mesic mowed meadow, 22
Jul. 2020, F. Fuljer (BRA CR34367); Kržeľ, Papradno, N49°17’40.49”
E18°20’15.30”, alt. 772 m, overgrown complex of meadows, 24 Jul.
2020, F. Fuljer (BRA CR34366); Čiakov, Kolárovice, N49°19’27.38”
E18°31’25.04”, alt. 674 m, overgrown meadow, 25 Jul. 2020, F. Fuljer
(BRA CR34365); Tomborov Salaš, Pšurnovice (Bytča), N49°14’0.45”
E18°31’57.01”, alt. 373 m, cow grazed meadow, 25 Jul. 2020, F. Fuljer
(BRA CR34364); Brezie, Petrovice, N49°15’41.07” E18°30’57.63”,
alt. 456 m, mowed meadow, 12 Sep. 2020, F. Fuljer (BRA CR34355);
Zákysučie, Krásno nad Kysucou, N49°22’44.18” E18°48’59.35”, alt.
559 m, overgrowing mesic heathland, 10 Oct. 2020, F. Fuljer (BRA
CR34356); Medvedie 2, Petrovice, N49°15’46.45” E18°30’51.77”,
alt. 466 m, small sized overgrown meadow, 26 Oct. 2020, F. Fuljer
(BRA CR34357); Jablunkovské medzihorie Mts., Poľana, Skalité,
N49°30’16.3” E18°55’32.9”alt. 730 m, mesophilic mowed meadow,
25 Jul. 2020, M. Zajac (BRA CR34381); Turzovská vrchovina Mts.,
Neohygrocybe pseudoingrata MZ479357 SVK
Neohygrocybe pseudoingrata MZ479345 SVK
Neohygrocybe pseudoingrata MZ479362 SVK
Neohygrocybe pseudoingrata MZ479343 SVK
Neohygrocybe pseudoingrata MZ479346 SVK
Neohygrocybe pseudoingrata MZ479344 SVK
Neohygrocybe pseudoingrata MZ479358 SVK
Neohygrocybe pseudoingrata MZ479356 SVK holotype
Neohygrocybe pseudoingrata MZ479355 SVK
Neohygrocybe pseudoingrata MZ479351 SVK
Neohygrocybe pseudoingrata MZ479360 SVK
Neohygrocybe pseudoingrata MZ479345 SVK
Neohygrocybe nitrata KP96581 GER
Neohygrocybe nitrata MZ479340 CZ
Neohygrocybe ingrata KF291225 UK
Neohygrocybe ingrata MZ479336 SVK
Neohygrocybe ingrata MZ479337 SVK
Neohygrocybe ingrata MZ479339 SVK
Neohygrocybe ingrata EU784317 UK
Neohygrocybe subovina KF291136 USA
Neohygrocybe subovina KF291140 USA
Neohygrocybe ovina KF291233 UK
Neohygrocybe ovina KF291228 UK
Neohygrocybe ovina MZ479341 SVK
Neohygrocybe ovina MZ479342 SVK
Cuphophyllus fornicatus EU784308 UK
Cuphophyllus fornicatus KF291123 DK
99
48
92
99
46
93
46 95
98
100
100
0.10
Fig. 1. Maximum likelihood tree obtained from the analysis of ITS sequences of Neohygrocybe and Cuphophyllus fornicatus as outgroup. Bootstrap
support values are indicated at the nodes.

© 2022 Westerdijk Fungal Biodiversity Instute
Neohygrocybe pseudoingrata sp. nov.
Editor-in-Chief
Prof. dr P.W. Crous,Westerdijk Fungal BiodiversityInstitute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.
E-mail:p.crous@westerdijkinstitute.nl
15
Fig. 2. Macromorphological characters of Neohygrocybe pseudoingrata. A. Basidiomata in the natural habitat, where the holotype was collected
(BRA CR33023, holotype). B. Dierent shapes of basidiomata (PHFF11143, paratype). C. The robust stature of N. pseudoingrata in the natural habitat
(PHFF11554, paratype). D. Basidiomata in the natural habitat (PHFF10723, paratype). E. Basidioma with brownish pileus, in the natural habitat
(PMZ554, paratype). F. Basidioma with greyish pileus, in the natural habitat (PHFF11080, paratype). G. Cross-secon of the well-grown basidioma,
hollow spe and adnexed lamellae visible (BRA CR33023, holotype). H. Closer, ventral view on the compressed spes and lamellae (BRA CR33023,
holotype). I. Closer view showing the colour, shape and smooth surface of the pileus (BRA CR33023, holotype). Scale bars = 20 mm.

© 2022 Westerdijk Fungal Biodiversity Instute
Fuljer et al.
Editor-in-Chief
Prof. dr P.W. Crous,Westerdijk Fungal BiodiversityInstitute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.
E-mail:p.crous@westerdijkinstitute.nl
16
Boháčovci, Korňa, N49°26’33.33” E18°31’49.19”, alt. 714 m, mesic
mowed meadow, 8 Sep. 2019, F. Fuljer (BRA CR34362); Ďurajčíkovci,
Korňa, N49°25’46.13” E18°31’4.40”, alt. 698 m, mesic mowed meadow,
8 Sep. 2019, F. Fuljer (BRA CR34379); Hlavice – Flintovci, Klokočov,
N49°27’52.4” E18°36’33.2”, alt. 720 m, mesic mowed meadow, 10
Sep. 2019, M. Zajac (BRA CR34382); Kysucká vrchovina Mts., Tatarovci
– Senkov, Povina, N49°18’08.7” E18°43’52.7”, alt. 633 m, mesic
mowed meadow, 11 Sep. 2019, Z. Václavová (BRA CR34384); Harvelka,
Nová Bystrica, N49°21’27.10” E19°8’50.17”, alt. 808 m, sheep grazed
pasture, 25 Sep. 2019, F. Fuljer & M. Zajac (BRA CR34378); Harvelka,
Nová Bystrica, N49°21’25.21” E19°8’4.39”, alt. 783 m, sheep grazed
pasture, 17 Sep. 2020, F. Fuljer (BRA CR34359); Brodenec, Snežnica,
N49°15’42.87” E18°47’4.41”, alt. 459 m, mesic mowed meadow, 7
Oct. 2020, F. Fuljer (BRA CR34360); Kysucké Beskydy Mts., Seranov
vlek, Skalité, N49°29’39.1” E18°57’48.8”, alt. 725 m, mesic mowed
meadow, 28 Sep. 2019, M. Zajac (BRA CR34383); Biele Karpaty Mts.,
Kopánka, Horné Orechové, N48°55’31.31” E18°1’59.62”, alt. 261 m,
cow grazed pasture, 20 Oct. 2020, F. Fuljer (BRA CR34361).
DISCUSSION
Due to the dull colouraon of the basidiomata and dry surfaces
of spe and pileus, this new waxcap clearly belongs to the genus
Neohygrocybe, as has been conrmed also by the phylogenec
analysis. It is a well recognisable species, characterised by
robust dull coloured basidiomata, nitrous smell, non-reddening
context, pale brownish and greyish, smooth or nely brillose
pileus, slightly greyish or brownish, contorted, compressed and
hollow spe and broadly ellipsoid to ellipsoid spores (Figs 2, 3).
Closely related species are N. ingrata, N. nitrata and N. ovina.
The most similar species is N. ingrata, in which the context
stains reddish. Young basiomata of N. pseudoingrata and N.
ingrata can be very similar, disnguished only by the reddening
context of N. ingrata. Neohygrocybe nitrata also has a nitrous
smell and also lacks the reddish reacon of the context, but it
is usually smaller (up to 60–70 mm high), with a more or less
squamulose dark brown pileus and thinner spe (up to 6 mm
diam) which is also dark brown. Neohygrocybe ovina is much
darker, with dark brown, dark grey or almost black spe, pileus
and lamellae and the context is strongly reddening, and the cap
may be squamulose. Several other Neohygrocybe-taxa have
been described from North and Central America, Australia, New
Zealand and China (many not yet combined into the genus)
such as Hygrocybe lepidopellis, H. cinerascens, H. mellita, H.
albomarginata, H. caespitosa, H. melleofusca, H. ovinoides, H.
fuligineosquamosa, H. waolipo, Neohygrocybe griseonigra, N.
innata, N. subovina, and N. squarrosa (Hesler & Smith 1963,
Pegler 1983, Horak 1990, Desjardin & Hemmes 1997, Cantrell
& Lodge 2004, Young 2005, Bessee et al. 2012, Wang et al.
2018). None of these have been sequenced, but all dier from
N. pseudoingrata in darker colouraon, spore morphology,
structure of pileus surface or colour changes. Cuphophyllus
species dier by deeply decurrent lamellae; C. fornicatus is the
single species of the genus that lacks decurrent lamellae and
strongly resembles N. pseudoingrata but for the nitrous smell.
Dull coloured Gliophorus species dier by lubricous cap and
spe surfaces. Pseudotricholoma metapodium is characterised
by amyloid spores, solid, non-compressed spe and lamellae
that are not veined.
The indicator value for valuable grasslands of N.
pseudoingrata is uncertain. Recorded collecons from
Slovakia and the Czech Republic are from mowed meadows or
extensively grazed pastures and were accompanied by various
CHEG fungi. Further research will reveal whether the species
is rare or only overlooked and misinterpreted. Based on the
numerous collecons from NW Slovakia it can be assumed that
N. pseudoingrata is probably common in Slovakia and should be
searched for in neighbouring countries.
ACKNOWLEDGEMENTS
The authors greatly thank M. Cechová, M. Kudrna, M. Mička, V. Rochová
and Z. Václavová for their eld assistance and collecons. V. Kautman
is acknowledged for help with macromorphology documentaon.
Research was funded by Operaonal Program of Integrated
Infrastructure, co-nanced with the European Fund for Regional
Development (EFRD) ITMS2014+313021W683: “DNA barcoding of
Slovakia (SK-BOL), as a part of internaonal iniave Internaonal
Barcode of Life (iBOL)”.
Conict of interest: The authors declare that there is no conict
of interest.
Fig. 3. Neohygrocybe pseudoingrata (BRA CR33023, holotype). A. Basidiospores. B. Pileipellis. Scale bars: A = 10 µm; B = 20 µm.

© 2022 Westerdijk Fungal Biodiversity Instute
Neohygrocybe pseudoingrata sp. nov.
Editor-in-Chief
Prof. dr P.W. Crous,Westerdijk Fungal BiodiversityInstitute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.
E-mail:p.crous@westerdijkinstitute.nl
17
REFERENCES
Adamčík S, Kautmanová I (2005). Hygrocybe species as indicators of
natural value of grasslands in Slovakia. Catathelasma 6: 25–34.
Bessee AE, Roody WC, Sturgeon WE, et al. (2012). Waxcap Mushrooms
of Eastern North America. Syracuse University Press.
Boertmann D (2010). The genus Hygrocybe. 2nd revised edion.
Svampetryk, Denmark.
Cantrell SA, Lodge DJ (2000). Hygrophoraceae of the Greater Anlles:
Hygrocybe subgenus Hygrocybe. Mycological Research 104: 873–
878.
Cantrell S, Lodge DJ (2004). Hygrophoraceae (Agaricales) of the Greater
Anlles: Hygrocybe subgenus Pseudohygrocybe secons Coccineae
and Neohygrocybe. Mycological Research 108: 1301–1314.
Desjardin DE, Hemmes DE (1997). Agaricales of the Hawaiian Islands. 4.
Hygrophoraceae. Mycologia 89: 615–638.
Ejrnæs R, Brunn HH (1995). Predicons of grassland quality
for environmental management. Journal of Environmental
Management 41: 171–183.
Fuljer F, Zajac M, Václavová Z, et al. (2020). Hygrocybe (genera
Hygrocybe, Gliophorus, Porpolomopsis and Cuphophyllus) in
northwestern Slovakia, Part III. Catathelasma 20: 5–55.
Geologická mapa Slovenska M 1:50 000 [online]. Braslava: Štátny
geologický ústav Dionýza Štúra, 2013. Available online on: hp://
apl.geology.sk/gm50js [last accessed 9 February 2021].
Grith GW, Braon JL, Easton GL (2004). Charismac megafungi – the
conservaon of waxcap grasslands. Brish Wildlife 15: 31–45.
Halbwachs H, Dennger BTM, Detheridge AP, et al. (2013). Hyphae of
waxcap fungi colonise plant roots. Fungal Ecology 6: 487–492.
Halbwachs H, Easton GL, Bol R, et al. (2018). Isotopic evidence
of biotrophy and unusual nitrogen nutrion in soil-dwelling
Hygrophoraceae. Environmental Microbiology 20: 3573–3588.
Hesler LR, Smith AH (1963). North American species of Hygrophorus.
The University of Tennessee Press, Knoxville.
Horak E (1990). Monograph of the New Zealand Hygrophoraceae
(Agaricales). New Zealand Journal of Botany 28: 255–309.
Læssøe T, Boertmann D (2008). A new alamellate Hygrocybe species
from Ecuador. Mycological Research 112: 1206–1209.
Kimura M (1980). A simple method for esmang evoluonary rate
of base substuons through comparave studies of nucleode
sequences. Journal of Molecular Evoluon 16: 111–120.
Kumar S, Stecher G, Li M, et al. (2018). MEGA X: Molecular Evoluonary
Genecs Analysis across compung plaorms. Molecular Biology
and Evoluon 35: 1547–1549.
Lodge DJ, Padamsee M, Matheny PB, et al. (2013). Molecular phylogeny,
morphology, pigment chemistry and ecology in Hygrophoraceae
(Agaricales). Fungal Diversity 64: 1–99.
Pantone Colour Finder: hps://www.pantone.com/color-nder#/
pick?panton eBook=pantoneSolidCoatedV3M2 [last accessed 9
February 2021].
Pegler DN (1983). Agaric ora of the Lesser Anlles. Kew Bullen
Addional Series IX: 1–668.
Pegler DN, Fiard JP (1978). Hygrocybe sect. Firmae (Agaricales) in
tropical America. Kew Bullen 32: 297–312.
Rotheroe M (2001). A preliminary survey of waxcap grasslands
indicator species in South Wales. In: Fungal Conservaon: Issues
and Soluons (Moore D, Nauta NN, Evans SE, et al. eds). Cambridge
University Press, UK: 120–135.
Seitzman BH, Ouimee A, Mixon RL, et al. (2011) Conservaon of
biotrophy in Hygrophoraceae inferred from combined stable
isotope and phylogenec analyses. Mycologia 103: 280–290.
Tamura K, Nei M (1993). Esmaon of the number of nucleode
substuons in the control region of mitochondrial DNA in humans
and chimpanzees. Molecular Biology and Evoluon 10: 512–526.
Tello SA, Silva-Flores P, Agerer R, et al. (2013). Hygrocybe virginea is
a systemac endophyte of Plantago lanceolata. Mycological
Progress 13: 471–475.
Vilgalys R, Hester M (1990). Rapid genec idencaon and mapping of
enzymacally amplied ribosomal DNA from several Cryptococcus
species. Journal of Bacteriology 172: 4239−4246.
Wang C-Q, Zhang M, Li T-H (2018). Neohygrocybe griseonigra
(Hygrophoraceae, Agaricales), a new species from subtropical
China. Phytotaxa 350: 64–70.
White TJ, Bruns T, Lee SB, et al. (1990). Amplicaon and direct
sequencing of fungal ribosomal RNA genes for phylogenecs. In:
PCR Protocols: A Guide to Methods and Applicaons (Innis MA,
Gelfand DH, Sninsky JJ, et al. eds.). Academic Press, New York:
315–322.
Young AM (2005). Fungi of Australia. Hygrophoraceae. ABRS, Canberra;
CSIRO Publishing, Melbourne.