Mycena fragosa (Mycenaceae), a new species from the fragas in northwestern Spain

Abstract

A new species of Mycena from northwestern Spain is described, illustrated and morphologically and molecularly compared to related species. The new species belongs to M. sect. Viscipelles, presenting as tiny in stature, with small spores, claviform to subglobose cheilocystidia with short excrescences, and a small disc at the base of the stipe, which is made up of long cystidia. Currently, it has been only found at three localities of Atlantic forest, where it grows on dead leaves of Alnus lusitanica, Acer pseudoplatanus, Salix atrocinerea, and Quercus robur acorn cupules. In addition, a key to the taxa of M. sect. Viscipelles, which are known in the Northern Hemisphere, is provided.

Full text

7
Fungi Iberici 3 : 7-18 (2023)
DOI : https://doi.org/10.51436/funiber/03.001
Mycena fragosa (Mycenaceae),
a new species from the fragas in northwestern Spain
VILLARREAL M.1, TRABA J.M.2, COUCEIRO A.3, NAVEIRA H.4 & VILA-SANJURJO A.5
1 Dpto. Ciencias de la Vida (Botánica), Facultad de Ciencias, Universidad de Alcalá, 28805, Alcalá de Henares, Madrid, Spain. E-mail:
mvillarrmycena@gmail.com
2 Grupo GIBE, Dpto. Bioloxía Celular e Molecular, Centro interdisciplinar de Química e Bioloxia (CICA), Universidade da Coruña,
Campus Zapateira s/n, 15071, A Coruña, Spain. E-mail: jose.maria.traba@udc.es
3 Asociación Micolóxica Andoa, Río Barcés 6, bajo, Barcala, 15660, A Coruña, Spain. E-mail: acnaveira69@gmail.com
4 Vicerrectorado de Política Cientíca, Investigación y Transferencia, Universidade da Coruña (UDC). Campus Zapateira s/n, 15071, A
Coruña, Spain. E-mail: horacio.naveira.fachal@udc.es
5 Vicerrectorado de Política Cientíca, Investigación y Transferencia, Universidade da Coruña (UDC). Campus Zapateira s/n, 15071, A
Coruña, Spain. E-mail: anton.vila@udc.es
Resumen
Una nueva especie de Mycena del noroeste de España es descrita, ilustrada y comparada con especies afines.
Esta especie pertenece a M. sect. Viscipelles y se caracteriza morfológicamente por tener un tamaño diminuto,
esporas pequeñas, queilocistidios claviformes a subglobosos con excrecencias cortas, un pequeño disco basal
espinoso en la base del estípite formado por cistidios largos y estipitipellis con caulocistidios lisos. Hasta el
momento, solo se conoce de tres localidades del bosque atlántico donde crece sobre hojas muertas de Alnus
lusitanica, Acer pseudoplatanus, Salix atrocinerea y cúpulas de bellota de Quercus robur. Además, se
proporciona una clave con los taxones conocidos para la sección en el hemisferio norte.
Palabras clave: Mycenaceae, Mycena sect. Viscipelles, taxonomía, morfología, filogenia, Europa.
Resumo
Uma nova espécie de Mycena do noroeste da Espanha é descrita, ilustrada e comparada com espécies próximas.
Pertencendo à M. sect. Viscipelles, caracterizase morfologicamente pelas dimensões reduzidas, esporos pequenos,
queilocistídios claviformes a subglobosos com excrescências curtas, um pequeno disco basal espinhoso na base do
estipe formado por cistídios longos e uma stipitipellis com caulocistídios lisos. Atualmente, só se conhece de três
locais da mata atlântica frutificando em folhas mortas Alnus lusitanica, Acer pseudoplatanus, Salix atrocinerea e
cúpulas de bolota de Quercus robur. Por fim, é fornecida uma chave para os táxones conhecidos no hemisfério
norte.
Palavras chave: Mycenaceae, Mycena sect. Viscipelles, taxonomia, morfología, filogenia, Europa.
Summary
A new species of Mycena from northwestern Spain is described, illustrated and morphologically and molecularly
compared to related species. The new species belongs to M. sect. Viscipelles, presenting as tiny in stature, with small
spores, claviform to subglobose cheilocystidia with short excrescences, and a small disc at the base of the stipe, which
is made up of long cystidia. Currently, it has been only found at three localities of Atlantic forest, where it grows
on dead leaves of Alnus lusitanica, Acer pseudoplatanus, Salix atrocinerea, and Quercus robur acorn cupules. In
addition, a key to the taxa of M. sect. Viscipelles, which are known in the Northern Hemisphere, is provided.
Keywords: Mycenaceae, Mycena sect. Viscipelles, morphology, taxonomy, phylogeny, Europe.
Publicado el 28 de noviembre de 2023
ISSN 2660-6534

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Villarreal M., Traba J.M., Couceiro A., Naveira H. & Vila-Sanjurjo A.
Introduction
In Galician, a fraga is a type of forest that is usually
isolated and difcult to access. Fragas are populated
by various species of deciduous trees, such as Quercus
robur L., Alnus lusitanica Vít, Douda & Mandák, Be-
tula alba L., Laurus nobilis L., Castanea sativa Mill.,
together with a rich undergrowth of ferns that include
Struthiopteris spicant (L.) F.W. Weiss., Culcita macro-
carpa C. Presl., Dryopteris afnis (Lowe) Fraser-Jenk.
or Woodwardia radicans (L.) Sm. Some Galician fragas
are considered the last remaining examples of virgin
Atlantic European forest. Of these, only small, isolated
spots remain today due to human activity. Claiming to
be the best-preserved coastal Atlantic forest in Europe,
and one of the very few forests of its kind on the Euro-
pean continent, the Natural Park Fragas do Eume covers
the steep valley created by the Eume river. During some
recent mycological samplings at this Natural Park and
adjacent areas, our team spotted an unknown species of
Mycena (Pers.) Rousell. The gathered specimens diffe-
red from other related species in having a tiny stature,
presence of a small basal disc, small spores, and near-
ly smooth cheilocystidia. Based on the morphological
study of living and dry material, as well as the use of
genetic data, we here describe them as a new species
within Mycena sect. Viscipelles Kühner.
Materials and methods
Morphological and material study
The samples were photographed macroscopically in situ
and from moist chamber cultures of Alnus and Acer dead
leaves. We used a Lumix TZ90 and an Olympus Tou-
gh TG6 digital camera, with a tripod under natural or
LED ring light. All macroscopic descriptions are based
on fresh material, which was subsequently dehydrated at
45°C in a Vidaxl food dehydrator or air dried, in prepa-
ration for herbarium conservation. Once dried, the spe-
cimens were sealed in ziplock plastic bags and labelled.
The photographic images were processed with Adobe
Photoshop. Colour codes were taken from MUNSELL
(1994). For microscopic characterisation, free-handmade
sections were made from dried fruitbodies under a Mo-
tic SFL11C-2LBB dissecting microscope, rehydrated in
water and mounted in 2 % Congo red to highlight all tis-
sues. Melzer’s reagent was used for checking amyloidity
of the spores and dextrinoidity of the tissues. Piximètre
5.10 R 1541 was used for spore measurement. Spores
were observed using an 100x oil immersion objective
in water, choosing the “classique” format to present the
results (http://ach.log.free.fr/Piximetre/Piximetre5.htm).
All cited specimens have been deposited in the herba-
rium of the University of Alcalá (AH) and one in Cou-
ceiro’s personal herbarium with the acronym ACN. For
microscopic study, the following equipment were used:
an Olympus CX21 optical microscope with a Koppace
microphotographic camera, an Olympus CX31 micros-
cope with a Lumix DMC fz300 camera, or an Olympus
BX53 microscope with DIC Nomarski and Moticam
5000 cooler camera, connected to a personal computer.
Terminology follows VELLINGA (1988).
Molecular analysis
Dried material was used for DNA extractions using the
Omega E.Z.N.A.® Fungal DNA Mini Kit, ref. D3390.
The internal transcribed spacer of the nuclear ribosomal
DNA (ITS) was targeted for molecular analysis because
it is recognised as the universal fungal barcode and is
the most sequenced region of the genus Mycena and
commonly used for systematics, phylogenetics, iden-
tification and species delimitation (SCHOCH & al.
2012). Amplication of the target region was done by
using primers ITS1F/ITS4 (GARDES & BRUNS 1993,
WHITE & al. 1990). PCR conditions were as follows:
5 min at 95 °C; 35 cycles of 30 s at 94 °C, 30 s at 52
°C, 1 min at 72 °C; and a nal 10 min step at 72 °C.
Amplied fragments were visualised by electrophore-
sis on 1% agarose gels, stained with RedSafe Nucleic
Acid Staining solution (Intron Byothecnology), and
visualised with a UV light transilluminator (Gel Doc
XR+ Bio-Rad). The amplied samples were puried by
ExoSAP-IT™ Express PCR Product Cleanup (Applied
Biosystems). Amplicons were sequenced at the Servi-
zos de Apoio á Investigación (Vicerrectorado de Política
Cientíca, Investigación y Transferencia, Universidade
da Coruña, UDC). Sequencing was performed using
the BigDye Terminator v3.1 Cycle Sequencing Kit
and BigDye XTerminator™ Purication Kit (Applied
Biosystems), followed by screening in an automatic
3500xL Genetic Analyzer (Applied Biosystems). The
consensus ITS sequence of both primer reads was obtai-
ned with Bioedit 7.2.5 (HALL 1999). Newly generated
sequences are included in Table 1. BLAST searches
(ALTSCHUL & al. 1990) were performed to prelimi-
narily verify the identity of the sequences. A multiple
sequence alignment was performed with the newly ge-
nerated sequences and the closest BLAST hits, using
the MAFFT 7 v7.475 (KATOCH & STANDLEY 2013)
and manually edited afterwards in MEGA XI (TAMU-
RA & al. 2021). The samples sequence, together with
the sequences retrieved as the closest BLAST hits, and
sequences of Panellus longinquus (Berk.) Singer and
Panellus violaceofulvus (Batsch) Singer were elected
as outgroup (DAUNER & al. 2021) for the analyses.
Phylogenetic relationships were estimated by using
maximum likelihood (ML) and Bayesian inference (BI)
approaches. ML analyses were performed in IQ-TREE
web server (NGUYEN & al. 2015; TRIFINOPOULOS
& al. 2016), with all options as default. The substitution
model was calculated by ModelFinder implemented

9
Mycena fragosa (Mycenaceae), a new species from the fragas in nor thwestern Spain
Fungi Iberici
in IQ-TREE web server KALYAANAMOORTHY &
al. (2017). The best tting model for ML analysis was
TPM2+F+I+G4. To determine node support, 1000 rapid
bootstrap replicates were performed. The BI analysis
was carried out in MrBayes 3.2.7 (RONQUIST & al.
2012). The substitution models were sampled within the
GTR space, using two analyses of four MCMC chains
over 4 million generations, starting with a random tree
and sampling one tree every 1000 generations. A burn-
in of 25% of the trees was selected and discarded from
each analysis. Branch support (posterior probability
values, BPP) was calculated using the consensus tree
following the “50% majority rule” of the remaining
trees. Branches were considered well-supported when
BS ≥ 97% and PP ≥ 0.95. The phylogram obtained was
visualized and edited with FigTree v1.4.4 (RAMBAUT
2019) and Inkscape vector graphics editor.
Results
The best ML tree and the tree of the BI analysis showed
a similar topology (Fig. 1). Mycena sect. Viscipelles s.
MAAS GEESTERANUS (1980) shows a higher in-
terspecic genetic distance, than that observed in other
sections of the genus, and its species form a sister clade
with Mycena sect. Exornatae Maas Geest., grouping in
a well-supported clade (BS = 99 %, PP = 0.99).
Taxonomy
Mycena fragosa M. Villarreal, Couceiro & Traba,
sp. nov.
MycoBank: MB 843223
Holotypus: SPAIN. A Coruña: Fragas do Eume,
Pontedeume, 43.424142,-8.113712, 14 m, on Alnus lu-
sitanica dead leaves, leg. A. Couceiro, M. Saavedra,
J.M. Traba & M. Villarreal, 6-XII-2021, AH56008, (ITS
GenBank accesion ON113883).
Figs. 2-5
Etymology: fragosa from Latin fragosa = rough,
intricate, full of cracks, weeds and brush, in reference
to habitat.
Diagnosis: Characterised by tiny to small basidio-
mata growing on dead leaves of Alnus lusitanica, Acer
pseudoplatanus, Salix atrocinerea and acorn domes of
Quercus robur, with a small pubescent basal disc; a dark
to light grey-brown pileus; with cheilocystidia, which
is nearly smooth or covered with short excrescences;
hyphae of the pileipellis embedded in gelatinous ma-
terial, but without separable pellicle; smooth caulo-
cystidia and elongated basal disc cystidia. Differing
from M. cyanorhiza Quél. in being smaller, having a
brown pileus, the absence of blue colours at the stipe
base, having a small disc and cheilocystidia with shor-
ter excrescences not embedded in gelatinous material.
Mycena fragosa differs from M. cyanorhiza by 109 bp
in the ITS1 and 118 bp in the ITS2 region (Blast 90.88
% similarity).
Basidiomata gregarious. Primordia 0.1-0.3 mm he-
mispheric to obtusely conic, initially white then very
dark brown to slightly lighter brown (7.5YR 3/1-3/2),
densely hirsute overall. Pileus 1-2.7 mm in diam., ini-
tially obtusely conic to hemispheric convex with age,
without umbo, the disc attened to shallowly depressed,
surface minutely white-pubescent, partially to entirely
glabrescent with age, dry, devoid of a separable gela-
tinous pellicle, sulcate-striate with striations distinctly
darker than the rest of the pileus, disc and striations
greyish brown (7.5YR 5/1-5/2) initially and light gray
(7.5YR 7/1-7/2) elsewhere, hygrophanous, fading with
moisture loss to watery white to pale gray and with or
without brown tones, the disc and striations often retai-
ning darker coloration. Lamellae ascending, narrowly
adnate, distant to subdistant, 7-11 reaching stipe, with
one series of lamellulae, moderately broad 0.2-0.4 mm,
pale gray to white; the edges paler, not separable as
a gelatinous, elastic-like thread. Stipe 3-22 × 0.2-0.6
mm, cylindrical, central, hollow, straight to exuous,
the base broader at the point of attachment with a small
pubescent basal disc present; surface entirely covered
with minute white-pubescence, glabrescent towards the
apex and more densely pubescent at the base. Context
very thin; odour and taste not recorded.
Basidiospores (6.2-)7.0-8.0(-8.3) × (3.4-)3.6-4.1(-
4.4) µm, Q = (1.7-)1.8-2.1(-2.2); N = 30, Me = 7.5 ×
3.8 µm; Qe = 2; ellipsoid to narrowly ellipsoid, smooth,
hyaline, strongly amyloid in Melzer´s reagent, thin wa-
lled. Basidia (16.8-)16.8-20.6 × 5.7-6.6 µm, 4-spored,
clavate, stout, with sterigmata 2.5-4 μm long; basidioles
similar. Cheilocystidia (17.7-)20.2-27.2(-27.8) × (5.7-
)5.8-11.7(-13.6) µm, claviform to subclaviform, smooth
or with 2-7 short apical excrescences, forming a steri-
le band. Pleurocystidia absent. Pileipellis an ixocutis
composed of narrow hyphae 2-6.5 μm, diverticulated
with simple excrescences 4-7 × 1.5-2.5 µm; terminal
cells 26.9-32.1 × 7.9-14.5 µm diverticulated, abundant
at the margin of the pileus with protruding lageniform to
cylindrical dermatocystidia up to 45 µm long. Hypoder-
mium composed of inated, globose to ellipsoid cells,
24-39 × 18-33 μm, thin-walled, brownish-vinescent
in Melzer’s reagent. Stipitipellis a cutis composed of
narrow and smooth hyphae 2.5-4.5 µm in diam. Cau-
locystidia (18.6-)25.0-53.5(-65.3) × (2-)2.9-5.2(-5.8)
µm, fusiform, smooth, all over the stipe. Cystidia of the
basal disc utriform, cylindrical to lageniform. Hyphae
of the stipititrama 21.8-22.4 × 10.9 µm in diam. Clamp
connections present, but not at all septa and hard to nd.

10
Villarreal M., Traba J.M., Couceiro A., Naveira H. & Vila-Sanjurjo A.
Habitat & Distribution: Gregarious on dead leaves
of Alnus lusitanica, Acer pseudoplatanus and Salix
atrocinerea and acorn domes of Quercus robur. Sep-
tember to March. Uncommon, detected only at three
sites (6 collections) located in riparious Atlantic forests
of A Coruña, Spain.
Additional material studied: SPAIN. A Coruña:
Burricios, 43.196977 -8.169361, elevation 199 m, leg.
A. Couceiro & J. M. Traba, 3-IX-2021, on acorn domes
of Quercus robur, AH56009; (ITS Genbank accession
ON113884). Fragas do Eume, Pontedeume, 43.424142
-8.113712, elevation 15 m, leg. A. Couceiro, J. M. Traba
& M. Villarreal, 15-I-2022, on dead leaves of Alnus
lusitanica, AH56010. Playa de Morouzos, Ortigueira,
43.699373 -7.843229, elevation 3 m, leg. A. Coucei-
ro, 15-III-2022, on dead leaves of Salix atrocinerea,
ACN2022031500. From batch moist chambers, on dead
leaves of Acer pseudoplatanus, collected 15-I-2022,
wetted 17-I-2022, harvested by M. Villarreal, 25-I-
2020, AH59679; ibidem 27-II-2022, AH59680.
Fig. 1. Phylogeny of Mycena fragosa and closely related species based on ITS region of the DNA. Branch lengths are proportional
to the number of substitutions per site as measured by the scale bar. Node support is indicated as bootstrap-ML values/posterior
probability from Bayesian analysis. Mycena fragosa is highlighted in bold. Thick branches indicate nodes with high phylogenetic
support in both analysis (BS ≥ 97 %, BPP ≥ 0.95).

11
Mycena fragosa (Mycenaceae), a new species from the fragas in nor thwestern Spain
Fungi Iberici
Fig. 2. A. Fragas do Eume, type locality, B-G. Mycena fragosa. Photographers: A, D, F from M. Villarreal, B, C, E from A. Couceiro
(holotype), G. from J.M. Traba (AH56009).

12
Villarreal M., Traba J.M., Couceiro A., Naveira H. & Vila-Sanjurjo A.
Table 1. Newly generated sequences in this study with voucher numbers, geographical origin, hosts, and GenBank
accession numbers.
Identication
(M. = Mycena)Voucher specimen Geographical
origin Host ITS GenBank
accession number
M. aciculata AH56012 Spain Betula pendula ON113879
M. clavularis AH56015 Spain Quercus pyrenaica ON323585
M. cyanorhiza AH57270 Spain Pinus sylvestris OR230014
M. fragosa AH56008 holotype Spain Alnus lusitanica ON113883
M. fragosa AH56009 Spain Acer pseudoplatanus ON113884
M. galericulata AH56010 Portugal Quercus ilex ON113880
M. pachyderma AH56013 Spain Quercus rotundifolia ON113882
M. pseudocyanorrhiza AH56014 France Castanea sativa ON113885
M. rhenana AH31358 Spain Alnus glutinosa ON113878
Fig. 3. A. Mycena fragosa (holotype). Macroscpic and microscopic characters. Primordium, B. Dermatocystidia in the pileipellis, C.
Basal disc at the base of the stipe, D. Cystidia of the basal disc. Scale bar: 10 μm.

13
Mycena fragosa (Mycenaceae), a new species from the fragas in nor thwestern Spain
Fungi Iberici
Fig.4. Mycena fragosa (holotype). Microscopic characters. A. Spores, B. Basidia, C-D. Cheilocystidia, E. Hyphae of the pileipellis, F.
Terminal cells of the pileipellis, G. Pileocystidia, H. Terminal cell of the pileipellis, I-J. Caulocystidia. Scale bar: 10 μm.

14
Villarreal M., Traba J.M., Couceiro A., Naveira H. & Vila-Sanjurjo A.
Fig. 5. Mycena fragosa (Holotype). Drawings of microscopic characters. A. Hyphae of the pileipellis, B. Terminal cells of the
pileipellis, C. Spores, D. Cheilocystidia, E. Basal disc cystidia, F. Basidia and basidioles, G. Caulocystidia. Scales bars: 10 μm.

15
Mycena fragosa (Mycenaceae), a new species from the fragas in nor thwestern Spain
Fungi Iberici
Discussion
MAAS GEESTERANUS (1980) placed, in Mycena
sect. Viscipelles Kühner: M. amicta (Fr.) Quél., M. cya-
norhiza, M. pachyderma Kühner and M. subcaerulea
(Peck) Sacc. This was in accordance with Kühner (1938)
who placed them in M. sect. Cyanescentes Kühner, which
was not a validly published name, and later became a
synonym of M. sect. Viscipelles (MAAS GEESTE-
RANUS 1992). In the Conspectus of the Mycenas of
the Northern Hemisphere (MAAS GEESTERANUS
1984), M. cyanorhiza and M. pachyderma were the only
species treated as members of M. sect. Viscipelles and
there was recognition of M. sect. Amictae Maas Geest.,
which comprised of M. amicta and M. subcaerulea. In
Europe, ROBICH (2003) described M. pseudocyano-
rrhiza Robich from Italy, and (COCHARD & al. 2016)
described M. agellata Cochard & P.A. Moureau from
France. In North America (PERRY & al. (2020) des-
cribed M. ulmi B.A. Perry & H.W. Keller and recently
two new chinese species has been described belonging
to M. sect. Amictae (NA & al. 2022): M. caeruleogrisea
Q. Na, Y.P. Ge & H. Zeng and M. caeruleomarginata
Q. Na & Y.P. Ge. The last described species, M. chlo-
rocyanea L.N. Liu from China, has been incomprehen-
sibly included in this section (LIU & al. 2022). In our
phylogenetic analysis, this species groups together with
the coffee plant-pathogen M. citricolor (Berk. & M.A.
Curtis) Sacc. in a well-supported clade. Both taxa share
many common morphological characteristics, such as
decurrent lamellae and the presence of rhizomorphs. As
far as we know, M. citricolor is still yet to be assigned
to any section of genus Mycena.
Mycena cyanorhiza is the type of M. sect. Viscipe-
lles and differs from M. fragosa as follows: M. cyanor-
hiza has a stipe with a blue base arising from a basal
path of radiating brils and cheilocystidia with longer,
simple or branched flexuous excrescences that are
embedded in gelatinous matter. In addition, it usually
fruits on conifer litter. Comparison of aligned M. fra-
gosa ITS sequence data indicates a 90.88% pairwise
similarity to M. cyanorhiza.
Mycena flagellata has not yet been assigned to
any section. According to our phylogenetic analysis,
this species appears to also belong to M. sect. Viscipe-
lles. This species is only known from the type locality
in France, where it grows on Pseudotsuga menziesii
(Mirb.) Franco debris and differs from M. fragosa in ha-
ving ampullaceous cheilocystidia with a long agellum.
Comparison of aligned and overlapping M. fragosa ITS
sequence data indicates 97.65% pairwise similarity to
M. agellata, but with a poor 31% query cover.
Mycena pachyderma, is a well-known corticolous
species in Europe, originally described in France by
KÜHNER (1938), where it grows on Ulmus and Sa-
lix bark. It can also fruit on Quercus, Eucalyptus, and
even on Juniperus bark. This species has also been
collected in Belgium and Germany (ARONSEN &
LÆSSØE 2016), Italy (LONATI 1998, ROBICH
2003), Norway (ARONSEN 2023) and Spain (ESTE-
VE-RAVENTÓS & VILLARREAL 2003, SÁNCHEZ
& PÉREZ-DE-GREGORIO 2013). Mycena pachy-
derma differs from M. fragosa in having subglobose
spores, cheilocystidia with exuous excrescences, and
preferring a corticolous habitat. PLAZA & al. (2021)
studied material of Mycena marocana Maas Geest. (=
M. clavularis s. MALENÇON & BERTAULT 1975),
collecting specimens from the mossy base of Eucalyp-
tus globulus in Córdoba (southern Spain). Whilst their
description fully coincided, with regards to the ampu-
llaceous dermatocystidia, sequencing data revealed its
homology to M. pachyderma, leading them to propose
a synonymy between both species, as previously su-
ggested by ESTEVE-RAVENTÓS & VILLARREAL
(2003) and SIQUIER & al. (2012). As a result, M. sect.
Exiguae Maas Geest. should be considered a synonym
of M. sect. Viscipelles. Mycena fragosa ITS sequen-
ce data indicates an 88.48% pairwise similarity to M.
pachyderma.
Mycena pseudocyanorrhiza, a rare species only
known from Italy, France, the Iberian Peninsula (PÉ-
REZ-DE-GREGORIO 2012), and the Canary Islands
(MIERSCH & DÄHNCKE 2010), has a too brownish
pileus like M. fragosa. However, M. pseudocyanorrhi-
za differs in being larger in size, lacking a basal disc,
having caulocystidia with blue contents in fresh and
dry specimens, and possessing broader spores. Whilst
ARONSEN (2023), on his website, has argued that the
proposed reasons to separate this species from M. cya-
norhiza are quite weak, our molecular results clearly
conrm that M. pseudocyanorrhiza should be consi-
dered a different species. Mycena fragosa ITS sequen-
ce data indicates a 91.21% pairwise similarity to M.
pseudocyanorrhiza.
The recently described M. ulmi differs from the new
species in having broader spores, cheilocystidia with
more numerous diverticula, and in preferring a cortico-
lous habitat. Our data, as those of PERRY & al. (2020),
place M. ulmi in an isolated position, relative to the rest
of the species of M. sect. Viscipelles, revealing little
with regards to the phylogenetic relatedness of this ta-
xon to the other members of the studied section. This
lack of phylogenetic resolution could be partly due to
the fact that the available sequence of M. ulmi type ma-
terial is too short, with almost the entire ITS1 region
missing.
Our phylogenetic analysis of the ITS sequences
supports the identity of all these taxa as independent

16
Villarreal M., Traba J.M., Couceiro A., Naveira H. & Vila-Sanjurjo A.
species and suggests a close phylogenetic relations-
hip between M. sect. Amictae and M. sect. Viscipelles,
both recognized by MAAS GEESTERANUS (1984,
1992) as independent. Our results also suggest that both
sections form a monophyletic group (BS = 97 %, BPP
= 0.99), a conclusion that appears consistent with the
treatment given by KÜHNER (1938) for “Mycena sect.
Cyanescentes” and MAAS GEESTERANUS (1980),
containing both sections.
Therefore, since both sections form a monophyletic
group, treatment in a single section is feasible and natu-
ralises the section. On the other hand, since all species
within M. sect. Amictae group in a clade within M. sect.
Viscipelles, we propose its consideration as a subsec-
tion, and so it will be termed M. subsect. Amictae:
Mycena subsect. Amictae (Maas Geest.) M. Villa-
rreal, comb. nov. MycoBank: MB 850930
≡Mycena sect. Amictae Maas Geest., Proc. Kon.
Ned. Akad. Wetensch. C. 87(2): 134. 1984. [basionym]
In addition to this, the characteristics described for
M. sect. Viscipelles, as described by MAAS GEESTE-
RANUS (1980), must be modied with the discovery
of M. fragosa, and its afliation to the M. sect. Viscipe-
lles, because this new species presents with a basal disc.
Therefore, we propose the emendation of M. sect. Vis-
cipelles to be dened by the following characteristics:
small to medium-sized carpophores, with pruinose to
minutely puberulous caps, covered or not with a separa-
ble gelatinous lm; an entirely puberulous to tomentose
stipe, with a whitish or bright blue base and, sometimes,
with a small basal disc; the absence of pleurocystidia
and the presence of long and narrow caulocystidia along
the stipe; the presence of short to long cheilocystidia,
which are (i) elongate cylindrical to somewhat fusiform
and smooth to (ii) versiform, covered with compara-
tively few simple exuous excrescences, that may or
may not be embedded in gelatinous matter; ellipsoid or
subglobose amyloid basidiospores. Hyphae of the pilei-
pellis embedded in gelatinous material with or without
pileocystidia. To date, M. sect. Viscipelles seems to be
absent in the Southern Hemisphere.
Artificial key to the species
of Mycena sect. Viscipelles
1. Cheilocystidia cylindrical and smooth .......2
2. Basidiospores subglobose (6-8 × 6-7(-8) μm) ..
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. subcaerulea
2. Basidiospores broadly ellipsoid to ellipsoid ..3
3. Caulocystidia of two types: (a) fusiform or cylin-
drical, 19-40 × 4-8 μm; (b) extremely long, cylindrical
(length > 100 μm) ......... M. caeruleomarginata
3. Caulocystidia of one type, fusiform, subcylindri-
cal to cylindrical (length < 100 μm) .............4
4. Pileus pale grey-brown or pale sepia-brown, so-
metimes with an olivaceous, greenish or bluish-green
shade; margin often bluish-green or rarely dingy citrine
to ochraceous yellow ..................M. amicta
4 Pileus sky blue, greyish blue with age; margin
blue when young, turning bluish-grey when old .....
. . . . . . . . . . . . . . . . . . . . . . . . . . . . M. caeruleogrisea
1. Cheilocystidia different shaped ............5
5. Basidiospores globose to subglobose (6.5-9.5 μm
diam.). Pileus and stipe white to pale grey .........
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. pachyderma
5. Basidiospores ellipsoid or narrowly ellipsoid. Pi-
leus with distinctive brownish colours ...........6
6. Stipe base with distinct blue colouration .....7
6. Stipe base lacking blue colouration .........8
7. Lamellar edge separable as a gelatinous elastic-li-
ke thread; stipe base with blue colouration in the stipe
esh. Stipe generally up to 32 µm in length. Basidios-
pores 3.6-5 μm in diam ............M. cyanorhiza
7. Lamellar edge not separable as a gelatinous elas-
tic-like thread; stipe base colouration due to presence
of blue granules and/or hairs. Stipe generally up to 75
mm in length. Basidiospores 6-7 μm in diam .......
. . . . . . . . . . . . . . . . . . . . . . . . M. pseudocyanorrhiza
8. Presence of basal disc. Basidiospores < 4.5 μm in
diam. and pileipellis with broader terminal cells .....
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. fragosa
8. Absence of basal disc. Basidiospores > 4.5 μm in
diam. and pileipellis without broad terminal cells ...9
9. Basidiospores 7.0-9 × 5.0-6.5 μm. Cheilocystidia
clavate to broadly clavate ............... M. ulmi
9. Basidiospores 6.7-11 × 3.6-5.3 μm. Cheilocysti-
dia fusiform ...................... M. agellata

17
Mycena fragosa (Mycenaceae), a new species from the fragas in nor thwestern Spain
Fungi Iberici
Acknowledgements
We thank Mr. Henri Noguere for making a collection
of Mycena pseudocyanorrhiza available to us. We also
thank Dr. Fernanda Rodríguez Fariña, Unidade de Bio-
loxía Molecular (SAI) Universidade da Coruña, who
assisted with the generation of part of the ITS sequen-
cing data. Finally, we thank Mr. Ben Garden and Dr.
Vasco Fachada for their kind help with the English and
Portuguese grammar, respectively..
References
ALTSCHUL SF, GISH W, MILLER W, MYERS EW
& LIPMAN DJ (1990) Basic local alignment search
tool. J. Mol. Biol. 215: 403-410. DOI: https://doi.
org/10.1016/S0022-2836(05)80360-2
ARONSEN A (2023) The Mycenas of Northern Europe.
Available at: https://mycena.no/. [consulted on 31
July 2023]
ARONSEN A & LÆSSØE T (2016) The Genus Mycena
s.l. Fungi of Northern Europe (Vol. 5). Narayana
Press. Gylling.
COCHARD H, GHYSELINCK D & MOREAU P-A
(2016) Mycena agellata sp. nov. Une nouvelle es-
pèce phylogénétiquement rattachée aux “mycènes
à pied bleu”. Bull. Soc. Mycol. France 132(3-4):
251-259.
DAUNER LAP, KARUNARATHNA SC, TIBPROM-
MA S, XU J & MORTIMER PE (2021) Biolumi-
nescent fungus Roridomyces viridiluminus sp.
nov. and the rst Chinese record of the genus Ro-
ridomyces, from Southwestern China. Phytotaxa
487(3): 233–250. DOI: https://doi.org/10.11646/
phytotaxa.487.3.4
ESTEVE-RAVENTÓS F & VILLARREAL M (2003)
Notas corológicas sobre algunos Agaricales reco-
gidos en el Parque Natural de Sanabria (Zamora),
Valle del Bibei (Zamora, Orense) y Parque Natural
de Montesino (Tras-os Montes, Portugal). Bol. Soc.
Micol. Madrid 27: 174-179.
GARDES M & BRUNS TD (1993) ITS Primers with
Enhanced Specicity for Basidiomycetes—Appli-
cation to the Identification of Mycorrhizae and
Rusts. Molec. Ecol. 2: 113-118. DOI: http://dx.doi.
org/10.1111/j.1365-294X.1993.tb00005.x
HALL TA (1999) BioEdit: A User-Friendly Biological
Sequence Alignment Editor and Analysis Program
for Windows 95/98/NT. Nucleic Acids Symposium
Series 41: 95-98.
KALYAANAMOORTHY S, MINH BQ, WONG TKF,
HAESELER A & JERMIIN LS (2017) Modelnder:
fast model selection for accurate phylogenetic esti-
mates. Nature Methods 14:587–589. DOI: https://
doi.org/10.1038/nmeth.4285
KATOH K & STANDLEY DM (2013) Multiple Se-
quence Alignment Software Version 7: Improve-
ments in Performance and Usability. Mol. Biol.
Evol. 30(4): 772-780. DOI: https://doi.org/10.1093/
molbev/mst010
KÜHNER R (1938) Le Genre Mycena (Fries), Etude
cytologique et systematique des especes d’Europe
et d’Amerique du Nord. Encycl. Mycol. 10: 1-710.
Paul Lechevalier. Paris.
LIU LN, ZHOU G-Y, TAN Z-M & TIAN Y-X (2022)
Two new Mycena (Mycenaceae, Agaricales) spe-
cies with rhizomorphs from subtropical areas of
China. Phytotaxa 576(1): 75-88. DOI: https://doi.
org/10.11646/phytotaxa.576.1.4
LONATI G (1998) Funghi rari o poco conosciuti. Myce-
na pseudocorticola Kühner - Mycena pachyderma
Kühner. Boll. Assoc. Micol. Ecol. Romana 43(1):
3-8.
MAAS GEESTERANUS RA (1980) Studies in Myce-
nas-15, A tentative subdivision of the genus Mycena
in the Northern Hemisphere. Persoonia 11: 93-120.
MAAS GEESTERANUS RA (1984) Conspectus of the
Mycenas of the Northern Hemisphere - 2, Sections
Viscipelles, Amictae, and Supinae. Proc. Kon. Ned.
Akad. Wetensch. C. 87(2): 131-147.
MAAS GEESTERANUS R.A (1992) Mycenas of the
Northern Hemisphere. II Conspectus of the Myce-
nas of the Northern Hemisphere. K. Ned. Akad.
Wet. 493 pp.
MALENÇON G & BERTAULT R (1975) Flore des
Champignons Superieurs du Maroc 2. Institut
Scientique Chérien. Rabat.
MIERSCH J & DÄHNCKE RM (2010) Zur Pilzora
der Kanaren-Insel La Palma. Häuge und bemer-
kenswerte Funde von Hemlingen (Mycena) und
helmlingsähnlichen Arten (Delicatula, Hemimyce-
na, Resinomycena, Roridomyces). Z. Mykol. 76(2):
217-236.
MUNSELL (1994) Soil Color Charts (revised edition).
Macbeth Division of Kollmorgen Instruments Cor-
poration. New Windsor. New York. USA.
NA Q, LIU Z, ZENG H, KE B, SONG Z, CHENG X,
GE Y (2022) Taxonomic studies of bluish Mycena
(Mycenaceae, Agaricales) with two new species
from northern China. MycoKeys 90: 119–145. DOI:
https://doi.org/10.3897/mycokeys.90.78880
NGUYEN L-T, SCHMIDT HA, HAESELER A &
MINH BQ (2015) IQ-TREE: A fast and effective
stochastic algorithm for estimating maximum li-
kelihood phylogenies. Mol. Biol. Evol. 32: 268-274.
DOI: https://doi.org/10.1093/ molbev/msu300
PÉREZ-DE-GREGORIO MÀ (2012) Mycena pseudo-
cyanorrhiza Robich, en la Península Ibérica. Lac-
tarius 21: 31-34.
PERRY BA, KELLER HW, FORRESTER ED & STO-
NE BG (2020) A new corticolous species of My-
cena sect. Viscipelles (Basidiomycota: Agaricales)

18
Villarreal M., Traba J.M., Couceiro A., Naveira H. & Vila-Sanjurjo A.
from the bark of a living american elm tree in Texas,
U.S.A. J. Bot. Res. Inst. Texas 14(2): 167-185. DOI:
https://doi.org/10.17348/jbrit.v14.i2.1000
PLAZA M, VILLARREAL M & MARCOS J (2021)
Primer registro de Mycena discobasis Métrod (Aga-
ricales, Mycenaceae) en Europa. Rev. Soc. Gad.
Hist. Nat. 15: 31-37.
RAMBAUT A (2019) Figtree v 1.4.4. http://tree.bio.
ed.ac.uk/software/gtree/
ROBICH G (2003) Mycena d’Europa. A.M.B. Fonda-
zione Centro Studi Micologici. Trento.
RONQUIST F, TESLENKO M, VAN DER MARK P,
AYRES D, DARLING A, HOHNA S, LARGE B,
LIU L & al. (2012) MrBayes 3.2: efcient Bayesian
phylogenetic inference and model choice across a
large model space. Syst. Biol. 61: 539-554. DOI:
https://doi.org/10.1093/ sysbio/sys029
SÁNCHEZ F & PÉREZ-DE-GREGORIO MÀ (2013)
Mycena pachyderma Kühner, una escurridiza espe-
cie cortícola. Micobotánica-Jaen 13(3): 1-10.
SCHOCH CL, SEIFERT KA, HUHNDORF S. &
SCHINDEL D (2012) Nuclear ribosomal inter-
nal transcribed spacer (ITS) region as a universal
DNA barcode marker for Fungi. Proc. Nat. Acad.
Sci. U.S.A. 109(16): 6241-6246. DOI: https://doi.
org/10.1073/pnas.1117018109
SIQUIER JLL, ESTEVE-RAVENTÓS F, SALOM J &
LLISTOSELLA J (2012) Mycena marocana, trova-
ta a Minorca (Isole Baleari, Spagna), e sue afnità
con M. pachyderma. Rivista Micol. 4: 313-323.
TAMURA K, STECHER G & KUMAR S (2021)
MEGA 11: Molecular Evolutionary Genetics Analy-
sis Version 11. Mol. Biol. Evol. 38(7): 3022-3027.
DOI: http://doi: 10.1093/molbev/msab120
TRIFINOPOULOS J, NGUYEN L-T, VON HAESE-
LER A & MINH BQ (2016) W-IQ-TREE: a fast
online phylogenetic tool for maximum likehood
analysis. Nucleic Acids Res. 44: W232-W235. DOI:
https://doi.org/10.1093/nar/gkw256
WHITE TJ, BRUNS T, LEE S & TAYLOR JW (1990)
Amplication and direct sequencing of fungal ribo-
somal RNA genes for phylogenetics: 315-322. In:
INNIS MA, GELFAND DH, SNINSKY JJ,
WHITE TJ (ed.). PCR protocols: a guide to
methods and applications. Academic Press Inc.,
New York.
VELLINGA EC (1988) Glossary: 54-64. In BAS C,
KUYPER TW, NOORDELOOS ME & VELLIN-
GA EC (ed.). Flora Agaricina Neerlandica 1. AA
Balkema. Rotterdam.