Content uploaded by Fermín Pancorbo
Author content
All content in this area was uploaded by Fermín Pancorbo on Oct 28, 2022
Content may be subject to copyright.
49Bol. Soc. Micol. Madrid 46. 2022
TYPIFICATION OF INOCYBE OBLECTABILIS F. MACROSPORA, AND
ADDITIONAL DATA ON THE PHYLOGENY OF THE TILIAE AND
PSEUDOHIULCA GROUPS IN EUROPE
F. ESTEVE-RAVENTÓS1, E. LARSSON2, F. PANCORBO3 & M. SCHOLLER4
1Departamento Ciencias de la Vida (Botánica), Facultad de Ciencias,
Universidad de Alcalá, Alcalá de Henares, 28805 Madrid, España
fernando.esteve@uah.com
2Biological and Environmental Sciences, University of Gothenburg and Gothenburg Global Biodiversity
Centre, Box 461, SE 40530 Göteborg, Sverige
ellen.larsson@bioenv.gu.se
3Sociedad Micológica de Madrid, Real Jardín Botánico,
c/ Claudio Moyano, 1, 28014 Madrid, España
fpancorbo@gmail.com
4Staatliches Museum für Naturkunde Karlsruhe,
Erbprizenstr. 13, F-76133 Karlsruhe, Deutschland
markus.scholler@smnk.de
Summary: ESTEVE-RAVENTÓS, F., E. LARSSON, F. PANCORBO & M. SCHOLLER (2022). Typication
of Inocybe oblectabilis f. macrospora, and additional data on the phylogeny of the Tiliae and Pseudohiulca
groups in Europe. Bol. Soc. Micol. Madrid 46: 49-63.
A lectotype of Inocybe oblectabilis f. macrospora (=I. robertii) is designated on the basis of original material of
Kühner from the Herbarium of Genève (G). Due to the failure to obtain ITS sequence data from it, an epitype
for this name is designated on the basis of a specimen with similar characters from the Gothenburg Herbarium
(GB). A phylogenetic analysis is undertaken to determine the taxonomic position of this taxon, which is inclu-
ded in what we provisionally have named the Tiliae/Dunensis/Pseudohiulca group (I. sect. Marginatae). The
diagnostic characters of the European species that belong in this clade (except those of the Dunensis group or
subclade) are discussed. The new combination I. tenuiorparietalis (=I. piceae var. tenuiorparietalis) is made
and I. pachycaulis is shown to be a synonym of I. tiliae. Inocybe pseudohiulca, I. piceae and I. kohistanensis
are shown to form an unresolved complex of species, based on ITS data from the type specimens and other
available sequence data. A lectotype is designated for I. pseudohiulca.
Key words: Molecular systematics, taxonomy, Agaricales, Inocybaceae, Europe, Type revision, typication.
Resumen: ESTEVE-RAVENTÓS, F., E. LARSSON, F. PANCORBO & M. SCHOLLER (2022). Tipicación
de Inocybe oblectabilis f. macrospora, y datos adicionales sobre la logenia de los grupos Tiliae y Pseudohiulca
en Europa. Bol. Soc. Micol. Madrid 46: 49-63.
Se designa válidamente un lectotipo para Inocybe oblectabilis f. macrospora (=I. robertii) sobre la base de
material original de Kühner procedente del Herbario de Ginebra (G). Debido a la imposibilidad de obtener
datos de la secuencia ITS de la misma, se designa también un epitipo para este nombre sobre la base de un
ejemplar con caracteres morfológicos y hábitat semejantes procedente del Herbario de Gothenburg (GB). Se
realiza un análisis logenético para certicar la posición taxonómica de este taxón, que queda incluido en lo
que provisionalmente denominamos grupo Tiliae/Dunensis/Pseudohiulca (I. sect. Marginatae). Se discuten
los caracteres diagnósticos de las diferentes especies europeas que componen este clado (excepto las del grupo
o subclado Dunensis). Se realiza la nueva combinación I. tenuiorparietalis (=I. piceae var. tenuiorparietalis)
y se propone I. pachycaulis como sinónimo de I. tiliae. Se muestra que Inocybe pseudohiulca, I. piceae e I.
kohistanensis forman un complejo de especies sin resolver, basándose en los datos de ITS de los especímenes
tipo y otras secuencias disponibles. Se designa un lectotipo para I. pseudohiulca.
Palabras clave: Sistemática molecular, taxonomía, Agaricales, Inocybaceae, Europa, revisión de tipos, tipi-
cación.
MICOLOGICA madrid 2022.indd 49 3/10/22 10:49
Bol. Soc. Micol. Madrid 46. 202250
F. ESTEVE-RAVENTÓS, E. LARSSON, F. PANCORBO & M. SCHOLLER
INTRODUCTION
In a previous work on nodulose and angular
spores of the genus Inocybe (Fr.) Fr. in the Com-
munity of La Rioja (ESTEVE-RAVENTÓS &
CABALLERO MORENO, 2009), the name I.
robertii Esteve-Rav. & A. Caball. was proposed
for the taxon described by KÜHNER (1933) as I.
oblectabilis f. macrospora. Most of the type spec-
imens of the prestigious French mycologist are
deposited in the Herbarium of Genève (G), and a
good number of them have already been studied or
reviewed both morphologically and molecularly by
several researchers. Regarding this taxon, in 2011
we decided to study the type material, of which
the only specimen with this epithet (G00111215,
Kühner “ebenda”) was sent to us; the morpholog-
ical data of this specimen are in perfect agreement
with those expressed by Kühner in the original de-
scription. This single exsiccatum, which undoubt-
edly corresponds to the type material of KÜHNER
(1933), was annotated as a “potential lectotype” in
the herbarium label (see Fig. 1). This ineffective
lectotypication was probably made by J. Poirier
(undated), although corroborating data do not ap-
pear on the label or in the information accompany-
ing the voucher.
The intention in this work is to publish an ef-
fective lectotypication of I. oblectabilis f. macro-
spora with this specimen. The different molecular
studies carried out on it, were unsuccessful in ob-
taining ITS sequence data for comparison. For this
reason, an epitype is here designated from several
collections of which sequences are available from
different European herbaria, gathered in similar
habitats and geographical regions. In this way, the
aim is to stabilize the taxonomic interpretation of
this taxon. All the collections studied coincide with
Kühner’s original description, both in morphologi-
cal and ecological aspects.
ESTEVE-RAVENTÓS & CABALLERO
MORENO (2009), in view of the morphological
evidence observed with respect to I. oblectabilis
(Britzelm.) Sacc. (M!), made the corresponding
combination at species level and gave the name I.
robertii (in honour of Robert Kühner), since the
epithet I. macrospora was already used for another
taxon. In their comments, they argued that this spe-
cies cannot be considered a synonym of I. piceae
Stangl & Schwöbel nor of I. pseudohiulca Küh-
ner, an opinion supported after the study that was
made of the type material of both taxa (M!, G!).
Inocybe pseudohiulca and I. piceae are two closely
related species that occur in Picea forests or stands
in central Europe (KÜHNER, 1933; STANGL &
SCHWÖBEL, 1985). This habitat is not related to
the mesophilous deciduous forests in continental
Europe, as is the case with I. oblectabilis f. macro-
spora (Fig. 2). In addition, there are other notable
differences in morphological and phylogenetical
aspects, and the results of the phylogenetic anal-
ysis presented here (see Fig. 3) support this con-
sideration.
Phylogenetically, I. robertii is not closely relat-
ed to I. oblectabilis, but it is included in what we
provisionally have named the Tiliae clade, a group
of species that share a robust appearance and large
heterodiametric spores. This clade or phyloge-
netic group was already presented in the study by
FRANCHI & al. (2016: 119), when the species I.
tiliae Franchi, M. Marchetti & Papetti was pro-
posed as new. The Tiliae clade includes a number
of species in Europe whose taxonomic characters
will be discussed below.
MATERIALS AND METHODS
Morphological methods.
Preparations of cystidia, basidia and basidio-
spores were mounted both in water and 5% ammo-
nium hydroxide and observed by light microscopy
with the aid of an oil immersion objective. Draw-
ings were made with the aid of a Zeiss drawing tube
under an oil-immersion objective and then con-
verted to vectors with Adobe Illustrator. Scanning
electron micrographs (SEM) were obtained with a
Zeiss DSM-950, applying the critical point tech-
nique described in MORENO & OLTRA (2010).
Measurements of microscopic elements were
aggregated in a spreadsheet that automatically
computes the 5% and 95% percentiles, minimum,
MICOLOGICA madrid 2022.indd 50 3/10/22 10:49
51Bol. Soc. Micol. Madrid 46. 2022
TYPIFICATION OF INOCYBE OBLECTABILIS F. MACROSPORA, AND ADDITIONAL DATA ON THE PHYLOGENY OF
THE TILIAE AND PSEUDOHIULCA GROUPS IN EUROPE
Fig. 1.- Lectotype of Inocybe oblectabilis f. macrospora G00111215. a. Spores. b. Caulocystidia. c. Pleurocystidia. d. Cheilocystidia. Scale: 10 µm.
MICOLOGICA madrid 2022.indd 51 3/10/22 10:49
Bol. Soc. Micol. Madrid 46. 202252
F. ESTEVE-RAVENTÓS, E. LARSSON, F. PANCORBO & M. SCHOLLER
Fig. 2.- Avenue de la Dame
Blanche at the beginning of
the 20th Century (Unknown
author, public domain, via
Wikimedia Commons). Type
locality of I. oblectabilis f.
macrospora.
Fig. 3.- Phylogram obtained based on ITS and LSU data showing the position of and relationships among taxa in focus of this study. Bootstrap
support values are indicated on branches. Type specimens are marked in bold. The country of origin is abbreviated by ISO Alpha-2 codes.
MICOLOGICA madrid 2022.indd 52 3/10/22 10:49
53Bol. Soc. Micol. Madrid 46. 2022
TYPIFICATION OF INOCYBE OBLECTABILIS F. MACROSPORA, AND ADDITIONAL DATA ON THE PHYLOGENY OF
THE TILIAE AND PSEUDOHIULCA GROUPS IN EUROPE
maximum, mean (avg), median (M) and standard
deviation (SD) for each parameter: [(min-)5%-avg-
95%(-max)]. Spores measurements include the
knobs. Cystidia were measured without crystals
and basidia without sterigmata.
The material has been deposited in the herbar-
ium of the University of Alcalá (AH), University
of Gothenburg (GB) and Staatliches Museum für
Naturkunde, Karlsruhe (KR). The type collections
studied were sent on loan from the Herbaria of the
Conservatoire et Jardin Botaniques de Genève (G)
and Staatliche Naturwissenschaftliche Sammlun-
gen Bayerns (SNSB), München (M).
Molecular methods.
At the University of Gothenburg the sequenc-
es for the complete ITS region and about 1,200
base pairs (bp) of the 5´end of the LSU (28S) of
the nuclear ribosomal DNA were generated using
the primers ITS1F (GARDES & BRUNS, 1993)
and LR21, LR0R, and LR7 (HOPPLE & VILGA-
LYS, 1999). Genomic DNA was extracted from the
specimens using the DNeasy Plant Mini Kit (Qia-
gen, Hilden) and PCR reactions were performed
with Illustra PuReTaq Ready to go PCR beads (GE
Healthcare, Buckinghamshire) using 0.5 μM of
each primer and 1–3μL of the DNA extracts. PCR
clean-up was carried out using the QIAquick PCR
purication kit (Qiagen, Hilden). Sequences were
generated by Macrogen Europe (Amsterdam, the
Netherlands) using primers ITS1, ITS4 (WHITE &
al., 1990), Ctb6 (https://nature.berkeley.edu/brun-
slab/) and LR5 and LR3R (HOPPLE & VILGA-
LYS, 1999). At the University of Alcalá de Henares
the DNA extractions were performed from dried
material using a NZY Plant/Fungi gDNA Isolation
Kit (NZYTech, Lisboa, Portugal). The amplied
regions were the ITS rDNA, which was ampli-
ed using primers ITS5 and ITS4 (WHITE & al.,
1990), and LSU rDNA, which was amplied using
primers LR0R and LR5 (VILGALYS & HESTER,
1990). Amplicons were sequenced at the Molecular
Biology Unit (Faculty of Environmental Scienc-
es, University of Alcalá de Henares) using primers
ITS2, ITS3, ITS4 and ITS5 (WHITE & al., 1990)
for ITS region; and primers LR0R, LR5 (VILGA-
LYS & HESTER, 1990) and LR21 (HOPPLE &
VILGALYS, 1994) for LSU. For old type speci-
mens the ITS1 and ITS2 regions were amplied
separately using primers ITS1F-ITS2 and ITS3-
ITS4 respectively (WHITE & al., 1990; GARDES
& BRUNS, 1993). PCR reactions and clean up fol-
lowed the methods described above. Primers used
for sequencing were ITS1, ITS2, ITS3 and ITS4
(WHITE & al., 1990).
Sequences were edited and assembled using Se-
quencher 5.1 (Gene Codes, Ann Arbor, Michigan)
or Geneious 5.1.7 (https://www.geneious.com).
Molecular analyses.
Data on sequences used in the phylogenetic
analyses and newly generated ITS and LSU se-
quence data for this work is presented in Table 1.
The ITS of the target species in this study were
blasted in GenBank (CLARK & al., 2016) and the
UNITE database (KÕLJALG & al., 2020) to seek
additional available sequence data. Inocybe gram-
mata Quél. was used for rooting of trees.
Alignment of the ITS and 28S region data set
was performed using the L-INS-i strategy imple-
mented in mafft 7.017 (KATOH & STANDLEY,
2013) and the alignment was adjusted using al-
iview 1.17.1 (LARSSON, 2014). For inference of
phylogenetic relationships of the dataset, heuristic
searches for the most parsimonious trees were per-
formed using PAUP* v. 4.0a under the maximum
parsimony (MP) criterion (SWOFFORD, 2003).
All transformations were considered unordered and
equally weighted. Variable regions with ambiguous
alignment were excluded, and gaps were treated
as missing data. Heuristic searches with 1000 ran-
dom-addition sequence replicates and TBR branch
swapping were performed. Relative robustness of
clades was assessed by the bootstrap (BT) method
using 1000 heuristic search replicates with 100 ran-
dom taxon addition sequence replicates and TBR
branch swapping.
The aligned ITS-LSU dataset consisted of 33
taxa and 2280 characters. After exclusion of am-
biguous data, mainly from the beginning and the
end of the dataset, 2177 characters remained for
the analysis. Of these, 1547 were constant, 24
MICOLOGICA madrid 2022.indd 53 3/10/22 10:49
Bol. Soc. Micol. Madrid 46. 202254
F. ESTEVE-RAVENTÓS, E. LARSSON, F. PANCORBO & M. SCHOLLER
were variable but parsimony uninformative, and
606 were parsimony informative. The MP analy-
sis yielded 2476 equally most parsimonious trees
(length = 878 steps, CI = 0.9100, and RI = 0.9687).
One of these trees is presented in Fig. 3. Bootstrap
analysis recovered 5 strongly supported terminal
clades within the ingroup, corresponding to I. pall-
ida/nobilis (96%), I. tenuiorparietalis (99%), I. til-
iae (98%), I. robertii (100%), and I. pseudohiulca/
piceae/kohistanensis (100%). No internal structure
was supported within the I. pseudohiulca/piceae/
kohistanensis clade, except for the three sequenc-
es of I. kohistanensis that received a BT support
of 85%, however the low number of substitution
differences between the three species and short
branch length suggests that they consist of species
complex. A BT value greater that 70% is considered
strong.
Table 1.
Data of sequenced collections used in this study; newly sequenced collections are marked in bold
Species Voucher Country Habitat ITS LSU
I. decemgibbosa JV8115 France Quercus KT958905 KT958905
I. decemgibbosa (as I. calida) BJ910802 Sweden Quercus AM882900 AM882900
I. grammata EL190-06 Sweden Betula, Salix KT958933 KT958933
I. grammata EL102B-06 Sweden Betula FN550885 FN550885
I. kohistanensis (isotype) LAH35002 Pakistan Cedrus deodara KP316244
I. kohistanensis (type) LAH35001 Pakistan Cedrus deodara KP316243
(NR_153155)
I. kohistanensis LAH35003 Pakistan Cedrus deodara KP316245
I. nobilis (Inocybe nobilis s.
Franchi et al.) Rebaudengo
27.10.1982 Italy Pinus pinaster?KX592683
I. oblectabilis JV32005 Italy Quercus ilex ON754370 ON754370
I. oblectabilis (epitype) M0222614 Germany Quercus, Ulmus,
Acer and Salix KM873365
(NR_153147)
I. pallida s. auct. MCVE 21493 Italy Quercus ilex subsp.
ilex JF908198
I. piceae EL215 08 Slovakia Picea abies ON754366 ON754366
I. piceae (holotype) M0138785 Germany Picea abies KX592682
I. piceae (paratype) M0021644 Germany Picea abies KX619421
I. pseudohiulca (lectotype) G053288 France Picea ON845750
I. pseudohiulca AH25452 Switzerland Picea abies ON809536
I. pseudohiulca AH48229 Spain Pinus uncinata ON809537 ON809537
I. pseudohiulca EL266-13 Sweden Salix, Dryas ON844187 ON844187
I. pseudohiulca OTU612 Switzerland Soil, alpine MT095731
I. robertii AH 24656 Spain Quercus robur and
Populus sp. ON754364 ON754364
MICOLOGICA madrid 2022.indd 54 3/10/22 10:49
55Bol. Soc. Micol. Madrid 46. 2022
TYPIFICATION OF INOCYBE OBLECTABILIS F. MACROSPORA, AND ADDITIONAL DATA ON THE PHYLOGENY OF
THE TILIAE AND PSEUDOHIULCA GROUPS IN EUROPE
TAXONOMY
Inocybe oblectabilis f. macrospora Kühner,
Bull. Soc. Mycol. France 49: 118. 1933. MycoBank
no.: 444996.
≡ I. robertii Esteve-Rav. & A. Caball., Fungi
non Delineati, Raro vel Haud Perspecte et Ex-
plorate Descripti aut Denite Picti 47: 95. 2009.
MycoBank no.: 548444. [“replacement name for
I. oblectabilis f. macrospora”].
Material studied: FRANCE: Grand Est:
Meuse, entre Nogent et Fontenay, au bord de l’Av-
enue de la Dame Blanche, among grass in urban
spot, 3–VIII–1931, leg. R. Kühner, Kühner “eben-
da” - G00111215 (lectotypus designated here!;
MBT: 10007880). Fig. 1. SWEDEN: Gotland:
Lärbro, in open deciduous forest near Tilia corda-
ta, in calcareous soil, 23–IX–1999, leg. I. Anders-
son, GB-0125088 (epitypus designated here!, that
supports the lectotype designated above; MBT:
10007881), ITS/LSU GenBank AM882979. Fig. 4.
Species description
Macroscopic description of the lectotype (See
KÜHNER, 1933: 118–119)
Microscopic description of the lectotype (Fig.
1)
Species Voucher Country Habitat ITS LSU
I. robertii AH 45182 Spain Quercus faginea
and Pinus
halepensis
ON754365 ON754365
I. robertii (as I. aff.
oblectabilis) (epitype) GB-0125088 Sweden Quercus robur AM882979 AM882979
I. robertii (as I. cf. oblectabilis) KRM0044689 Germany Quercus robur MT005874
I. robertii (as I. cf. oblectabilis) KRM0044734 Germany Quercus robur MT005876
I. tenuiorparietalis EL408-17 Sweden Conifer forest with
Helianthemum ON754369 ON754369
I. tenuiorparietalis (as I.
decipiens) GB-0125094 Sweden Betula, Pinus AM882832 AM882832
I. tenuiorparietalis (as I.
decipienss) GB-0125095 Sweden Betula pubescens AM882831 AM882831
I. tenuiorparietalis (as I.
decipienss) GB-0125088 Sweden Betula, Corylus AM882833 AM882833
I. tenuiorparietalis (as I.
piceae var. tenuiorparietalis,
holotype)
M0216705 Sweden Pinus sylvestris ON754368 ON754368
I. tiliae EL257 17 Sweden
Deciduous forest
with Quercus,
Corylus, Tilia,
Betula
ON754367 ON754367
I. tiliae (as I. pachycaulis
holotype) M0216741 Italy Quercus suber and
Pinus sp. ON754371
I. tiliae (holotype) AMB n. 18103 Italy Tilia KX592681
MICOLOGICA madrid 2022.indd 55 3/10/22 10:49
Bol. Soc. Micol. Madrid 46. 202256
F. ESTEVE-RAVENTÓS, E. LARSSON, F. PANCORBO & M. SCHOLLER
Fig. 4.- Epitype of Inocybe robertii GB-0125088. a. Spores. b. Caulocystidia. c. Pleurocystidia. d. Cheilocystidia. Scale: 10 µm.
MICOLOGICA madrid 2022.indd 56 3/10/22 10:49
57Bol. Soc. Micol. Madrid 46. 2022
TYPIFICATION OF INOCYBE OBLECTABILIS F. MACROSPORA, AND ADDITIONAL DATA ON THE PHYLOGENY OF
THE TILIAE AND PSEUDOHIULCA GROUPS IN EUROPE
Basidiospores nodulose, ochraceous, provided
with numerous (11–18) knobs, –2.4 µm in height,
heterodiametric, (9.5–)10–11–12(–12.5) × (7–)7–
7.8–8.8(–9) µm. Q = (1.16–)1.28–1.4–1.57(–1.64)
(21 spores). Basida hyaline, claviform, 4-spored.
Lamellar edge more or less concolorous. Cheilo-
cystidia 49.4–60 × 19.9–21.2 µm, abundant, well
differentiated, hyaline, crystalliferous at the apex,
broadly fusiform; paracystidia claviform. Pleuro-
cystidia abundant, similar to cheilocystidia, 55.2–
70 × 16.6–24 µm, hyaline or showing a glaucous
intracellular content, thick walled (2–3) µm, hard-
ly yellowing or very pale yellowish in ammonia
media. Caulocystidia morphologically similar to
cheilocystidia 42.2–63.3 × 15.4–18 µm, sometimes
showing a pale brownish intracellular content, crys-
talliferous, abundant in the upper half of the stipe,
mixed with paracaulocystidia hyaline and clavi-
form. Clamps present.
Microscopic description of the epitype (Fig.
4)
Basidiospores distinctly nodulose, ochraceous,
provided with numerous (11–18) knobs, –2.4 µm
in height, with prominent apicula, heterodiametric,
(9.5–)9.8–10.4–11(–11) × (6.8–)7.1–7.6–8.4(–9)
µm. Q = (1.16–)1.25–1.3–1.48(–1.5) (16 spores).
Basidia hyaline, claviform, 4-spored. Lamel-
lar edge concolorous. Cheilocystidia 50.2–62.3
× 12.3–22.7 µm, abundant, protruding, hyaline,
crystalliferous at the apex, broadly fusiform to
subcylindrical; paracystidia not abundant, hyaline,
similar in shape and size to basidia. Pleurocystid-
ia abundant, similar to cheilocystidia, (52–)52.6–
60.5–67(–69.7) × (15.1–)15.6–18.9–24.1(–30)
µm, often showing a refringent, oily and glaucous
content, thick walled (2–3.5) µm, hardly reacting
to ammonia or very pale yellowish. Caulocystid-
ia abundant, hyaline, morphologically similar to
cheilocystidia (48.5–)49.9–57.5–67.6(–70.3) ×
(15–)16–18.1–20.4(–21) µm, sometimes showing
a brownish vacuolar content, crystalliferous, abun-
dant and present all over the stipe surface, mixed
with numerous hyalines, clavate paracaulocystidia.
Clamps present.
Habitat and distribution: Inocybe robertii is
most probably a widely distributed taxon in conti-
nental Europe, although it can be found more rare-
ly in very humid western Mediterranean environ-
ments (AH 45182 was found in a Quercus faginea
forest); these collections (including the types) come
from deciduous Quercus forests on calcareous soils
and also from urban areas under oaks (Q. robur)
and lime trees (Tilia sp.). It is distributed through-
out Europe in these own habitats, although we only
know from certainty records from Germany, Spain
and Sweden.
Additional material studied: GERMANY:
Baden-Württenberg: Karlsruhe, Downtown area,
Stadtgarten, S. Stadtgartensee, an der Brücke near
Quercus robur, 9–VIII–2016, leg. T. Bernauer, KR-
M-0074689 (ITS GenBank MT005874). Ibidem,
same habitat, 21–IX–2016, leg. A. Schneider, KR-
M-0044734 (ITS GenBank MT005876). SPAIN:
Andalucía: Jaén, Santiago-Pontones, La Quebra-
da, 38.00881317, -2.87697472, 697 m, in mixed
forest of Quercus faginea and Pinus halepensis, in
calcareous soil, 13–XI–2013, leg. J.F. Mateos & F.
Pancorbo, AH 45182 (MERINO & al., 2015). País
Vasco: Vizcaya, La Calera del Prado, 13–X–1998,
in calcareous soil under Quercus robur and Populus
sp., leg. F. Esteve-Raventós, AH 24656.
DISCUSSION
Inocybe robertii is part of a group of phyloge-
netically related species which we refer temporarily
as the Tiliae clade, a group including approximately
four to six species in Europe (Fig. 3). In a tradi-
tional taxonomic classication as that proposed by
BON (1998), they are part of I. (sect. Marginatae
Kühner) subsection Oblectabiles Bon. For BON
(loc. cit.), the subsection Oblectabiles includes
those species with nodulose to subangular spores,
caulocystidia present at least on the upper 2/3 of
the stipe, generally clearly bulbous (more rarely
subbulbous to claviform, sometimes marginate),
and with a more or less marked pinkish hue. The
phylogenetic analysis presented here (see Fig. 3)
MICOLOGICA madrid 2022.indd 57 3/10/22 10:49
Bol. Soc. Micol. Madrid 46. 202258
F. ESTEVE-RAVENTÓS, E. LARSSON, F. PANCORBO & M. SCHOLLER
groups in the Tiliae clade species which have in
common robust fructications (stipe reaching at
least 1 cm in thickness), pileus ranging from pal-
lid ochraceous to buff yellowish or even chestnut
brown color, pink coloring in the stipe, and large
heterodiametric spores, with an average size ex-
ceeding 10 µm in length.
The Tiliae and Piceae clades were already
highlighted in the phylogenetic tree presented by
FRANCHI & al. (2016: 119), being paraphyletic of
the Dunensis clade. The last includes strictly sabuli-
colous species with heterodiametric spores, but not
showing distinct pink to reddish tones in the stipe;
for this reason, BON (loc. cit.) considered I. dunen-
sis P.D. Orton in the subsection Praetervisae Bon.
The phylogenetic tree generated in this work
(Fig. 3), shows that I. robertii (= I. oblectabilis f.
macrospora) is related to the species discussed be-
low, all belonging to the Tiliae and Piceae clades:
1) Inocybe tiliae: this is a recently described
species, which has probably been confused before
with I. oblectabilis. It was originally found in north-
ern Italy (Brescia) in urban areas under lime trees
(Tilia sp.), and is characterized (see FRANCHI &
al., 2016) by the pinkish tones manifested on the
stipe and, above all, for its heterodiametric spores
(Spavg: 10 × 7.4 µm) with 10–15 well-marked and
high knobs [1.2–2(–3) µm]; its cystidia tend to be
subcylindrical to narrowly fusiform, 11–15 µm in
width, are not distinctly ventricose and sometimes
show an elongated neck; this shape differs from the
mostly broadly ventricose in I. robertii. The Italian
authors indicate an urban habitat under lime trees,
but we have studied several collections from hu-
mid Mediterranean and continental environments
whose sequences match I. tiliae, and which were
found in Quercus or deciduous mixed forests in
natural habitats.
In addition, the sequence that we have obtained
from the ITS region of the holotype of I. pachy-
caulis E. Ludw. (M!) was found to be identical to
that of I. tiliae. Inocybe pachycaulis was described
by LUDWIG (2017: 474), from material from Sar-
dinia (Italy), from mixed forest of Quercus suber
and Pinus sp. It differs by a marked pink color on
its voluminous stipe and spores similar to I. tili-
ae (Spavg: 10. 4 × 7.5), with numerous and well-
marked knobs; the cystidia in the holotype are
somewhat longer than those reported for I. tiliae
(55–65 × 15–17 µm) but show a similar morpholo-
gy, with a clear tendency to be narrowly fusiform,
more rarely sublageniform.
2) Inocybe nobilis (R. Heim) Alessio s.
Franchi & al.: in the same work by FRANCHI &
al. (2016), the study of a collection deposited in
the Herbarium of E. Rebaudengo (Ceva, Italy), de-
termined by C.L. Alessio as I. nobilis (R. Heim)
Alessio, is presented. From this collection (H. Re-
baudengo, 27.10.1982), there is an ITS sequence
(GenBank KX592683), from Marina di Bibbona
(Livorno, Italy) in mediterranean coastal forest,
probably under maritime pines. This collection
does not correspond to and is later than the one
originally mentioned in the work of ALESSIO &
REBAUDENGO (1980: 328, Pamparato, Castello),
and for which no material seems to exist. HEIM’s
(1931) interpretation of I. brosa var. nobilis R.
Heim corresponds to an enigmatic taxon, very pale,
with claviform to subbulbous stipe, of a high mon-
tane environment under conifers. The description
of ALESSIO & REBAUDENGO (loc. cit.) is not
accompanied by habitat data clearly supporting its
taxonomic correspondence. The iconography of
this work (Tab. XC) suggests that it could corre-
spond or be related to I. oblectabilis, because of
the brownish-pink shades of the basidiomata and
the presence of a clearly marginate bulb. However,
there are many doubts about a correct taxonomic
identity.
Consequently, we think that the interpretation
of FRANCHI & al. (1996) of I. nobilis does not
clearly correspond to the enigmatic interpretation
of HEIM (1931) nor to the “confusing” interpreta-
tion of ALESSIO & REBAUDENGO (1980). Our
opinion is that this taxon needs a taxonomically
clarifying status and since it is supported by sev-
eral molecular sequences, it should be described
as a new species if it were not con-specic with I.
pallida Velen.
3) Inocybe pallida Velen. s. E. Bizio & Mar-
chetti: there is a sequence in GenBank (JF908198),
MICOLOGICA madrid 2022.indd 58 3/10/22 10:49
59Bol. Soc. Micol. Madrid 46. 2022
TYPIFICATION OF INOCYBE OBLECTABILIS F. MACROSPORA, AND ADDITIONAL DATA ON THE PHYLOGENY OF
THE TILIAE AND PSEUDOHIULCA GROUPS IN EUROPE
determined as I. pallida, which is deposited in the
Venice Herbarium (MCVE no. 21493, Loc.: Finale
Ligure, Italy, Det. E. Bizio & M. Marchetti, under
Quercus ilex subsp. ilex, E. Bizio pers. comm.). Its
ITS in BLAST is practically the same or very close
(98.95 %) to the sequence of I. nobilis s. Franchi
& al. (KX592683). These data support the exist-
ence and preference of this taxon for sandy soils
in mediterranean environments, especially coastal
ones. Both I. nobilis s. Franchi & al. and I. pallida
s. Bizio & Marchetti, seem to represent small ge-
netic variations of the same species. Moreover, they
coincide in their genetic characters with several
collections we have studied from ibero-mediterra-
nean areas. Regardless of the preferences for sandy
soils, it is characterized by the rather short cystidia
of 45–55(–60) ˟ 15–22(–25) µm, with a marked
claviform to fusiform tendency (see FRANCHI
& al. 2016: 111) and their heterodiametric spores
provided with very obtuse and low (< 1µm) knobs,
sometimes even with a subpolygonal outline. VE-
LENOVSKY’s (1920) original interpretation of I.
pallida does not seem to correspond (stipe 1–2 mm
wide!). Velenovsky’s type material is deposited at
PR-PRC and was studied by KUYPER (1985); this
type material has been preserved in formaldehyde
and unfortunately no DNA is expected to be ob-
tained from it (ESTEVE-RAVENTÓS & al., 2018:
226).
4) Inocybe piceae var. tenuiorparietalis E.
Ludw.: this taxon was described by LUDWIG
(2017) from material collected in Skåne (Sweden)
under conifers. It is here shown to correspond to the
interpretation that some Nordic mycologists have
had of I. decipiens Bres. or of I. oblectabilis. The
ITS sequence was obtained from Ludwig’s holo-
type (M!) and coincides with several sequences
deposited in GenBank. In our phylogeny (Fig. 3)
they form a strongly supported clade, suggesting it
represents an independent species. The holotype of
I. decipiens (S!), which was previously studied by
J. Vauras in 1993, shows different more elongated
and heterodiametric spores, with a polygonal out-
line of soft angles, as well as caulocystidia only
present in the upper third of the stipe that gradually
deteriorate and take on the appearance of caulocys-
tidioid tortuose hairs in the lower half (see PICÓN,
2007: 64). In the absence of a molecular study of
the Bresadola holotype, our hypothesis is that I.
decipiens could belong to a group of strictly sa-
bulicolous species showing polygonal spores and
rareed caulocystidia even in the upper half of the
stipe [e.g. I. paucicystidiosa (Bon) Franchi & M.
Marchetti and I. velata Franchi & M. Marchetti].
Inocybe piceae var. tenuiorparietalis is genetically
closer to I. tiliae and I. robertii than the I. pseudohi-
ulca/piceae/kohistanensis clade (Fig. 3).
Inocybe tenuiorparietalis (E. Ludw.) E. Larss.,
Esteve-Rav. & Pancorbo, comb. nov. MycoBank
no.: 844400. (Fig. 5)
Basionym: Inocybe piceae var. tenuiorparietalis
E. Ludw., Pilzkompendium 4: 487. 2017
5) Inocybe pseudohiulca/I. piceae/I. kohis-
tanensis clade: Inocybe pseudohiulca is a montane
to boreo-alpine species (see KÜHNER, 1933; FA-
VRE, 1955; HORAK, 1987 and BON, 1997)) from
continental Europe, relatively frequent in the Picea
forests of the central region of the continent (spe-
cially the Alps) and also reaching the boreal and
alpine locations with Dryas and dwarf Salix spp. in
calcareous soils (JACOBSON & LARSSON, 2012:
1001). It can be confused with I. piceae, which was
originally described from an urban park (Stadtwald,
aprox. 500 m alt.) in Augsburg (Germany) under
Picea (STANGL & SCHWÖBEL, 1985). The two
are genetically very closely related (Fig. 3). They
differ in the ITS1 by a single 2bp insertion/dele-
tion event, and in the ITS2 by one substitution and
a single 6bp insertion/deletion event, though can
be separated by some morphological characters
(STANGL, 1989). Inocybe piceae shows a more
accentuated brown to chestnut color in the pileus
and stipe, no distinct whitish veil remains on the
pileus of young basidiomata and the spores are
more heterodiametric in outline, with somewhat
more prominent and pointed knobs (M!, G!) (Fig.
6). It is also evident that I. kohistanensis S. Jabeen,
I. Ahmad & A.N. Khalid native to the montane
Cedrus deodara forests of Kohistan, Pakistan, is
MICOLOGICA madrid 2022.indd 59 3/10/22 10:49
Bol. Soc. Micol. Madrid 46. 202260
F. ESTEVE-RAVENTÓS, E. LARSSON, F. PANCORBO & M. SCHOLLER
closely related (Fig. 3) (JABEEN & al., 2016). In-
ocybe kohistanensis differs in morphology from the
previous species, not only in its Asian distribution
and habitat, but also in the spores that according to
the original description, show considerably fewer
number of knobs (8–9). It differs from I. pseudohi-
ulca in the ITS1 region by a single 1bp insertion/
deletion event and one substitution, and in the ITS2
by four substitutions. Based on the phylogenetic
analyses presented here (Fig 3) and by FRANCHI
Fig. 5. Inocybe tenuiorparietalis. Collected on Öland, Klosterholmen (Sweden), 6 October 2017, EL408-17.
Fig. 6. Spores SEM. a. Inocybe piceae (M 0138785 holotype). b. Inocybe pseudohiulca (G053288, lectotype).
MICOLOGICA madrid 2022.indd 60 3/10/22 10:49
61Bol. Soc. Micol. Madrid 46. 2022
TYPIFICATION OF INOCYBE OBLECTABILIS F. MACROSPORA, AND ADDITIONAL DATA ON THE PHYLOGENY OF
THE TILIAE AND PSEUDOHIULCA GROUPS IN EUROPE
& al. (2016), we conclude that the synonymy be-
tween I. robertii and I. piceae, as currently reected
by LUDWIG (2017: 484), in MykoBank (www.
mycobank.org) and Index Fungorum (http://www.
indexfungorum.org/), is incorrect. A probable rea-
son for this confusion is that STANGL & SCHWÖ-
BEL (1985) described I. piceae as a new species
and not as a new name for I. oblectabilis f. macro-
spora, on the incorrect assumption that it was both
a species and a name that would replace the homon-
ymy resulting from the potential combination of I.
oblectabilis f. macrospora. However, the authors
never studied the type material of I. oblectabilis f.
macrospora, a deciduous forest species. We here
clarify the taxonomic status of I. robertii in relation
to I. piceae. However, the I. pseudohiulca/piceae/
kohistanensis clade, where no internal structure
was supported based on available data, except for
the three sequences of I. kohistanensis (BT 85%), is
in need of further attention. The low number of sub-
stitution differences between the three species and
the in comparison seemingly high within species
variation may suggest incomplete lineage sorting
and the clade can be regarded as a species complex.
We designate here as lectotype of I. pseudohiul-
ca, among the syntypes deposited in G (KÜHNER,
1933: 107), the collection G053288 - “Kühner type
p.1”, MBT: 10007882, from which we have ob-
tained the ITS1/ITS2 sequence.
ACKNOWLEDGEMENTS
The authors would like to thank the curators of
the Herbaria G (Genève), GB (Göteborg), KR-M
(Karlsruhe) and M (Munich) for loans of type col-
lections. We would also like to thank Javier Rejos,
curator of the Herbarium AH (Alcalá de Henares),
for his constant help in requesting collections for
study. To Gabriel Moreno and Alberto Altés for
their help in obtaining the SEM photos. Yolanda
Turégano, Francisco Gasulla and Lidia Plaza have
been a very important part in the generation of se-
quences of Inocybe material; likewise, we would
like to thank Enrico Bizio (Venice, Italy) for his
help and positive disposition to collaborate altru-
istically, sending us descriptions and very inform-
ative photos; to Luis Parra for his valuable help
and nomenclatural suggestions. Finally, to Brian
Abrams for his help in the linguistic correction of
the text.
This work was nancially supported by FED-
ER/Ministerio de Ciencia, Innovación y Universi-
dades - Agencia Estatal de Investigación (Spain)/
Project CGL2017-86540-P. The Swedish Tax-
onomy Initiative, SLU Artdatabanken, Uppsala,
Sweden (E. Larsson). The KLIMOPASS Projekt
“Exotische Gehölze und Diversität der Ektomyk-
orrhiza-Pilze im urbanen Grünächenbereich” -
nanced by Landesanstalt füe Umwelt, Messungen
und Naturschutz Baden-Württemberg, Germany
(M. Scholler 2017).
BIBLIOGRAPHY
ALESSIO, C.L. & E. RABAUDENGO (1980). In-
ocybe. Iconographia Mycologica Vol.29, Suppl.
3. Museo Tridentino di Scienze Naturali, Trento.
BON, M. (1997). Clé monographique des Inocy-
bes alpins. Bull. Féder. Mycol. Dauphiné-Savoie
144: 71–109.
BON, M. (1998). Clé monographique du genre
Inocybe (Fr.) Fr. (3ème partie: espèces gibbos-
porées = sous-genre Clypeus Brtiz., = Genre As-
trosporina Schroet). Doc. Mycol. 28(111): 1–45.
CLARK, K., I. KARSCH-MIZRACHI, D.J. LIP-
MAN, J. OSTELL & E. SAYERS (2016).
Genbank. Nucl. Acids Res. 44, https://doi.
org/10.1093/nar/gkv1276. Database issue:
D67–D72.
ESTEVE-RAVENTÓS, F. & A. CABALLERO
MORENO (2009). Especies nuevas e intere-
santes del género Inocybe (Inocybaceae, Basidi-
omycetes) en la Comunidad de La Rioja (España
peninsular). Taxones con esporas nodulosas (I).
Fungi non Delineati XLVII. Edizione Candus-
so, Alassio. 128 p.
ESTEVE-RAVENTÓS, F., D. BANDINI, B.
OERTEL, V. GONZÁLEZ & G. MORENO
(2018). Advances in the knowledge of the Ino-
MICOLOGICA madrid 2022.indd 61 3/10/22 10:49
Bol. Soc. Micol. Madrid 46. 202262
F. ESTEVE-RAVENTÓS, E. LARSSON, F. PANCORBO & M. SCHOLLER
cybe mixtilis group (Inocybaceae, Agaricomy-
cetes), through molecular and morphological
studies. Persoonia 41: 213–236. https://doi.
org/10.3767/persoonia.2018.41.11
FAVRE, J. (1955). Les champignons supérieurs de
la zone alpine du Parc National Suisse. Ergebn.
Wiss. Untersuch. Schweiz. Nationalparks 5:
1–212.
FRANCHI, P., M. MARCHETTI & C. PAPETTI
(2016). Inocybe tiliae, una nuova specie della
Sezione Marginatae. Riv. Micol. 59(2): 99–121.
GARDES M. & BRUNS, T.D. (1993). ITS primers
with enhanced specicity for basidiomycetes -
application to the identication of mycorrhizae
and rusts. Mol. Ecol. 2(2): 113–118. https://doi.
org/10.1111/j.1365-294X.1993.tb00005.x.
HEIM, R. (1931). Le genre Inocybe précédé d’une
introduction general à l’étude des Agarics
Ochrosporés. P. Lechevalier & Fils, Paris. 431
p. + 35 pl.
HOPPLE, J.S. JR. & R. VILGALYS (1999) Phy-
logenetic relationships in the mushroom genus
Coprinus and dark-spored allies based on se-
quence data from the nuclear gene coding for
the large ribosomal subunit RNA: divergent do-
mains, outgroups, and monophyly. Molec. Phy-
logen. Evol. 13: 1–19. https://doi.org/10.1006/
mpev.1999.0634
HORAK, E. (1987). Astrosporina in the alpine
zone of the Swiss National Park (SNP) and ad-
jacent regions. Arctic Alpine Mycol. 2: 205–234.
JABEEN, S., I. AHMAD, A. RASHID & A.N.
KHALID (2016). Inocybe kohistanensis, a new
species from Swat, Pakistan. Turkish J. Bot. 40:
312–318. https://doi.org/10.3906/bot-1501-17
JACOBSON, S. & E. LARSSON (2012). Inocybe
(Fr.) Fr. In: KNUDSEN, H. & J. VESTERHOLT
(eds.), Funga Nordica (2nd ed.): 981–1021.
Nordsvamp, Copenhagen.
KAOTH, K. & STANDLEY, D.M. (2013). MAFFT
multiple sequence alignment software version
7: improvements in performance and usabili-
ty. Molec. Biol. Evol. 30: 772–780. https://doi.
org/10.1093/molbev/mst010
KÕLJALG, U., H.R. NILSSON, D. SCHIGEL,
L. TEDERSOO, K.H. LARSSON, T.W.
MAY, A.F.S. TAYLOR, T.S. JEPPESEN, T.G.
FRØSLEV, B.D. LINDAHL, K. PÕLDMAA,
I. SAAR, A. SUIJA, A. SAVCHENKO, I.
YATSIUK, K. ADOJAAN, F. IVANOV, T.
PIIRMANN, R. PÖHÖNEN, A. ZIRK & K.
ABARENKOV (2020). The Taxon Hypothe-
sis Paradigm-On the Unambiguous Detection
and Communication of Taxa. Microorganisms
8(12): 1910. https://doi.org/10.3390/microor-
ganisms8121910
KÜHNER, R. (1933). Notes sur le genre Inocybe.
Bull. Soc. Mycol. France 49: 81–121.
KUYPER, T.W. (1985). Studies in Inocybe – I. Re-
vision of the new taxa of Inocybe described by
Velenovský. Persoonia 12(4): 375–400.
LARSSON, A. (2014). AliView: a fast and light-
weight alignment viewer and editor for large
data sets. Bioinformatics 22: 3276–3278.
https://doi.org/10.1093/bioinformatics/btu531
LUDWIG, E. (2017). Pilzkompendium 4. Abbil-
dungen. Cortinariaceae I – Cortinarius (part
I), Galerina, Hebeloma, Hebelomina, Inocybe,
Phaeogalera. Fungicon Verlag, Berlin.
MERINO, D., M.A. RIBES, T. ILLESCAS, F.
PANCORBO, S. TELLO, J.F. MATEO, J.C.
CAMPOS, G. SÁNCHEZ & J. CUESTA
(2015). Algunas especies recolectadas con mo-
tivo de las XXI Jornadas de la Confederación
Europea de Micología Mediterránea, CEMM,
organizadas por la Asociación Botánica y Mi-
cológica de Jaén, en noviembre de 2013, en el
Parque Natural de las Sierras de Cazorla, Segura
y Las Villas. Anales XXI Jornadas CEMM 21:
73–92.
MORENO, G. & M. OLTRA (2010). Notas sobre
los géneros Badhamia, Badhamiopsis y Crateri-
um (Myxomycetes) en España. Bol. Soc. Micol.
Madrid 34: 161–197.
PICÓN, R.M. (2007). Macromicetos de las dunas
del litoral cantábrico II. Zizak 4: 51–73.
STANGL, J. (1989). Die Gattung Inocybe in Bay-
ern. Hoppea 46: 5–388
STANGL, J. & H. SCHWÖBEL (1985). Höcker-
ig-sporige Risspilze aus dem Formenkreis der
Inocybe oblectabilis (Britz.) Sacc. 1895. Int. J.
Mycol. Lichenol. 2: 53–74.
MICOLOGICA madrid 2022.indd 62 3/10/22 10:49
63Bol. Soc. Micol. Madrid 46. 2022
TYPIFICATION OF INOCYBE OBLECTABILIS F. MACROSPORA, AND ADDITIONAL DATA ON THE PHYLOGENY OF
THE TILIAE AND PSEUDOHIULCA GROUPS IN EUROPE
SWOFFORD, D.L. (2003). PAUP*. Phylogenetic
analysis using parsimony (* and other meth-
ods). Version 4. Sinauer Associates. Sunderland,
MA.
VELENOVSKÝ, J. (1920). Houby, Vols. 1–2.
Nákladem “Ĉeské Botanické Spoleĉnosti” V.
Praze.
VILGALYS, R., HESTER, M. (1990) Rapid genet-
ic identication and mapping of enzymatically
amplied ribosomal DNA from several Cryp-
tococcus species. J. Bacteriol. 172(8): 4238–
4246. https://doi.org/10.1128/jb.172.8.4238-
4246.1990
WHITE, T.J., T. BRUNS, S. LEE & J. W. TAYLOR
(1990). Amplication and direct sequencing of
fungal ribosomal RNA genes for phylogenetics.
In: INNIS, M.A., D.H. GELFAND, J.J. SNIN-
SKY & T.J. WHITE (eds.), PCR protocols: A
guide to methods and applications: 315–322.
Academic Press, USA.
MICOLOGICA madrid 2022.indd 63 3/10/22 10:49
