Looking for Lepiota psalion Huijser & Vellinga (Agaricales, Agaricaceae)

Abstract

Lepiota psalion is fully described based on a recent collection from Sardinia (Italy) and the holotype. NrITS-and nrLSU-based phylogeny demonstrates that sequences deposited in GenBank as "L. psalion" and generated from two Dutch and one Chinese collections are not conspecific with the holotype and represent two distinct, undescribed species. These species are here proposed as Lepiota recondita sp. nov. and Lepiota sinorecondita ad int.

Full text

Looking for Lepiota psalion Huijser & Vellinga 45
Looking for Lepiota psalion Huijser & Vellinga
(Agaricales, Agaricaceae)
Alfredo Vizzini1,2, Alessia Tatti3, Henk A. Huijser4, Jun F. Liang5, Enrico Ercole1
1 Department of Life Sciences and Systems Biology, University of Torino, Viale P.A. Mattioli 25, I-10125,
Torino, Italy 2 Institute for Sustainable Plant Protection (IPSP)-CNR, Viale P.A. Mattioli 25, I-10125,
Torino, Italy 3 Department of Environmental and Life Science, Section Botany, University of Cagliari, Viale S.
Ignazio 1, I-09123, Cagliari, Italy 4 Frederikstraat 6, 5671 XH Nuenen, e Netherlands 5 Research Institute
of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, 510520, China
Corresponding author: Alfredo Vizzini (alfredo.vizzini@unito.it)
Academic editor: T. Lumbsch |Received21 February 2019| Accepted 11 April 2019| Published 9 May2019
Citation: Vizzini A, Tatti A, Huijser HA, Liang JF, Ercole E (2019) Looking for Lepiota psalion Huijser & Vellinga
(Agaricales, Agaricaceae). MycoKeys 52: 45–69. https://doi.org/10.3897/mycokeys.52.34021
Abstract
Lepiota psalion is fully described based on a recent collection from Sardinia (Italy) and the holotype.
NrITS- and nrLSU-based phylogeny demonstrates that sequences deposited in GenBank as “L. psalion”
and generated from two Dutch and one Chinese collections are not conspecic with the holotype and
represent two distinct, undescribed species. ese species are here proposed as Lepiota recondita sp. nov.
and Lepiota sinorecondita ad int.
Keywords
Agaricomycetes, Basidiomycota, cryptic species, hymeniform pileus covering, taxonomy
Introduction
Recent molecular analyses have indicated that the genus Lepiota (Pers.) Gray is a para-
phyletic assemblage that is monophyletic only if it is considered together with spe-
cies of Cystolepiota Singer, Echinoderma (Locq. ex Bon) Bon, Melanophyllum Velen.,
Copyright Alfredo Vizzini et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
MycoKeys 52: 45–69 (2019)
doi: 10.3897/mycokeys.52.34021
http://mycokeys.pensoft.net
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RESEARCH ARTICLE

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
46
and Pulverolepiota Bon (Johnson 1999; Vellinga 2003, 2004; Vellinga et al. 2011).
Consequently, according to the modern concept of Vellinga (2003, 2004), the ge-
nus Lepiota s.l. includes the pale-spored members of the Agaricaceae Chevall., which
are circumscribed by having non-metachromatic, dextrinoid, and usually binucleate
spores, cheilocystidia usually present, pleurocystidia absent, a regular hymenophoral
trama, and clamp-connections usually present. e structure of the pileus covering has
been shown to be a key character to divide the genus into operative, morphology-based
sections (Vellinga and Huijser 1999; Vellinga 2001, 2003, 2010).
Species of Lepiota with a hymeniform pileus covering were distributed by Bon
(1993) over three dierent sections, Cristatae (Kühner ex Wasser) Bon, Integrellae
(Kühner ex Bon) Bon and Lilaceae Bon, based mainly on dierent spore shapes (either
ellipsoid or spurred) and spore nuclear number (mononucleate vs binucleate); all spe-
cies were included by Vellinga and Huijser (1999) and Vellinga (2001) in an emended
large section Lilaceae.
According to recent molecular analyses, the species with a hymeniform pileus cov-
ering do not form a monophyletic lineage (Vellinga 2003, 2004, 2010; Vizzini et al.
2014a, b; Justo et al. 2015; Qasim et al. 2015; Hosen et al. 2016), even though most
of them (with dierent spore shapes and nuclear number) fall in a clade (named clade
3 by Vellinga 2003) which also includes taxa as L. albogranulosa T. Qasim & A.N.
Khalid, L. cystophoroides Joss. & Riousset, L. luteophylla Sundb., and L. scaberula Vel -
linga with a hymeniderm giving rise to loose globose elements (a transition between
hymeniderm and epithelium, Vellinga 1988).
During a 3-year survey of macrofungi in the Botanical Garden of Cagliari (Sar-
dinia, Italy), a collection of a Lepiota with a hymeniform pileus covering was recorded.
It showed striking morphological anities with L. psalion Huijser & Vellinga. e pre-
sent paper fully describes this collection using morphological features and molecular
data, and infers, through sequencing of the holotype, the phylogenetic placement of
L.psalion. Additionally, two morphologically allied taxa, Lepiota recondita sp. nov. and
L. sinorecondita ad int. are described.
Materials and methods
Morphology
Macroscopic description was based on detailed eld notes of fresh basidiomes. Colour
terms in capital letters (e.g., Pale Cinnamon-Pink, Plate XXIX) are those of Ridgway
(1912). HTML alphanumeric colour codes (https://html-color-codes.info/) were
obtained using GIMP (GNU Image Manipulation Program, https://www.gimp.org/)
with the “Color Picker” tool on photographs taken in natural light of fresh basidiomes.
Micromorphological features were observed on dried material; sections were rehydrated
in water or 5% KOH and mounted separately in ammoniacal Congo Red, Cotton

Looking for Lepiota psalion Huijser & Vellinga 47
Blue, Cresyl Blue, and Melzer’s reagent. Measurements of the microscopic features of
Lepiotapsalion and L. recondita were made by photographing all the elements occurring in
the visual eld of an Optika B-383 PLi light microscope. Measurements were performed
using the Piximètre 5.9 R 1530 software (http://ach.log.free.fr/Piximetre/) at 1000×
magnication. e microphotographs were taken by an Optikam B5, 5 MP× camera.
When possible, dimensions of the microscopic elements are given as: (minimum–)
average minus standard deviation – average plus standard deviation (–maximum) of
length × (minimum–) average minus standard deviation – average plus standard devia-
tion (–maximum) of width. Spore dimensions do not include the hilar appendix. e
width of each basidium was measured at the widest part, and the length was meas-
ured from the apex (sterigmata excluded) to the basal septum. e DNA uorescent
dye 4′,6-diamidino-2-phenyl-indoldihydrochloride (DAPI) was used to stain nuclei in
spores following Horton (2006). e number of nuclei in spores were then determined
using a Leica TCS-SP2 confocal microscope. Samples were excited with 405 nm light
and uorescence was recorded at 440–500 nm. e following abbreviations are used: l
= number of lamellulae between each pair of lamellae reaching the stipe; the notation
[X, Y, Z] indicates that measurements were made on X randomly selected spores (taken
from spore-prints), in Y samples from Z collections; Q = the spore quotient (length/
width ratio); Qav = the average spore quotient. Terminology for descriptive terms is
according to Vellinga (1988, 2001). Herbarium abbreviations follow iers (2019,
continuously updated). Author citations follow the Index Fungorum – Authors of
Fungal Names (http://www.indexfungorum.org/authorsoungalnames.htm).
DNA extraction, PCR amplification and DNA sequencing
Total DNA was extracted from seven dry basidiomes (Tab. 1): two basidiomes (labelled
as “a” and “b”) from the same L. psalion CAG P.11_9/7.68 collection, one basidiome
from the L. psalion holotype (WU 5152), two basidiomes from two collections of the
new species L.recondita, and two basidiomes from two collections of L. sanguineofracta
Vizzini (TO-HG2916, holotype and TO-HG2917). DNA extraction and PCR
amplications were performed as described by Alvarado et al. (2015). Primers ITS1F
and ITS4 (White et al. 1990; Gardes and Bruns 1993) were used for the nrITS
region; primers LR0R and LR5 (Vilgalys and Hester 1990) were used for the nrLSU
(28S) rDNA, and nally EF1-983F and EF1-1567R (Rehner and Buckley 2005) for
the translation elongation factor 1-α (tef1-α) gene. Chromatograms were checked
searching for putative reading errors, and these were corrected. e PCR products were
puried with the Wizard SV Gel and PCR Clean-UP System (Promega) following
manufacturer’s instructions and sequenced forward and reverse by MACROGEN Inc.
(Seoul, Republic of Korea). Sequences were checked and assembled using Geneious v.
5.3 (Drummond et al. 2010) and submitted to GenBank (http://www.ncbi.nlm.nih.
gov/genbank/). Accession numbers are reported in Table 1.

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
48
Table 1. Taxa, vouchers and GenBank accession numbers used in the molecular analyses. Newly se-
quenced collections are in bold.
Species Collection No. Origin GenBank accession No.
nrITS nrLSU
Chamaemyces fracidus .W. Kuyper 960 (L) Belgium AY176343 AY176344
Cystolepiota cystophora MCVE 56163 Italy GQ141550 –
Cystolepiota seminuda 4-X-1989, H.A. Huijser s.n. (herb. Huijser) e Netherlands AY176350 –
MCVE 9247 Italy JF907983 –
Lepiota a. grangei TENN 064380, ECV4063 USA –MF797685
Lepiota acutesquamosa DUKE-JJ177 USA –U85293
Lepiota albogranulosa LAH. NO. 10152012, Holotype Pakistan LK932284 –
LAH. NO. 9992012 Pakistan LK932285 –
Lepiota apatelia 26-IX-1990, H.A. Huijser (herb. Huijser) e Netherlands AY176462 –
04-X-1991, H.A. Huijser (herb. Huijser) e Netherlands GQ203819 –
Lepiota aspera E.C. Vellinga 2233 (L) e Netherlands AY176354 –
GLM 45944 Germany –AY207219
Lepiota bengalensis Iqbal 825 GDGM 45684 Holotype Bangladesh KU563148 KU563150
Iqbal 860 Paratype Bangladesh KU563149 –
Lepiota brunneoincarnata DB4157 Hungary –MK278258
NL-5409 Hungary –MK278260
Lepiota castanea TENN 064371, ECV4016 USA –MF797675
NL-2980 Hungary –MK278259
Lepiota castaneidisca E.C. Vellinga 2594 (UC) USA AF391055 –
E.C. Vellinga 2410 (UC) USA AF391064 –
E.C. Vellinga 2805 (UC) USA GQ203808 –
E.C. Vellinga 2756 (UC) USA GQ203816 –
Lepiota cf. aspera MFLU 09-0061 ailand –HM488788
Lepiota cf. cristata E.C. Vellinga 2515 (UC) USA AF391052 –
E.C. Vellinga 2677 (UCB) USA AY176466 –
E.C. Vellinga 2714 (UC) USA GQ203807 –
Lepiota clypeolaria E.C. Vellinga 1683 (L) Germany AY176361 –
TENN 064372, ECV4003 USA –MF797684
VPI-OKM22029 South Korea –U85291
CBS 146.42 Sweden –MH867601
Lepiota coloratipes 9-X-1991, H.A. Huijser (herb. Huijser) e Netherlands AF391066 –
MCVE 16888 Italy FJ998406 –
Zhu L. Yang 4790 China KC819621 –
Zhu L. Yang 4951 China KC819622 –
SAV F-3212 Spain KC900376 –
SAV F-3213, Holotype Spain KC900377 –
NL-5353 Hungary –MK278270
Lepiota cortinarius NL-1602 Hungary –MK278262
Lepiota cristata 22-IX-1993, H.A. Huijser (herb. Huijser) e Netherlands AF391042 –
20-IX-1989, H.A. Huijser (L) e Netherlands AF391043 –
9-VII-1998, Z.L. Yang 2238 (HKAS) China AF391044 –
8-XII-2000, E.C. Vellinga 2611 (UC) USA AF391045 –
30-I-1993, D.E. Desjardin 5658 (SFSU) USA AF391050 –
24-IX-2000, S. Clark (coll. P.B. Matheny 1958) (WTU) USA AF391051 –
AFTOL-ID 1625, ECV 2449 (UC) USA –DQ457685
E.C. Vellinga 2780 (UC) USA GQ203806 –
E.C. Vellinga 2750 (UC) USA GQ203815 –
DUKE1582 USA –U85292
420526MF0542 China–MH141343
420526MF0550 China –MG712361
Lepiota cristatoides 5-IX-1996, H.A. Huijser s.n. (herb. Huijser) e Netherlands AY176363 –
Lepiota cystophoroides E.C. Vellinga 2142 (L) France AF391031 –
Lepiota erminea NL-3095 Hungary –MK278263

Looking for Lepiota psalion Huijser & Vellinga 49
Species Collection No. Origin GenBank accession No.
nrITS nrLSU
Lepiota felina VPI-OKM20596 USA U85330 U85295
NL-4207 Slovakia –MK278264
Lepiota geogenia MEL 2358504 Australia –JX179270
MEL:2358503 Australia –JX179271
Lepiota griseovirens MCVE 13747 Italy FJ998403 –
Lepiota hymenoderma E.C. Vellinga 2017 (L) e Netherlands AF391083 –
Lepiota laevigata FP2012-11-02 Hungary –MK278266
Lepiota lilacea E.C. Vellinga 2451 (UCB) USA AY176379 –
E. Brown (coll. E.C. Vellinga 1873) (L) United Kingdom GQ203820 –
Lepiota luteophylla H.V. Smith 284 (MICH) USA AY176475 –
Lepiota maculans TENN 064381 USA –HQ832458
Lepiota mandarina HKAS 50028 China –KM214816
Lepiota neophana E.C. Vellinga 2602 (UCB) USA AY176492 –
E.C. Vellinga 3947 (UC) USA GQ203812 –
rh24 08/27/07 (ISC) USA GQ375546 –
rh39 08/11/07 (ISC) USA GQ375547 –
E.C. Vellinga ecv3955 (UC) USA –HM488785
Lepiota ochraceofulva E.C. Vellinga 2267) (L) e Netherlands AF391032 –
E.C. Vellinga 2273 (L) e Netherlands AY176386 –
Lepiota ochraceofulva NL-2973 Hungary –MK278267
Lepiota ochraceoumbonata Murhula Cizungu 39 Gabon –MK278268
Lepiota oreadiformis FO 46679 Germany –AF291344
Lepiota phaeoderma E.C. Vellinga 3000 (UC) USA GQ203810 –
Lepiota psalion WU 5152 Holotype AUSTRIA MG581687 MG581699
Lepiota psalion
basidiome a
CAG P.11_9/7.68 Italy MG581688 –
Lepiota psalion
basidiome b
CAG P.11_9/7.68 Italy MG581689 MG581700
Lepiota psalion (L.
recondita)
15-IX-1999, H.A. Huijser (herb. Huijser) hah6153 e Netherlands AY176390 –
3-VIII-1999, H.A. Huijser s.n. (herb. Huijser) e Netherlands –AY176391
H.A. Huijser (herb. Huijser) hah6177 e Netherlands GQ203823 –
Lepiota psalion (L.
sinorecondita ad interim)
HMJAU3799 China GU199362 GU199355
Lepiota pseudohelveola GLM 45945 Germany –AY207220
Lepiota pyrochroa E.C. Vellinga 2006 (L) e Netherlands AY176477 –
Lepiota recondita TR gmb 01481, paratype e Netherlands MK508899 MK508901
TR gmb 01482, holotype e Netherlands MK508900 MK508902
Lepiota rhodophylla E.C. Vellinga 2610 (UCB) USA AY176480 –
Lepiota sanguineofracta TO-HG2916, Holotype Italy KF879620 MG581701
TO-HG2917 Italy KF879621 MG581702
Lepiota scaberula E.C. Vellinga 2307 (UC) USA AF391029 –
E.C. Vellinga 2595 (holotype) (UC) USA AF391030 –
UC1999143 USA –MK278271
Lepiota subcastanea HKAS 45633 China –KM214817
Lepiota subgranulosa ANGE253 (JBSD, duplicate in MEXU) e Dominican
Republic
KR022007 –
Lepiota subalba E.C. Vellinga 2242 (L) e Netherlands AY176489 –
Lepiota subincarnata E.C. Vellinga 2234 (L) e Netherlands AY176491 –
VPI-OKM22153 South Korea –U85294
NL-2022 Hungary –MK278273
Lepiota thiersii E.C. Vellinga 2590 (UCB) USA AY176485 –
E.C. Vellinga 2589 (UC) USA GQ203817 –
Lepiota xanthophylla TUB 011553 Germany –DQ071712
Uncultured
Basidiomycota
Environmental sample, man22_soil_G02 USA GU328508 –

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
50
Sequence alignment, dataset assembly and phylogenetic analysis
Sequences obtained in this study were compared to those available in the GenBank
(http://www.ncbi.nlm.nih.gov/) and UNITE (http://unite.ut.ee/) databases by using
the Blastn algorithm (Altschul et al. 1990).
Based on the BLASTn results (sequences were selected based on the greatest
similarity) and outcomes of recent phylogenetic studies incorporating Lepiota
sequences (Vellinga 2003, 2004, 2010; Vizzini et al. 2014a, b; Justo et al. 2015;
Qasim et al. 2015; Hosen et al. 2016) sequences were retrieved from GenBank for
the comparative phylogenetic analysis. e nrITS and nrLSU datasets were analysed
separately. e combined nrITS/nrLSU phylogeny was not inferred as most Lepiota
collections in GenBank are not provided with both molecular markers (Table 1).
Although tef1-α sequences were generated for L.psalion, they were not included in
phylogenetic analyses because comparable sequences for most Lepiota taxa are currently
unavailable in public databases, and, in this case, only the Blastn results were provided
in the Results. In the nrITS dataset, besides Lepiota species with a hymeniform pileus
covering, eight species (indicated by an asterisk in Fig. 1) representative of the major
clades in Lepiota as delimited by Vellinga (2003) were chosen for comparison. e
nrLSU dataset consists of all the Lepiota s.l. collections determined at species level
present in GenBank. Alignments were generated for each nrITS and nrLSU dataset
using MAFFT (Katoh et al. 2002) with default conditions for gap openings and gap
extension penalties. e two alignments were imported into MEGA v. 6.0 (Tamura
et al. 2013) for manual adjustment. e best-t substitution model for each single
alignment was estimated by the Bayesian information criterion (BIC) with jModelTest
2 (Darriba et al. 2012). e GTR + G model was chosen for the nrITS alignment and
the TrN+I+G for the nrLSU alignment. e nrITS dataset was partitioned into ITS1,
5.8S and ITS2 subsets. Chamaemyces fracidus (AY176343 and AY176344) was used
as an outgroup taxon in both the nrITS and nrLSU analyses because it is basal in the
Agaricaceae (Vellinga 2004, 2010).
Phylogenetic hypotheses were constructed with Bayesian inference (BI) and Maxi-
mum likelihood (ML) criteria. e BI was performed with MrBayes v. 3.2.6 (Ronquist
et al. 2012) with one cold and three incrementally heated simultaneous Monte Carlo
Markov chains (MCMC) run for 10 million generations, under the selected evolution-
ary model. Two simultaneous runs were performed independently. Trees were sampled
every 1,000 generations, resulting in overall sampling of 10,001 trees per single run;
the rst 2,500 trees (25%) were discarded as burn-in. For the remaining trees of the
two independent runs, a majority rule consensus tree showing all compatible partitions
was computed to obtain estimates for Bayesian posterior probabilities (BPP).
ML estimation was performed with RAxML v. 7.3.2 (Stamatakis 2006), with
1,000 bootstrap replicates (Felsenstein 1985) using the GTRGAMMA algorithm
to perform a tree inference and search for a good topology. Support values from
bootstrapping runs (MLB) were mapped on the globally best tree using the “-f a”
option of RAxML and “-x 12345” as a random seed to invoke the novel rapid

Looking for Lepiota psalion Huijser & Vellinga 51
bootstrapping algorithm. BI and ML analyses were run on the CIPRES Science
Gateway web server (Miller et al. 2010). Only BPP and MLB values over 0.70 and
50%, respectively, are reported in the resulting trees (Figs 1, 2). Pairwise % identity
values (P%IV) of the sequences were calculated using MEGA v. 6.0 (Tamura et al.
2013). Alignments and phylogenetic trees are available at TreeBASE (www.treebase.
org) under ID S22021.
Results
Molecular analysis
e PCR product was 476–729 bp (nrITS) and 894–1128 bp (nrLSU). e nrITS
data matrix comprised 68 sequences (including 63 from GenBank). is dataset was
814 bp long and contained 545 (66.9 %) variable sites. e nrLSU data matrix com-
prised 45 sequences (including 39 from GenBank). is dataset was 953 bp long and
contained 335 (35.2%) variable sites.
As both Bayesian and Maximum likelihood analyses produced a consistent topol-
ogy, only the Bayesian trees with both BPP and MLB values are shown (Figs 1, 2).
In both the nrITS and nrLSU analyses (Figs 1, 2), the sequences of the holotype
of L. psalion and of the Sardinian collection clustered together in a strongly supported
clade (BPP = 1.00, MLB = 100% and BPP = 1.00, MLB = 99%, respectively). e
sequences of this clade show a P%IV of 98.9% for the nrITS and of 99.6% for the
nrLSU. According to the nrITS analysis, which is based on a larger taxon sampling
(Fig. 1), L. psalion is sister (BPP = 1.00; MLB = 85%) to L. coloratipes Vizzini, J.F.
Liang, Jančovičová & Zhu L. Yang. e Blastn results of the tef1-α sequences obtained
from the two Sardinian specimens of CAG P.11_9/7.68 (MG597229 and MG597230)
show an identity value of 83% with Lepiota phaeoderma Vellinga (GQ375549), 81%
with Coniolepiota spongodes (Berk. & Broome) Vellinga (HM488881, HM488883 and
HM488884) and with Lepiota neophana Morgan (GQ375550 and GQ375551).
Both the nrITS and nrLSU analyses (Figs 1, 2) highlight the presence of sequences
in GenBank from Dutch [GQ203823, AY176390 (nrITS), the Netherlands, Limburg
province, Valkenburg, Schaelsberg, H.A. Huijser (herb. Huijser), 15-IX-1999, and
AY176391 (nrLSU), ibidem, H.A. Huijser (herb. Huijser), 23-VIII-1999] and Chi-
nese collections [GU199362 (nrITS) and GU199355 (nrLSU), China: Jilin province,
Changchun, Jinyuetan Park, herb. HMJAU3799] which are named as “Lepiota psalion”,
but are clearly distinct from the holotype and the Sardinian collection of L. psalion.
e Dutch “Lepiota psalion” sequences form a strongly supported clade (BPP = 1.00
and MLB = 100% in the nrITS analysis; BPP = 1.00 and MLB = 99% in the nrLSU
analysis) with sequences from the two collections of L. recondita (recondita clade). e
sequences of this clade show a P%IV of 99.3% for both the nrITS and the nrLSU. e
Chinese “Lepiota psalion” is sister (BPP = 1.00 and MLB = 98% in the nrITS analysis;
BPP = 1.00 and MLB = 94% in the nrLSU analysis) to the recondita clade.

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
52
Figure 1. Bayesian phylogram obtained from the general nrITS sequence alignment of Lepiota spp. Here there are included Lepiota species with a hymeniform
pileus covering, eight species representative of the major clades in Lepiota (indicated by *), and Chamaemyces fracidus as an outgroup taxon. Support values in either
the Bayesian (Posterior Probabilities values [BPP]) or Maximum likelihood (ML Bootstrap percentage [MLB]) analyses are indicated. Only BPP values over 0.70 (in
bold) and MLB values over 50% are given above clade branches. Newly sequenced collections are in bold.

Looking for Lepiota psalion Huijser & Vellinga 53
Taxonomy
Lepiota psalion Huijser & Vellinga, in Vellinga & Huijser, Belg. J. Bot. 131(2):
203 (1999) [1998]
Figs 3–6
Description. Macrocharacters (Fig. 3). Pileus 8–36 mm wide, at rst slightly obtusely
campanulate, hemispherical-trapezoid or broadly conical, later plano-convex to ap-
planate-expanded, subumbonate, with a shallow umbo; not hygrophanous; margin
Figure 2. Bayesian phylogram obtained from the general nrLSU sequence alignment of Lepiota spp.
Chamaemyces fracidus was used as an outgroup taxon. Support values in either the Bayesian (Posterior
Probabilities values [BPP]) or Maximum likelihood (ML Bootstrap percentage [MLB]) analyses are in-
dicated. Only BPP values over 0.70 (in bold) and MLB values over 50% are given above clade branches.
Newly sequenced collections are in bold.

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
54
not striated, slightly exceeding the lamellae when young, sinuous-undulate, entire or
slightly fringed with age, with minute adhering remnants of partial veil when young;
surface dry, at rst smooth, later irregularly cracking around centre into concentric
non-uplifted squamules; cream to pinkish-light brown at centre [*Vinaceous-bu
Figure 3. Lepiota psalion. Fresh basidiomes (CAG P.11_9/7.68) a Basidiomes in situ b–d Details of
pileus surface, stipe and annulus. Scale bars: 10 mm (a); 5 mm (b–d). Photographs by A. Tatti.

Looking for Lepiota psalion Huijser & Vellinga 55
Figure 4. Lepiota psalion. Holotype (WU 5152) a Labels and collection b Four basidiomes from the
collection. Scale bar: 10 mm. Photographs: a by W. Till; b by A. Vizzini.

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
56
(Plate XL 17’’’.c-y./d) HTML d3b094 to Orange-Cinnamon (Plate XXIX 13’’.ou-o.)
or Ochraceous-Tawny (Plate XV 15’.y-o./i) HTML bc7e4d], paler towards the margin
[Pale Cinnamon-Pink (Plate XXIX - 13’’.oy-o./f) HTML e5d6c3 to Pale Smoke-Gray
(Plate XLVI 21’’’’.o-y./d) HTML cdc9c6]. Stipe 22–33 × 1.5–2 mm, central, cylindri-
cal, usually regular, but sometimes also slightly exuous, hollow; shiny, at rst white,
soon becoming pink-brown [Tilleul-Bu (Plate XL - 17’’’.c-y./f), HTML c3b092 to
*Drab Gray (Plate XLVI 17’’’.o-y./d) HTML bda599] starting from the base and pro-
Figure 5. Lepiota psalion. Microscopic features (CAG P.11_9/7.68) a–b Elements of the pileus covering
c Cheilocystidia d Elements of the annulus e–f Spores. a–d in ammoniacal Congo red e in 5% KOH f in
Melzer’s reagent. Scale bars: 10 μm (a–d); 5 μm (e–f). Photographs by A. Tatti.

Looking for Lepiota psalion Huijser & Vellinga 57
Figure 6. Lepiota psalion. Microscopic features (CAG P.11_9/7.68) a Elements of the pileus covering
bCheilocystidia c Spores d Basidia e Elements of the annulus. Scale bars: 20 μm (a, e ); 10 μm (b , d );
5μm (c). Drawings by A. Tatti.

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
58
gressing upward; minutely silky brillose along all length; with whitish [Pale pinkish
bu (Plate XXIX 17’’.o-y./f) HTML ede2d4], ascending and often incomplete annu-
lus on the upper part of the stipe, sometimes disappearing in age; often with minute
white rhizomorphs. Lamellae 2–3(4) mm wide, l = 1–3(4), free, crowded, at rst white,
soon with evident pinkish tints [Cream-Bu (Plate XXX 19’’ .yo-y /d) HTML dfc38c
to Clay-Color Plate (XXIX 17’’ .o-y.) HTML ce9b44]; edge nely granulose. Context
elastic, whitish, pink-brown towards the stipe base; without specic smell and taste.
Spore-print pale cream.
Microcharacters (Figs 5, 6). Spores [700, 6, 2] (2.7–)3.5–4.3(–4.9) × (2.0–)2.6–
3.2(–3.9) μm, on average 3.9 × 2.9 μm, Q = (1.03–)1.23–1.49(–1.78), Qav = 1.36,
from broadly ellipsoid to ellipsoid, hyaline, thin-walled, smooth, not verruculose in
Melzer’s reagent, binucleate, not metachromatic in Cresyl Blue, nonamyloid, non-
dextrinoid, cyanophilic in Cotton Blue (Figs 5e, f, 6c). Basidia mainly 4-spored,
(15.5–)17.1–21(–22.0) × (4.2–)4.7–5.8 (–6.0) μm (n = 54), rarely 1- or 2-spored,
clavate, hyaline, thin-walled; sterigmata (2.6–) 3.0–4.2 (–4.9) × (0.5–)0.6–1.1(–1.2)
μm (n = 67) (Fig. 6d). Lamella edge sterile. Cheilocystidia (10.0–)13.7–21.1 (–26.3)
× (4.6–)6.2–8.7(–10.0) μm (n = 84), numerous and crowded, hyaline, thin-walled,
various in shape, mostly clavate to subutriform, occasionally subfusiform, subcapitu-
late (Figs 5c, 6b). Pleurocystidia absent. Pileus covering a (140.7–)153.7–179.1(–201.1)
μm (n = 16) thick hymeniderm with transition to an epithelium (Figs 5a,b, 6a), with
up to 2(or 3) colourless elements on top of each other; terminal elements not tightly
packed, (10.4–)18.0–53.6(–62.3) × (3.9–)7.7–19.3(–24.0) μm (n = 62), vesiculose,
sphaeropedunculate to clavate-pyriform, utriform; slightly thick-walled (walls ca 0.5
μm), with walls embedded in a thin gelatinous matrix; subpellis composed of densely
arranged and branching cylindrical hyphae, (21.3–)49.0–108.5(–136.8) × (3.8–)4.5–
8.8(–9.7) μm (n = 38). Pileitrama of cylindrical hyphae, (33.1–)42.1–93.2(–111.8)
× (2.7–)4.3–9.8(–14.4) μm (n = 45). Hymenophoral trama subregular, consisting of
cylindrical hyphae (33.8–)36.5–64.4(–83.1) × (6.0–)7.6–15.8(–17.3) μm (n = 61).
Stipe covering consisting of cylindrical hyphae, (23.8–)80.1–214.4(–370.8) × (2.6–
)5.4–12.1(–15.4) μm (n = 58). Stipe trama consisting of cylindrical hyphae, (21.8–
)58.5–178.9(–302.7) × (2.5–) 3.3–11.6(–12.5) μm (n = 32). Caulocystidia absent.
Partial veil (annulus) composed of cylindrical elements, (21.1–)27.5–52.7(–94.7) ×
(2.2–)2.9–4.8(–8.5) μm (n = 36) with terminal clavate elements, (12.4–)17.9–34.0(–
40.3) × (8.4–)10.6–17.7(–19.8) μm (n = 60) (Figs. 5d, 6e). Clamp-connections present
and abundant everywhere.
Ecology and distribution. Gregarious on bare soil, in gardens and parks; so far
known only from the type locality (Austria) and Sardinia (Italy).
Collections examined. Italy, Sardinia, Cagliari, Botanical Garden, 6 basidiomes
growing among the Searsia/Rhus sp. litter, calcareous soil, 17 January 2017, Alessia
Tatti and Giacomo Calvia (CAG P.11_9/7.68). Austria, Wien-Lobau, N. Uferhaus, 23
August 1985, Anton Hausknecht (WU 5152, holotype) (Fig. 4).

Looking for Lepiota psalion Huijser & Vellinga 59
Lepiota recondita Tatti, Huijser & Vizzini, sp. nov.
MycoBank No: MB 829963
Figs 7–9
Holotype. e Netherlands, prov. Limburg, Valkenburg, Schaelsberg, 02 September
2004, Henk A. Huijser (TR gmb 01482).
Etymology. From the Latin “reconditus”, meaning hidden, forgotten, which refers
to its resemblance with L. psalion with which it was confused.
Diagnosis. It is distinguished from Lepiota psalion by larger spores (3.7–)4.4–
5.4(–5.9) × (2.4–)2.9–3.6(–4.3) μm, versiform cheilocystidia and dierent nrITS and
nrLSU sequences.
Description. Macrocharacters (Fig. 7). Pileus 9–26 mm wide, at rst slightly ob-
tusely campanulate, hemispherical-trapezoid or broadly conical, later plano-convex to
applanate-expanded, subumbonate, with a shallow umbo; not hygrophanous; margin
not striated, slightly exceeding the lamellae when young, sinuous-undulate, entire or
slightly fringed with age, with minute adhering remnants of partial veil when young;
surface dry, at rst smooth, later irregularly cracking around centre into concentric
non-uplifted squamules; pinkish-light brown at centre from [Light Pinkish Cinnamon
(Plate XXIX, 15’’.Y-O./d) HTML f19b5f] to [Mikado brown (Plate XXIX 13’’.OY-
O./i), HTML 9f5425] or [Sayal Brown (Plate XXIX, 15’’.Y-O./i) HTML bc662d],
paler towards the margin: [Capucine Blu (Plate III, 13.OY-O./f) HTML fee6cc] or
[Orange Pink (Plate II, 11.ORANGE/f) HTML ecc8a3]. Stipe 26–47 × 1.5–3 mm,
central, cylindrical, at rst white, becoming pink-brown with manipulation [Pink-
ish Cinnamon (Plate XXIX, 15’’.Y-O./b) HTML e1934f]; minutely silky brillose
along all length; with whitish, ascending and often incomplete annulus on the up-
per part of the stipe, sometimes disappearing in age; often with minute white rhizo-
morphs. Lamellae free, crowded, l = 1–3, at rst white, soon with evident yellowish
tints [Catrige Bu (Plate XXX 19’’ .yo-y /f ) HTML cdaf68] becoming [Honey Yellow
(Plate XXX 19’’.YO-Y) HTML de9e42] when dry. Context elastic, whitish, smell weak,
Lepiotacristata-like, taste not recorded. Spore-print whitish.
Microcharacters (Figs 8, 9). Spores [350, 6, 2] (3.7–)4.4–5.4(–5.9) × (2.4–)2.9–
3.6(–4.3) μm, on average 4.8 × 3.3 μm, Q = (1.1–)1.3–1.7(–2.0), Qav = 1.5, from
subglobose to oblong, mainly ellipsoid, hyaline, thin-walled, smooth, not verrucu-
lose in Melzer’s reagent, binucleate, not metachromatic in Cresyl Blue, nonamyloid,
non-dextrinoid, cyanophilic in Cotton Blue (Figs 8f, 9c). Basidia mainly 4-spored,
(15.8–)17.4–25.4(–28.6) × (5.7–)6–7.3(–8.8) μm (n = 60), sometimes 1–2-spored,
clavate, hyaline, thin-walled (Fig. 9d); sterigmata (1.9–)2.4–4.2(–4.8) × (0.4–)0.6–
1.2(–1.5) μm (n = 70). Lamella edge sterile. Cheilocystidia (20.1–)25.4–44(–50.0) ×
(3.2–)7.2–10.4(–12.0) μm (n = 66), numerous and crowded, hyaline, thin-walled,
various in shape, mostly clavate, cylindrical-clavate, sphaeropedunculate to submon-
iliform, occasionally pyriform, cylindrical (Figs 8b–d, 9b). Pleurocystidia absent.

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
60
Pileus covering hymenidermic: terminal elements not tightly packed, (17–)24.7–
51.1(–59.6) × (8.1–)10–14(–27.3) μm (n = 70), vesiculose, sphaeropedunculate to
clavate-pyriform (Figs 8a, 9a); slightly thick-walled (walls ca 0.5 μm), with walls
embedded in a thin gelatinous matrix; subpellis composed of densely arranged and
branching cylindrical hyphae, (40.6–)47.0–118.3(–156.2) × (5.8–)7.6–16.2(–17.1)
μm (n = 20) and containing scattered ramied oleiferous hyphae, (1.5–)1.8–5.3(–
8.0) μm wide (n = 30). Hymenophoral trama subregular, consisting of ovate hyphae
(20.9–)21.1–40.3(–42) × (7–)9.6–13(–14.5) μm (n = 12). Stipe covering and trama
indistinguishable, consisting of cylindrical hyphae, (55.3–) 67.0–165.7 (–213.0) ×
(5.5–)7.6–15.0(–21.0) μm. Caulocystidia absent. Partial veil (annulus) composed of
cylindrical elements, (7.2–)22.3–59(–70.0) × (2.0–)2.5–4.2(–4.7) μm (n = 20) with
terminal clavate elements, (10.1–)12.4–26.7(–38.1) × (7.0–)9.5–16.7(–28.4) μm (n
= 40) (Figs 8e, 9e). Clamp-connections present and abundant everywhere.
Figure 7. Lepiota recondita. Fresh basidiomes a–b (TR gmb 01482, holotype) c (TR gmb 01481, para-
type). Scale bars= 10 mm. Photographs by H.A. Huijser.

Looking for Lepiota psalion Huijser & Vellinga 61
Figure 8. Lepiota recondita. Microscopic features (in ammoniacal Congo red, TR gmb 01482, holotype)
a Elements of the pileus covering b–d Cheilocystidia e Elements of the annulus f Spores. Scale bars:
10μm (a–e); 5 μm (f). Photographs by A. Tatti.
Ecology and distribution. Gregarious on rich in nutrients and lime (marl) bare
soil, in a mixed deciduous forest; so far known only from the type locality.
Collections examined. e Netherlands, Limburg province, Valkenburg,
Schaelsberg, man-made (anthropized) hilly grove with mainly deciduous trees (Quercus,
Fagus, Corylus, Fraxinus, Robinia, Prunus, Sambucus), together with Lepiota tomentella,
L. poliochloodes, Melanophyllum eyrei, and Limacella ochraceolutea, 22 September 2001,
Henk A. Huijser (TR gmb 01481, paratype); ibidem, 02 September 2004, Henk A.
Huijser (TR gmb 01482, holotype).

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
62
Figure 9. Lepiota recondita. Microscopic features (TR gmb 01482, holotype) a Elements of the pi-
leus covering b Cheilocystidia c Spores d Basidia e Elements of the annulus. Scale bars: 20 μm (a , e);
10μm(b, d); 5 μm (c). Drawings by A. Tatti.

Looking for Lepiota psalion Huijser & Vellinga 63
Lepiota sinorecondita ad interim
Fig. 10
Description. e specic epithet is a combination of Medieval Latin “sino” (which
means Chinese) and “recondita”, referring to the strong anity of the Chinese taxon
to the European L. recondita.
Basidiomata small (Fig. 10a). Pileus 9–17 mm wide, expanding to convex with
obtuse umbo; at centre on umbo smooth, dark yellowish brown to dark brown, around
umbo split up into pale brown concentrically arranged patches on dirty white to cream
background, paler and smaller towards margin. Stipe 35–37 × 1–4 mm, subcylindrical
or attenuate, slightly inated at base; hollow, dirty white and glabrous at the apical
part, surface whitish, covered white, tomentose at lower part, with white mycelial cords
at base; annulus membranous, superior, whitish on upper surface, with small yellowish
brown to brownish squamules on lower whitish surface. Lamellae free, cream, yellow
to brown when dry, crowded with lamellulae, edge wavy.
Spores [60,3,1] (4.0–)4.5–5.5 × 2.5–3.0(–3.5) μm, Q = 1.50–1.80(–1.83), Qav =
1.64 (Fig. 10b), ellipsoid to oblong in side and front view, without suprahilar depres-
sion, sometimes with straight adaxial side; hyaline, smooth, non-dextrinoid, congo-
philous but very weakly, slightly reddish purple in Cresyl Blue. Basidia 17–22 × 5–6
Figure 10. Lepiota sinorecondita (HMJAU 3799) a Basidiome b Spores c Cheilocystidia d Elements of
the pileus covering. Scale bars: 10 mm (a); 5 μm (b); 20 μm (c–d). Drawings by J.F. Liang.

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
64
μm, narrowly clavate or subcylindrical, 4-spored. Lamella edge sterile. Cheilocystidia
21–40 × 6–13 μm, clavate to narrowly clavate, rarely broadly clavate, colourless, hya-
line, thin-walled (Fig. 10c). Pleurocystidia absent. Pileus covering a hymeniderm made
up of broadly clavate, clavate to obpyriform terminal elements, 18–50 × 10–20 μm,
with pale yellowish brown intracellular pigment (Fig. 10d). Clamp-connections pre-
sent in all tissues.
Collection examined. China, Jilin Province, Changchun City, Jinyuetan Park, 7
July 2005, Wang Jianrui (HMJAU 3799).
Ecology and distribution. Solitary, terrestrial, on the ground in a larch forest in
summer and autumn. So far known only from China.
Discussion
Distinguishing characters of L. psalion and allied species
e morphological dierences among the Lepiota species with hymeniform pileus cov-
ering are often subtle (Vellinga and Huijser 1999; Vellinga 2010), but nrlTS sequence
data support the morphologically recognized species (Vellinga 2010; Vizzini et al.
2014a, b; Justo et al. 2015; Qasim et al. 2015; Hosen et al. 2016).
Lepiota psalion is distinguished by having a non-smooth pileus with concentric
non-uplifted squamules, a distinct annulus, and mostly clavate cheilocystidia (Vellinga
and Huijser 1999; Vellinga 2001; our observations). e annulus is quite evanescent
(Fig. 3) mainly because it is predominantly composed of inated elements (Figs 5d, 6e).
Lepiota “cf. rupes f. phaeophylla” sensu Winterho and Bon (1994) and L. rupes
sensu Babos (1974), Wasser (1980), and Krieglsteiner (1991), all with a distinct an-
nulus, are probably referable to L. psalion (Vellinga and Huijser 1999; Vellinga 2001),
but see below.
e phylogenetically closest species are L. coloratipes (= L. rupes ss. Auct. europ.
non ss. orig.) and L. sanguineofracta (Fig. 1). Lepiota coloratipes diers from L. psalion
in having a usually smooth pileus surface, a very evanescent partial veil not forming an
annulus but leaving brillose remnants on stipe surface, a stipe with reddish tinges at
base, the presence of oil droplets in all tissues (including spore surface), the hymeni-
form pileus covering consisting of very tightly arranged clavate to sphaeropedunculate
elements, the presence of uninucleate spores which are often verruculose in Melzer’s
reagent, versiform cheilocystidia (mostly lageniform or lecythiform), and the presence
of caulocystidia (Bon 1981, 1993; Candusso and Lanzoni 1990; Vellinga and Huijser
1999; Vellinga 2001; Vizzini et al. 2014b). Lepiota sanguineofracta, recently described
from Italy, is characterized by a micaceous but not squamulose pileus surface with
distinct green tinges when mature, a fugacious partial veil not forming an annulus, a
stipe with reddish tinges towards the base, the context smelling of dried rose petals,
basidiome surfaces and context strongly reddening on handling, binucleate spores, and
versiform cheilocystidia (clavate to subutriform, subfusiform) (Vizzini et al. 2014a).

Looking for Lepiota psalion Huijser & Vellinga 65
e other morphologically allied species of Lepiota with a hymeniform pileus
covering, ellipsoid spores, and a well-formed annulus, phylogenetically far from
L. psalion (Figs 1, 2), show distinctive morphological traits: L. apatelia Vellinga &
Huijser, L.cristatoides Einhell. (both from Europe), and L. thiersii Sundb. (from western
North America) have no cheilocystidia (Einhellinger 1973; Sundberg 1989; Vellinga and
Huijser 1999; Vellinga 2001, 2010; Hausknecht and Pidlich-Aigener 2005; Kosakyan et
al. 2008; Mertens 2010; Gierczyk et al. 2011). Lepiota neophana (including var. europaea
Bizio & Migl. and f. papillata Migl. & L. Perrone) shows a smooth pileus surface with
a bu to dark-brown and umbonate centre, very rare clamp-connections in the pileus
trama and no cheilocystidia (Anonymous 1992; Bizio et al. 1993; Vellinga and Huijser
1999; Vellinga 2010). Finally, pale collections of L. lilacea Bres. are distinguished by
whitish lamellae, an annulus with lilac-brown tinges on the lower part and margin, and
metachromatic (in Cresyl Blue) up to 6 μm long spores (Bon 1981, 1993; Migliozzi and
Clericuzio 1989; Candusso and Lanzoni 1990; Vellinga 2001).
The Lepiota psalion complex
Lepiota psalion was established by Vellinga and Huijser (1999) based on an Austrian col-
lection made by A. Hausknecht on 23 August 1985 (WU 5152) and determined by M.
Bon as L. rupes fo. annulata ined. (Fig. 4a). e extended description they provided is het-
erogeneous: the macromorphology was taken from Krieglsteiner (1991) who described a
German collection as L. rupes, collection considered by Vellinga and Huijser as L. psalion,
while the micromorphology was based on the analysis of the holotype made by the same
Dutch mycologists. NrITS and nrLSU sequences later deposited in GenBank as L. psalion
were generated by Vellinga (2004, 2010) not from the holotype, but from three Dutch col-
lections (vouchers 23-VIII-1999, 15-IX-1999, and hah6177, H.A. Huijser, herb. Huijser).
When the Sardinian specimens were collected, they were morphologically attrib-
uted to L. psalion, but when they were sequenced to obtain molecular evidence, they
did not cluster either with the Dutch collections or with a collection named L. psalion
from China (herb. HMJAU3799; Liang et al. 2011) (tree not shown). Consequently,
we decided to request the holotype collection from WU and sequenced it. Phyloge-
netic analyses highlighted that Sardinian collection and the holotype are conspecic
(Figs 1, 2) and sister to L. coloratipes (Fig. 1). Molecular data so conrm L. psalion
as independent species in the genus Lepiota; Dutch and Chinese collections are two
distinct and yet undescribed new species, phylogenetically close (BPP = 0.97; MLB =
91%) to L. thiersii (Fig. 1). Unfortunately, the collections of the Dutch taxon whose
sequences are deposited in GenBank were subsequently lost (Vellinga, pers. comm.)
but, based on two newly sequenced additional collections from the same original area
of the Dutch taxon, the new species L. recondita is here described. As only one collec-
tion (consisting of three basidiomes) is available for the Chinese taxon, it was decided
to propose it only as an ad interim species. Further collections will be necessary to
describe it as a new species.

Alfredo Vizzini et al. / MycoKeys 52: 45–69 (2019)
66
Lepiota psalion, L. recondita, L. “sinorecondita”, L. apatelia, and L. thiersii consti-
tute a homogeneous morphology-based but not monophyletic group, here named the
“L. psalion complex”, which is circumscribed by a set of shared characters: a pileus
surface breaking into small squamules, well-formed white partial veil (usually forming
an annulus, but see L. apatelia), hymeniform pileus covering, and ellipsoid spores.
An identication key for the taxa belonging to this complex is proposed below.
Key to the species of the Lepiota psalion complex
1 Cheilocystidia absent .................................................................................. 2
– Cheilocystidia present ................................................................................ 3
2 Smell farinaceous, annulus often adhering to pileus margin (as velar rem-
nants), spores weakly dextrinoid ..................................L. apatelia (Europe)
– Smell L. cristata-like, annulus usually ascending on stipe, spores non-dextri-
noid .................................................................. L. thiersii (North America)
3 Spores ellipsoid, on average = 3.9 μm long, Qav = 1.36 ................................
..................................................................................... L. psalion (Europe)
– Spores ellipsoid to oblong, on average > 4.0 μm long, Qav > 1.4 ................4
4 Cheilocystidia versiform, spores ellipsoid, Qav = 1.5, annulus entirely
smooth ......................................................................L. recondita (Europe)
– Cheilocystidia mainly clavate, spores oblong, Qav = 1.64, annulus covered by
minute yellowish brown squamules on lower surface.....................................
................................................................. L. sinorecondita ad int. (China)
Acknowledgements
We thank Irmgard Greilhuber and Walter Till (University of Vienna) for sending us
photographs and part of the holotype collection of Lepiota psalion, Giacomo Calvia
(University of Cagliari) for his assistance in collecting specimens in the Botanical Gar-
den of Cagliari, Marco Floriani (Pergine Valsugana, Trento) for depositing the collec-
tions of the new species in TR, and Else Vellinga (University of California - Berkeley)
for her suggestions. AT also thanks the University of Cagliari and, in particular, Gian-
luigi Bacchetta, director of the Hortus Botanicus Kalaritanum, for allowing sampling
of the studied material and Annalena Cogoni, the person in charge of the Herbarium
CAG, for allowing us access to fungarium material.
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