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Systematics and Biodiversity
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ht t p: / / www. t andf online. com/ loi/ t sab20
Crassisporium and Romagnesiella: two new genera of
dark-spored Agaricales
a
b
c
a
P. Brandon Mat heny , Pierre-Art hur Moreau , Alf redo Vizzini , Emma Harrower , Andre De
d
e
Haan , Marco Cont u & Mariano Curt i
f
a
Depart ment of Ecology and Evolut ionary Biology, Hesler 332, Universit y of Tennessee,
Knoxville, TN 37996-1610, USA
b
Facult é des Sciences Pharmaceut iques et Biologiques, Universit é Lille Nord de France,
F-59006 Lille, France
c
Dipart iment o di Scienze della Vit a e Biologia dei Sist emi, Universit à di Torino, Viale
Mat t ioli 25, 10125 Torino, It aly
d
Leopoldst raat 20/ 3, B-2850 Boom, Belgium
e
Via Marmilla12, I-07026 Olbia, It aly
f
Via Tit o Nicolini 12, I-02030 Pozzaglia Sabina, It aly
Published online: 30 Oct 2014.
To cite this article: P. Brandon Mat heny, Pierre-Art hur Moreau, Alf redo Vizzini, Emma Harrower, Andre De Haan, Marco
Cont u & Mariano Curt i (2014): Crassisporium and Romagnesiella: t wo new genera of dark-spored Agaricales, Syst emat ics and
Biodiversit y, DOI: 10. 1080/ 14772000. 2014. 967823
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Systematics and Biodiversity (2014)
Research Article
Crassisporium and Romagnesiella: two new genera of dark-spored
Agaricales
Downloaded by [University of Tennessee, Knoxville] at 13:56 31 October 2014
P. BRANDON MATHENY1*, PIERRE-ARTHUR MOREAU2, ALFREDO VIZZINI3, EMMA HARROWER1,
ANDRE DE HAAN4, MARCO CONTU5 & MARIANO CURTI6
1
Department of Ecology and Evolutionary Biology, Hesler 332, University of Tennessee, Knoxville, TN 37996-1610, USA
Faculte des Sciences Pharmaceutiques et Biologiques, Universite Lille Nord de France, F-59006 Lille, France
3
Dipartimento di Scienze della Vita e Biologia dei Sistemi, Universita di Torino, Viale Mattioli 25, 10125 Torino, Italy
4
Leopoldstraat 20/3, B-2850 Boom, Belgium
5
Via Marmilla12, I-07026 Olbia, Italy
6
Via Tito Nicolini 12, I-02030 Pozzaglia Sabina, Italy
2
(Received 2 February 2014; accepted 5 August 2014)
A systematic study of a rare and enigmatic European species, Galerina clavus Romagn., is presented. Phylogenetic
analyses show it to be most closely related to Pachylepyrium carbonicola (A.H. Sm.) Singer and P. funariophilum (M.M.
Moser) Singer (Strophariaceae). Investigation of additional species of Pachylepyrium suggests this genus is polyphyletic as
the type species, P. fulvidula (Singer) Singer, is nested in the Tubariaceae Vizzini based on multigene phylogenetic
analyses. Pachylepyrium nubicola Singer is allied with Pholiota (Fr.) P. Kumm. based on high ITS similarity, and P.
carbonicola and P. funariophilum, together with G. clavus, form a clade among a consortium of Strophariaceae Singer &
A.H. Sm. and Hymenogastraceae Vittad. As a result, we propose Romagnesiella gen. nov. to accommodate G. clavus, for
which a taxonomic description is given and lectotype and epitype are designated. The genus Crassisporium gen. nov. is
proposed to encompass Pachylepyrium funariophilum (of which P. carbonicola is considered a younger taxonomic
synonym), P. chilense M.M. Moser, and P. squarrulosum Singer. Crassisporium is distinguished from Romagnesiella by its
thick-walled basidiospores and occurrence in burnt habitats. The identities of the morphologically similar Tubaria
umbonata S. Lundell, T. embolus (Fr.) Sacc. and T. minima J.E. Lange are also discussed.
Key words: Agaricoid clade, carbonicolous fungi, Hymenogastraceae, Pachylepyrium, Strophariaceae, taxonomy, taxon
sampling, types
Introduction
Considerable progress has been made to assess phylogenetic
relationships in the Agaricales (Binder, Larsson, Matheny,
& Hibbett, 2010; Garnica, Weiss, Walther, & Oberwinkler,
2007; Matheny, et al., 2006; Moncalvo et al., 2002), the
largest order of mushroom-forming fungi with some 13 500
described species (Kirk, Cannon, Minter, & Stalpers, 2008).
However, continued assessment of evolutionary relationships within the order is necessary. For instance, taxa from
the tropics and southern hemisphere are in need of better
integration into more inclusive molecular systematic treatments (Matheny et al., 2009, Rees, Midgley, Marchant, Perkins, & Orlovich, 2013), and some species are known only
from type collections, of insufficient age for adequate gene
*Correspondence to: P. Brandon Matheny. Email: pmatheny@
utk.edu
ISSN 1477-2000 print / 1478-0933 online
Ó The Trustees of the Natural History Museum, London 2014. All Rights Reserved.
http://dx.doi.org/10.1080/14772000.2014.967823
sampling, missing or unavailable (Ammirati, Parker, &
Matheny, 2007; Baroni & Matheny, 2011).
The genus Galerina Earle (Agaricales), typified by
G. vittiformis (Fr.) Singer, traditionally encompasses saprotrophic dark-spored agarics often with small and slender
fruit bodies with a bell-shaped pileus (mycenoid or collybioid in habit), straight pileal margin, attached lamellae,
presence of veil, and an ochre to rusty brown spore
deposit. Spores of Galerina are typically yellow to dark
tawny in KOH (potassium hydroxide), amygdaliform to
elliptic, often verruculose or rugulose, and lack a welldefined germ pore. The spores of many species of Galerina are also characterized by a smooth region above the
apiculus on the adaxial side of the spore (this is known as
a plage) (Bon, 1992; Gulden, 2012; Watling & Gregory,
1993). The first (and only) detailed phylogenetic assessment of Galerina strongly suggests the genus is
Downloaded by [University of Tennessee, Knoxville] at 13:56 31 October 2014
2
P. Brandon Matheny et al.
Fig. 1 2. Fruit bodies of Galerina clavus in situ (PAM06090110). Photo by P.-A. Moreau. Scale bar D 10 mm. (Fig. 2) Fruit bodies of
Pachylepyrium carbonicola in situ (PBM2293, WTU). Scale bar D 10 mm. Photo by P.B. Matheny.
polyphyletic (Gulden, Stensrud, Shalchian-Tabrizi, &
Kauserud, 2005).
Galerina clavus Romagn. (Fig. 1) is a small inconspicuous species published in 1944 by Romagnesi (1942) from
Europe that displays a combination of anomalous characters for the genus: namely, its naucorioid habit (small
size, pileus with a decurved margin), smooth spores without a plage, and absence of a veil. The combination of
these traits cast doubts on an alliance with Galerina (de
Haan & Walleyn, 2009; Moreau, 2009). Smith & Singer
(1964) treated G. clavus in their world monograph of
Galerina but placed it, together with the South American
species G. fuegiana Singer, in an isolated section Pseudotubaria A.H. Sm. & Singer. This classification has been used
by Bon (1992), Horak (2005), Moser (1978, 1983) and
Singer (1986). Molecular systematic studies of Galerina
and other dark-spored agarics (Aime, Vilgalys, & Miller,
2005; Garnica, Weiss, Walther, & Oberwinkler, 2007;
Gulden, Stensrud, Shalchian-Tabrizi, & Kauserud, 2005;
Matheny et al., 2006, 2007a; Moncalvo et al., 2002;
Petersen, Knudsen, & Seberg, 2010; Walther, Garnica, &
Weiß 2005) have not included G. clavus, and thus its systematic position remains ambiguous.
Based on preliminary phylogenetic analysis of nuclear
ribosomal RNA (rRNA) gene sequences, samples of G.
clavus clustered together with sequences of the North American species Pachylepyrium carbonicola (A.H. Sm.) Singer
(Fig. 2). The genus Pachylepyrium Singer (Agaricales, Strophariaceae; type: P. fulvidula (Singer) Singer), however,
contains seven accepted species that differ from G. clavus
by their thick-walled spores typically with a germ pore and
presence of a veil. Furthermore, most species are carbonicolous (Claridge, Trappe, & Hansen, 2009; McMullan-Fisher
et al., 2011), fruiting among burnt debris or on burnt ground
in co-occurrence with bryophytes (viz. Funaria Hedwig) or
on wood (lignicolous) (Moser, 2000; Singer, 1986). Thus, a
substantial taxonomic emendation would be required to
place G. clavus within Pachylepyrium.
To confirm phylogenetic and taxonomic relationships to
other species of Pachylepyrium (viz. the type species of the
genus, P. fulvidula), we produced molecular data from four
of seven type collections of species accepted in this genus.
We also studied a fifth type collection morphologically
and several collections of European naucorioid fungi showing similar characters to G. clavus, including Tubaria umbonata S. Lundell, T. minima J.E. Lange and T. embolus (Fr.)
Sacc.
Collections of Pachylepyrium are not common (Moser,
2000), probably owing to their specific habitat (mostly
burnt areas), which is in decline in regions of Europe
(Veerkamp 1998). While dense taxon sampling from broad
geographic areas is a laudable goal, our focus is to produce
a contemporary taxonomic revision based on available
type materials. To accomplish this and a more thorough
systematic comparison with G. clavus, we carried out a
multi-gene phylogenetic analysis with an emphasis on the
Agaricoid clade (Matheny et al., 2006) to investigate the
relationship of G. clavus to Pachylepyrium.
Materials and methods
Morphological analysis
Collections of fruit bodies were studied from the personal
herbarium of M. Contu and material preserved at IB, K,
LIP, MICH, MPU, PC and TENN. Herbarium designations follow Thiers (continuously updated). Colour designations in the format ‘(5E7)’ refer to plate, column and
row of Kornerup & Wanscher (1967). Microscopic observations were made in 5% KOH, Melzer’s reagent and
water mounts. Spores were measured on a Moticam 1000
video camera connected to a Nachet Andromede 0181
compound microscope or on a Nikon Eclipse 80i using
NIS Elements (D) imaging software. First and ninth deciles (D1, 9) and average values (italicized and in bold) are
presented according to Fannechere (2005, 2009).
Two new genera of dark-spored Agaricales
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Type collections examined for molecular
and taxonomic analyses
To evaluate the relationship between Galerina clavus
and Pachylepyrium, we performed molecular and/or morphological annotations of five Pachylepyrium species,
including type collections and the type species of the genus
(P. fulvidula). The type of G. clavus is missing. Pachylepyrium types studied by us include: P. carbonicola
AHS44640 (holotype of Kuehneromyces carbonicola A.H.
Sm., MICH); P. funariophilum IB 1949/0008 (holotype of
Pholiotina funariophila M.M. Moser); P. nubicola Singer
K (M 181790) (holotype); P. fulvidula T1495 (isotype of
Phaeomarasmius fulvidulus, MICH); and P. chilense M.M.
Moser M3269 (paratype, MICH) (see also Appendix 1,
online supplemental material, which is available from the
article’s Taylor & Francis Online page at http://dx.doi.org/
10.1080/14772000.2014.967823). We obtained a loan of
part of the holotype (IB) of P. chilense, but the material
was inadequate for destructive sampling. Material of representative G. clavus sequenced included PAM06090110
(LIP) and Contu 15122007 (pers. herb.). To assess the taxonomic relationship of G. clavus to Tubaria umbonata and
T. embolus, we examined the isotype of T. umbonata (ex
Fungi exsiccati Suecici 2041 (PC)) and accessions labelled
as ‘Galerina embolus’ (Fr.) P.D. Orton (Bon 741120, Bon
70624) at LIP. Other taxa selected for phylogenetic analyses
are listed in Appendix 1 (see supplemental material online).
DNA extraction, PCR and Sanger sequencing
Procedures for DNA extraction, PCR and Sanger sequencing
follow those outlined in Matheny et al. (2007b); Matheny,
Austin, Birkebak, & Wolfenbarger (2010) except where
mentioned below. For collections older than 30 years, we
used an E.Z.N.A. High Performance (HP) Fungal DNA kit
(Omega Bio-Tek, Norcross, Georgia, USA). We sampled the
ITS region, including the 5.8S gene, using primers ITS1F
and ITS4 (Gardes & Bruns 1993; White, Bruns, Lee, &
Taylor, 1990); the 50 end of the nuclear 25S large subunit
ribosomal RNA gene region (nLSU) using primers LR0R
and LR7 or LR5 (Vilgalys & Hester, 1990); almost the entire
nuclear 18S small subunit ribosomal RNA gene (nSSU)
between primers PNS1 and NS8 (O’Donnell, Cigelnik, &
Benny, 1998; White, Bruns, Lee, & Taylor, 1990); and the
most variable region of rpb2 between conserved domains 6
and 7 using primers b6F and b7.1R (Matheny, 2005). All
new sequences have been deposited in GenBank (shown in
bold in Appendix 1, see supplemental material online).
DNA alignments and phylogenetic analyses
We manually aligned 151 of our 153 new nLSU, nSSU,
5.8S and rpb2 sequences from 42 taxa, including type collections of Pachylepyrium funariophilum and P. fulvidula
(type species of Pachylepyrium) with alignments
3
produced by Matheny et al. (2006) in MacClade 4.08
(Maddison & Maddison, 2005). We were only able to
obtain ITS sequences from type collections of Pachylepyrium nubicola and Pachylepyrium carbonicola; thus,
these were not added to our alignment due to insufficient
variation across the 5.8S locus. Integration of ITS1 and
ITS2 spacer sequences was not possible due to their high
substitution rates. However, we did add sequences from
two conspecific collections of P. carbonicola determined
as such by A.H. Smith (sequences of which did not differ
from the holotype). The datasets were pruned to exemplars of the Tricholomatoid clade and all members of the
Agaricoid clade following Matheny et al. (2006). To these
we added rRNA and/or rpb2 sequences of Leucoagaricus
barssii (Zeller) Vellinga (Matheny et al., 2007b) and
Pseudoclitocybe cyathiformis (Bull.: Fr.) Singer from
Binder, Larsson, Matheny, and Hibbett (2010) and rRNA
and/or rpb2 sequences of Squamanita paradoxa
(A.H. Sm. & Singer) Bas, Mycocalia denudata (Fr. &
Nordholm) J.T. Palmer and Nidula niveotomentosa
(Henn.) Lloyd from Matheny & Griffith (2010). LSU and
5.8S sequences (AF261513, EF051055, EF051060) of
‘Pachylepyrium funariophilum’ from Moncalvo et al.
(2002) and ‘Tubaria minima’ of Matheny et al. (2007a)
were also added.
Alignments were concatenated in MacClade in a noninterleaved format with the final supermatrix composed of
170 taxa. Sixty-five taxa (38%) lacked rpb2 sequences, 21
(12%) lacked 5.8S sequences and 27 (16%) lacked SSU
sequences. Several studies (Wiens, 2006; Wiens & Moen,
2008; Wiens & Tu, 2012) demonstrate that incorporation
of incompletely sampled taxa in supermatrices improves
phylogenetic accuracy, if the overall number of characters
is sufficiently large, thus supporting a supermatrix
approach. The concatenated alignment and tree files have
been submitted to TreeBASE (S15353).
We converted the concatenated supermatrix from nexus
format to a relaxed phylip format in Seaview version
4.2.4 (Gouy, Guindon, & Gascuel, 2010) after inspection
for strongly supported conflict (>70%) between rRNA
and rpb2 gene trees following Matheny (2005) using Maximum Likelihood (ML) bootstrapping as indicated below.
The resulting concatenated rRNA and rpb2 phylip file
contained 4508 total sites: 1451 sites from LSU, 1782
sites from SSU, 158 sites from the 5.8S gene and 1117
sites from the rpb2 gene region between conserved
domains 5 and 7 after trimming staggered ends. A partition text file was created to model the rRNA gene regions
(positions 1 3391) separately from first, second and third
codon positions of rpb2 (positions 3392 4508) to allow
separate GTRGAMMA models for each partition following model selection in Matheny et al. (2006) and
recommendations made in the RAxML user manual (Stamatakis, 2006). Thus, four unique partitions were established with one for the rRNA gene regions and three
separate partitions for each rpb2 codon position.
Downloaded by [University of Tennessee, Knoxville] at 13:56 31 October 2014
4
P. Brandon Matheny et al.
RAxML version 7.2.8 was used to generate 1000 rapid
bootstraps and a final ML tree with all free model parameters estimated by the program. The same partitions were
invoked using the parallel version of MrBayes 3.1.2 (Altekar, Dwarkadas, Huelsenbeck, & Ronquist, 2004; Ronquist
& Huelsenbeck, 2003) for a Bayesian inference of the
phylogeny with each partition modelled according to
GTRCICG following Matheny et al. (2006). This analysis
entailed two independent runs for 50 million generations
sampling trees and other parameters every 5000 generations on the Newton High Performance Computing cluster
at the University of Tennessee. The average standard deviation of split frequencies was used as a metric to determine
an appropriate burn-in. Trees were viewed in FigTree version 1.4.0 (Rambaut, 2009). Pseudoclitocybe cyathiformis
was used for rooting purposes based on Binder et al.
(2010). ML bootstrap proportions are referred to as MLBP
and Bayesian posterior probabilities as BPP.
Results
Pachylepyrium is polyphyletic
152 ITS, 168 nLSU, 151 sSSU and 105 rpb2 sequences,
153 of which are new, were analysed for this study. The
average standard deviation of split frequencies reached
less than 0.01 by the 27 795 000th generation in the
Bayesian inference analysis of the four-gene region supermatrix. We sampled trees every 5000 steps (producing a
total of 10 001 trees for each of the two 50 million generation runs); thus, we conservatively burned the first 6001
trees, including the initial starting tree. Posterior probabilities were calculated from a total sample of 8000 trees
(4000 from each run). Comparison of the rRNA-only ML
phylogeny with that of the rpb2 ML phylogeny (data not
shown) revealed no significantly supported conflicts.
The genus Pachylepyrium is polyphyletic (Fig. 3). The
type species, P. fulvidula, clusters with strong support (80%
MLBP, 0.99 BPP) among a grade of lineages that includes
Phaeomarasmius Scherff., Flammulaster Earle and Phaeomyces E. Horak in the Tubariaceae. Pachylepyrium carbonicola and P. funariophilum cluster with strong support with
Galerina clavus (90% MLBP, 1.0 BPP) forming a weakly
supported sister group to exemplars of the Strophariaceae
and Hymenogastraceae. ITS sequences of type collections
of Pachylepyrium carbonicola and P. funariophilum differ
only at two positions excluding polymorphic sites. A blastn
analysis of the ITS sequence of the holotype of Pachylepyrium nubicola strongly suggests this species belongs to the
genus Pholiota (96% similar to Ph. terrestris HQ604756,
95% similar to Ph. gummosa JF908581 and 95% similar to
numerous other ITS sequences of Pholiota).
Two collections (TENN053270, TENN05174) labelled
Pachylepyrium funariophilum are incorrectly identified
and not identical to each other. TENN053270, from
Washington state, likely represents a species of Psilocybe
as first suggested by Walther, Garnica, and Weiß (2005),
whereas TENN051714, collected in North Carolina, is an
unidentified species of Deconica. Morphological examination of both collections affirms these results.
The monophyly of the Agaricoid clade of Matheny
et al. (2006) is for the first time highly supported (77%
MLBP, 1.0 BPP). Monophyly of the Agaricoid clade was
recovered with significant BPP in Garnica et al. (2007)
and Matheny et al. (2006), but with poor maximum parsimony bootstrap support. The Hydnangiaceae is indicated
as the sister group to the rest of the Agaricoid clade but
with high BPP only (0.99). A grouping of the Cortinariaceae, Bolbitiaceae, Tubariaceae, Inocybaceae, Crepidotaceae, Strophariaceae, Hymenogastraceae, and Agrocybe
erebia receives poor bootstrap support but high BPP
(0.99). Taken together, eight of 12 families in the Agaricoid clade (Inocybaceae, Tubariaceae, Bolbitiaceae, Cortinariaceae, Agaricaceae, Psathyrellaceae, Nidulariaceae,
Hydnangiaceae) receive strong statistical support in our
analyses (MLBP >70% and BPP >0.95). The Hymenogastraceae and Crepidotaceae are supported with only
high BPPs (0.99). The Squamanitaceae is recovered as
monophyletic, but with poor support. Similar to Matheny
et al. (2006) the Hymenogastraceae and Strophariaceae
are recovered as sister groups with a high posterior probability. Unlike Matheny et al. (2006) samples of Gymnopilus, which previously were placed in an isolated position
with the Agaricoid clade, now cluster with samples of
Galerina in the Hymenogastraceae but without strong
support.
The lineage containing Pachylepyrium funariophilum
and P. carbonicola is proposed as a new genus based on
molecular, morphological and ecological distinctions
between it and the lineage containing Galerina clavus,
and due to the placement of the type of Pachylepyrium in
the Tubariaceae. A separate genus is proposed to accommodate G. clavus due to differences in morphology and
ecology with respect to Pachylepyrium funariophilum and
P. carbonicola. All three taxa, however, are united by
their basidiospores that darken to various shades of reddish brown in KOH. The spores of Pachylepyrium fulvidula are brownish yellow to yellowish brown in water
mounts and darken to brown (not reddish brown) in KOH.
Taxonomy
Crassisporium Matheny, P.-A. Moreau & Vizzini
gen. nov.
MYCOBANK No: MB 807853.
TYPE SPECIES: Pholiotina funariophila M.M. Moser,
1954.
ETYMOLOGY: crassus, Latin, means thick, and sporium,
Latin, spore; in reference to the thick-walled basidiospores (gender: neuter).
Downloaded by [University of Tennessee, Knoxville] at 13:56 31 October 2014
Two new genera of dark-spored Agaricales
Agaricoid
clade
Galerina sp. PR6575
Galerina marginata
Galerina semilanceata
Galerina atkinsoniana
Gymnopilus spectabilis
Gymnopilus sapineus
Galerina sp. NLB293
Psilocybe cyanescens
77
Psilocybe subaeruginosa
Psilocybe cubensis
Psilocybe stuntzii
Psilocybe
s ocybe
y caerulipes
ca
ae u p
pes
‘Pa
achylepy
pyyrium funariophilum’
fun
nariophil
p lum’ TENN
TENN053270
N053270
‘Pachylepyrium
ll t
Anamika angustilamellata
Hebeloma affine
Hebeloma velutipes
Hebeloma olympianum
Undet Hymenogastraceae sp. PBM3420
Undet Hymenogastraceae sp. PBM3116
Alnicola escharioides
Flammula alnicola
Hymenogastraceae
Phaeocollybia festiva
Hypholoma fasciculare
Hypholoma subviride
Strophariaceae
Hypholoma australe
Hypholoma sublateritium
“Naematoloma” longisporum
Stropharia ambigua
Stropharia rugosoannulata
Pholiota aff. astragalina
Nivatogastrium nubigenum
Pholiota multicingulata
Pholiota squarrosa
Agrocybe smithii
Agrocybe praecox
Agrocybe pediades
Agrocybe rivulosa
Deconica montana
“Pachylepyrium
funariophilum”
“Pa
achylepy
y pyyrium fun
nariophillum” TENN
TENN051714
N051714
“Psilocybe
P
silvatica”
Deconica sp. PBM3781
Kuehneromyces
y
rostratus
Pachylepyrium
carbonicola
P
achylep
pyrium ca
arbonicola
a TENN02878
TENN028785
85 (=type)
P
achylep
h l pyrium
i
ca
arbonicola
b i la TENN02878
TENN028784
84 (=type)
Pachylepyrium
carbonicola
Crassisporium
P
Pachylepyrium
achylep
hyl pyyrium
i
fun
ffunariophilum
nariophilu
i philum Moser49
Moser49/08
9/08 (type)
gen. nov.
Pachylepyrium
Pa
achylepyr
y p
pyrium funariophilum
funa
ariophilum
p m Moser49/222
Pachylepyrium
carbonicola
Pa
achylepyrrium carb
bonicola PB
PBM2293/PBM1411
BM2293/PBM1411
Galerina clavus C15122007
Romagnesiella gen. nov.
Galerina clavus PAM06090110 (epitype)
Crepidotus variabilis
Crepidotus sp. PBM3463
Crepidotus cf. applanatus
Simocybe sp. PBM3031
Simocybe serrulata
Pleuroflammula praestans
Pleuroflammula flammea
Crepidotaceae
Pleuroflammula tuberculosa
Inocybe lilacina
Inocybe aff. asterospora
Inocybe mutata
Inocybe rimosoides
Inocybe unicolor
Inocybaceae
Inocybe myriadophylla
Tubaria serrulata
Tubaria confragosa
Tubaria vinicolor
Tubaria furfuracea
Tubaria sp. PBM3355
Tubaria sp. BM378_17
Phaeomyces dubiosus
Flammulaster sp. PBM3449
p PBM1871
Flammulaster sp.
Pachylepyrium
fulvidula
P
achyle
epyrium
m fulvidu
ula Okada
Okada170163
a170163
Pachyle
h lepyrium
i m fulvidula
fulvidu
f l idulla T14955 ((type)
Pachylepyrium
Tubariaceae
i
Phaeomarasmius proximans
Agrocybe erebia
Conocybe apala
Conocybe smithii
Bolbitius vitellinus
undet Bolbitiaceae
Pholiotina filaris
sp. PBM3032
Descolea maculata
Descolea recedens
Descolea phlebophora
Descolea tenuipes
Panaeolus papilionaceus
Panaeolus sphinctrinus
Bolbitiaceae
Panaeolina foenisecii
Cortinarius aurilicis
Cortinarius sodagnitus
Cortinarius iodes
Cortinarius bolaris
Cortinariaceae
Cortinarius violaceus
Agaricus bisporus
Agaricus campestris
Agaricus sylvaticus
Hymenagaricus taiwanensis
Chlorophyllum agaricoides
Undet. Agaricaceae sp. RC_Mart06_016
Leucoagaricus barssii
Lycoperdon pyriforme
Langermannia gigantea
Macrolepiota procera
Macrolepiota dolichaula
Lepiota cristata
Verrucospora flavofusca
Coprinus comatus
Agaricaceae
Lepiota maculans
Tulostoma macrocephala
Psathyrella spadicea
Psathyrella rhodospora
Lacrymaria velutina
Psathyrella candolleana
Psathyrella gracilis
Coprinopsis atramentaria
Coprinopsis cinerea
Coprinellus disseminatus
Psathyrellaceae
Mythicomyces corneipes
Cyathus striatus
Nidulariaceae
Crucibulum laeve
Nidula niveotomentosa
Mycocalia denudata
Squamanita paradoxa
Squamanitaceae
Cystoderma amianthinum
Laccaria bicolor
Laccaria pumila
Laccaria ochropurpurea
Laccaria amethystina
Hydnangiaceae
Hydnangium carneum
5
0.07 expected substitutions
per site
Fig. 3. Phylogeny of the Agaricoid clade based on a Maximum Likelihood and Bayesian Inference analysis of a supermatrix of four
nuclear gene regions (5.8S rRNA, LSU-rRNA, SSU-rRNA and rpb2 conserved domains 5 7). Thickened branches indicate ML bootstrap support >70% and Bayesian posterior probability >0.95. Nodes that receive Bayesian posterior probabilities >0.95 but with
<70% ML bootstrap are indicated by small black-filled circles. Clade nomenclature follows Matheny et al. (2006). Grey shaded taxon
labels indicate placement of species of Pachylepyrium or collections mislabelled Pachylepyrium.
6
P. Brandon Matheny et al.
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0.07 expected substitutions
per site
Clitocybe aff. fellea PBM3028
Callistosporium graminicolor
Catathelasma ventricosum
Cleistocybe carneogrisea
Catathelasma clade
Cleistocybe vernalis
Entoloma canescens
Inocephalus sp. GD_b
Entoloma strictius
Entoloma sericeum
Entoloma sinuatum
Entoloma “prunuloides”
Clitopilus prunulus
Entolomataceae
Rhodocybe mundula
“Leucopaxillus albissimus”
Tricholoma inamoenum
Tricholoma myomyces
Tricholoma palustre
Tricholoma matsutake
Tricholoma saponaceum
Tricholomataceae
Porpoloma sp. PR3995
Termitomyces microcarpus
Termitomyces sp. ZA164
Tephrocybe boudieri
Calocybe carnea
Calocybe gangraenosa
Ossicaulis lignatilis
Tricholomella constricta
Asterophora lycoperdoides
Lyophyllum aff. decaste
Lyophyllum decastes
Lyophyllaceae
Lyophyllum sp. PBM2688
Clitocybe candicans
Clitocybe p.p.
Clitocybe subditopoda
Lepista personata
Lepista sordida
Clitocybe dealbata
Clitocybe nebularis
Lepista irina
Clitocybeae
Clitocybe adirondackensis
Pseudoclitocybe cyathiformis
Outgroups
Infundibulicybe gibba
Fig. 3. (Continued)
DIAGNOSIS: Basidiomata naucorioid, pileus hygrophanous, veil present. Lamellae attached (adnate). Basidiospores smooth, ovate or subangular, wider in face view
than in profile, thick-walled (>0.5 mm thick) and with a
broad or conspicuous germ pore (often >0.5 mm wide),
brownish yellow to golden yellow in water mounts,
becoming rusty brown to reddish brown or rich reddish
cinnamon in KOH. Pleurocystidia and chrysocystidia
absent, cheilocystidia present. Pileipellis a cutis, not
gelatinized. Hymenophoral trama regular to subregular,
subhymenium not gelatinized. Clamp connections present. Carbonicolous. Typus: Pholiotina funariophila M.M.
uhner & Romagnesi, Bull. Soc. nat. Oyonnax
Moser, in K€
8: 43, 1954.
Crassisporium chilense (M.M. Moser) Matheny, P.-A.
Moreau & Vizzini, comb. nov.
MYCOBANK no: MB 807854.
BASIONYM: Pachylepyrium chilense M.M. Moser,
Hoppea 61: 268, 2000, holotype seen. Chile.
Remarks
The carbonicolous habit and the smooth thick-walled basidiospores (c. 1.0 mm thick) with a distinct broad germ pore
and rust brown colouration in KOH support transfer to
Crassisporium. The species has been recorded from highelevation (550 m) Nothofagus forests in Chile and differs
from P. funariophilum by the somewhat smaller spores
(7.0 8.5 £ 4.5 6.0 mm versus 7.5 10.0 £ 5.5 7.0 mm)
and a more weakly developed and ochraceous veil, which
is more strongly developed and white in P. funariophilum
(Moser, 2000). Unfortunately, material sent to us from the
holotype collection was not sufficient for examination and
DNA extraction. However, upon examination of materials
labelled Pachylepyrium funariophilum located at MICH,
we found the spores of one collection (M3269, Validivia,
Chile, including one small fruit body) to measure 7.0 8.5
£ 4.5 6.0 mm. Based on the taxonomic key below, this
material keys out to Crassisporium chilense due to the
smaller spores and occurrence in Chile. Moser (2000) mistakenly cites two different collections (M3208 in the Latin
diagnosis and M3269 in the German description) as the
isotype of Pachylepyrium chilense. We suspect the designation of M3269 as isotype is an error as the referenced
collection date (3 Mar. 1963) in Moser’s publication does
not match that for M3269 (31 Mar. 1963). Under this scenario M3269 is the paratype. Unfortunately, the collection
date on the holotype packet (IB 1963/0210) is 30 Mar.
1963 (unlike the protologue), but the locality is exactly that
of M3269. Permission to extract DNA from the one small
fruit body of M3269 (paratype) was not granted owing to
the inadequate condition of this material.
Crassisporium funariophilum (M.M. Moser) Matheny,
P.-A. Moreau & Vizzini, comb. nov.
(Fig. 2, as Pachylepyrium carbonicola).
MYCOBANK No: MB 807855.
Two new genera of dark-spored Agaricales
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BASIONYM: Pholiotina funariophila M.M. Moser, in
K€
uhner & Romagnesi, Bull. Soc. nat. Oyonnax 8: 43,
1954, holotype seen. Austria.
Pachylepyrium funariophilum (M.M. Moser) Singer,
in Singer & Moser, Mycopath. Mycol. Appl. 26(2-3): 171,
1965.
D Kuehneromyces carbonicola A.H. Sm., Beihefte zur
Sydowia 1: 53, 1957. Holotype seen. Idaho.
Pachylepyrium carbonicola (A.H. Sm.) Singer,
Sydowia 11: 321, 1958 [1957].
Pholiota subangularis A.H. Sm. & Hesler, The North
American Species of Pholiota: 44, 1968.
Remarks
Crassisporium funariophilum is geographically widespread occurring in Europe, northern Africa and western North America (where it has been referred to as
Pachylepyrium carbonicola and Pholiota subangularis)
and may be expected elsewhere. Singer & Moser
(1965) and Singer (1969) also report it from Argentina,
but this material has not been revised in light of
description of P. chilense (see above). Moser (2000)
describes collections of P. carbonicola with a white
fugacious veil and similar ecology to P. funariophilum,
but with somewhat larger spores (8.2 12.1 £
6.5 8.3 mm) than for P. funariophilum (7.6 10.0 £
5.3 7.1 mm). However, pairwise comparison of ITS
sequences from the type collections of C. funariophilum and P. carbonicola differ at only two nucleotide
positions (excluding three polymorphic sites among the
five sequences considered) strongly suggesting the two
species are conspecific. As such, P. funariophilum has
nomenclatural priority.
Crassisporium squarrulosum (Singer) Matheny, P.-A.
Moreau & Vizzini, comb. nov.
MYCOBANK no: MB 807856.
BASIONYM: Pachylepyrium squarrulosum Singer, Beih.
Nova Hedwigia 29: 281, 1969, holotype not seen. Chile.
Remarks
We have not studied material of C. sqarrulosum, but the
thick spore wall with a truncate germ pore and intense
‘ferruginous’ colouration in KOH described by Singer
(1969) are consistent with placement in Crassisporium
rather than with the type of Pachylepyrium in the Tubariaceae or with Romagnesiella. The species is associated
with burnt debris and occurs at high elevations (1000 m)
in Chile. The type (M 6550) is reportedly at SGO. The
species differs most readily from C. chilense by the
7
flocculose-squarrose pileus surface and longer spores
(12.0 14.0 £ 6.5 8.0 mm).
Pholiota nubicola (Singer) Matheny & P.-A. Moreau,
comb. nov.
MYCOBANK No: MB 807857.
BASIONYM: Pachylepyrium nubicola Singer in Dennis,
Kew Bull. 15(1): 139, 1961, holotype seen. Venezuela.
Remarks
The ITS sequence produced from the holotype strongly
suggests that Pachylepyrium nubicola is a species of Pholiota. Consistent with this placement are the caespitose
and lignicolous habit, paler (yellowish) pigmented basidiospores with a thinner wall than in Crassisporium,
strongly gelatinized pileipellis composed of coarsely
incrusted yellowish hyphae, and gelatinized subhymenial
trama. Add to this the slightly phaseoliform spores with a
distinct germ pore (0.8 1.0 mm wide) and the squamulose
stipe covering, it is not surprising P. nubicola would be
closely related to Ph. gummosa (Lasch: Fr.) Singer as
described by Holec (2001).
In contrast to the protologue, our examination of the
type revealed a gelatinized pileipellis and cylindric to
subphaseoliform non-dextrinoid basidiospores, these
with a distinct germ pore. The spores measure 7.5 8.8
£ 4.5 4.8 mm, which is consistent with the protologue. The basidia measure 17 28 £ 7 8 mm with
yellowish contents when mature. The lamellar edge
was observed to be sterile and yellow but without
reviving elements. The presence or absence of chrysocystidia could not be confirmed, but given the high
sequence similarity to ITS sequences labelled Ph. gummosa and Ph. terrestris, we predict chrysocystidia will
be found in this species.
A taxonomic key to species of Crassisporium
1(a) Pileus surface flocculose-squarrose, spores 12 14 £
6.5 8 mm. . . .................................C. squarrulosum Singer
1(b) Pileus surface glabrous or with marginal fibrils,
spores 7 11.5 £ 5.5 7 mm. . .. . .. . .. . .. . . ..................... .2
2(a) Spores mostly 8 11.5 £ 5.5 7 mm, in north temperate forests of Europe, North Africa and western North
America (also reported from southern South America, but
this is likely C. chilense); veil well developed, white. . .. . .. . .. . .. . .. . . .. C. funariophilum (M.M. Moser) Singer
2(b) Spores mostly 7 9 £ 4.5 6 mm, in Nothofagus
forests in southern South America; veil weakly developed,
ochraceous. . .. . .. . .. . .. . .. . .. . . ... C. chilense M.M. Moser
8
P. Brandon Matheny et al.
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Romagnesiella Contu, P.-A. Moreau, Vizzini & A. de
Haan, gen. nov.
MYCOBANK No.: MB 519559.
TYPE SPECIES: Galerina clavus Romagn., 1944 [1942].
ETYMOLOGY: named in honour of Henri Romagnesi,
French mycologist (1912 1997) (gender: feminine).
DIAGNOSIS: Basidiomata naucorioid, lamellae distant,
adnate to subdecurrent; pileus dry, not hygrophanus; stipe
smooth, without a partial veil. Basidiospores smooth,
more or less ovate, not subangular, yellow in water
mounts, reddish ochre in KOH, not dextrinoid, germ pore
absent; necrobasidia numerous; cheilocystidia present,
edges of lamellae smooth and (sub)sterile, pleurocystidia
present but dispersed and infrequent, pileipellis filamentous, hymenophoral trama regular, clamp connections frequent. On unburnt soil or sand among mosses and grasses.
Typus: Galerina clavus Romagn., Bull. Trimest. Soc.
Mycol. France 58(4): 149 (1944 [1942]).
Romagnesiella clavus (Romagn.) Contu, P.-A. Moreau,
Vizzini & A. de Haan, comb. nov.
(Figs 1, 4 8).
MYCOBANK No.: MB 519560.
BASIONYM: Galerina clavus Romagn., Bull. Trimest.
Soc. Mycol. France 58(4): 149, 1944 [1942], lectotype
designated here (Fig. 14, p. 145, Romagnesi (1944)
[1942], MBT177567); epitype designated here (P.-A.
Moreau 06090110, LIP, MBT177568). Switzerland.
Naucoria clavus (Romagn.) K€uhner & Romagn., Fl.
Anal. Champ. Sup.: 239 (1953, comb. inval., Art. 33.4).
MISAPPLICATIONS: Tubaria minima J.E. Lange sensu
Moreau in Matheny et al. (2007a: 571); Galerina embolus
Figs. 4 8. Anatomical features of Romagnesiella clavus (PAM06090110, epitype). (Fig. 4) Spores. (Fig. 5) Basidia and subhymenium.
(Fig. 6) Cheilocystidia. (Fig. 7) Pleurocystidium. (Fig. 8) Pileipellis. Scale bars D 10 mm.
Two new genera of dark-spored Agaricales
(Fr.) Sacc. sensu Orton (1960: 239), sensu de Haan &
Walleyn (2009: 64).
BIBLIOGRAPHY: Romagnesi (1942: 144, protologue);
K€
uhner & Romagnesi (1953: 239; description); Smith &
Singer (1964: 336); de Haan & Walleyn (2009: 64, 66:
description, picture); North African collections: Hausknecht & Zuccherelli (1993: 47), Moreau (2009: 199).
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Description
Pileus 5 9 (12) mm diam, hemispheric-umbonate then §
depressed around umbo, margin early inrolled becoming
shortly crenulate when old, even, not striate, densely furfuraceous-micaceous, grey-brown with somewhat purplish tones when fresh, paler at margin, quickly fading
from margin to uniformly fleshy-ochre, without any trace
of veil. Lamellae adnexed-ventricose at first, becoming
shortly uncinate in age, distant with 14 16 L reaching
stipe, interspersed by 1 2 series of lamellulae, dull rusty
ochre even when young; edges smooth but (sub)sterile,
pale yellow. Stipe 15 25 £ 1 mm, flexuose, slightly
attenuate at base and inflated at apex, pruinose-floccose
just below lamellae, fibrillose below then glabrous against
a uniform dirty brown ground colour, slightly purplish
when young; no perceptible trace of veil (primordia not
observed). Context dark brown when fresh, pale ochre
when dry. Odour and taste fungoid, not remarkable.
Basidiospores (5.6) 6.2 6.7 7.3 (8.5) £ (3.6)
3.9 4.2 4.4 (5.0) mm, Q D 1.51 1.62 1.73 (n D 48),
ovate to obovate but longer spores more fusiform, smooth,
germ pore absent; bright yellow in water, amber yellow in
Melzer’s, warm reddish ochre in KOH, wall thickened up
to 0.3 (0.5) mm; content with a large central droplet, often
elongate. Basidia four-spored, 28 36 £ 7 9 mm, broadly
clavate, with long sterigmata, content often microguttulate;
necrobasidia abundant, with reddish-brown content. Cheilocystidia 22 45 £ 5.5 7 mm, cylindrical-flexuose with
slightly thickened yellowish wall, mixed with fascicles of
terminal hyphae issued from trama with pear-shaped to
subglobose terminal cells, 9 14 mm wide, lamella edge
fertile to locally substerile. Pleurocystidia 38 42 £
7.5 13 mm, cylindrical to subutriform, not very distinct
but not rare. Hymenophoral trama regular, with strongly
encrusted hyphae, 3 5 mm wide. Pileipellis a superficial
layer of short cells, these lobate, digitate, puzzle-like, fusiform or pyriform, 12 16 mm wide, more or less erected to
nearly hymeniform towards margin, pale in KOH, smooth,
issued from hyphae of subpellis; subpellis filamentous,
coarsely encrusted, thick-walled, deep yellow to reddish
brown in KOH, continuous with pileus context. Stipitipellis
a cutis with sparse to fasciculate (at apex) caulocystidia
measuring 16 25 £ 5 12 mm, cylindrical to clavate-pyriform, very rare below apex; superficial hyphae slender,
2 3.5 mm wide. Clamp connections frequent.
9
Habitat and distribution: Often on calcareous, mineral-rich, sandy or alluvial substrates in pioneer or disturbed habitats including fixed coastal dunes and banks of
trails or paths among mosses and grasses. Less frequent in
secondarized dunes, scattered and never abundant. Europe
(Belgium, France, Switzerland) and reported from Italy,
the Netherlands, and North Africa. Fruiting Sept. Nov.
Material studied: BELGIUM. Antwerpen: AntwerpenLinkeroever, Het Rot, 4 Sep 2004; 10 specimens, among
grass and mosses (Tortula ruralis and Ceratodon purpureus) on sandy, calcareous soil, herb. A. de Haan n 04101;
Antwerpen-Linkeroever, Blokkersdijk, 9 Sep 2004, 2
specimens, among mosses on sandy, calcareous soil, herb.
A. de Haan n 04113. Namur: Oignies-en-Thierache,
l’Estache, 23 Sep 1999, 1 specimen on wet calcareous
soil, herb. A. de Haan n 99100. Oost-Vlaanderen: Zwijnaarde, Rijvisschepark, 15 Oct 1989, about ten carpophores, in bare spot in mossy lawn, on sandy slightly
loamy soil, leg. P. Van der Veken, herb. A. de Haan n
89017; same location, leg. P. Van der Veken, 4 specimens, 29 Oct 2003, herb. A. de Haan n 03088. WestVlaanderen: De Panne, Calmeijnbos, 3 Nov 1997, 4 specimens, on humus-rich, calcareous soil, herb. A. de Haan n
97088; Oostduinkerke, Doornpanne, 1 Nov 2001, 2 specimens, among moss and lichens, on calcareous dune sand,
herb. A. de Haan n 01080 (as ‘Galerina embolus’).
FRANCE. Pas-De-Calais: Equihen-Plage, dunes d’Ecault,
7 November 2004, five specimens in Phleo-Tortuletum
with Calamagrostis epigeos, calcareous fixed dune, leg.
A. Brabant & P.-A. Moreau, 7 Nov 2004, herb. P.-A. Moreau n 04110710 (LIP); same location, along a sandy path
amongst Calamagrostis epigeos, fixed calcareous dunes
with Hippophae rhamnoides, leg. C. Hannoire & P.-A.
Moreau, 31 Oct 2008, herb. P.-A. Moreau n 08103102
(LIP). Seine: Paris, bois de Vincennes, 1 Oct 1932, herb.
R. K€uhner (G, as ‘Tubaria oligophylla’, ined.). ITALY:
Sardinia, prov. Olbia, Golfo Aranci, Golfo di Marinella,
in troops on sandy soil in a coastal grassland, leg. M.
Contu, 15 Dec 2007, herb. M. Contu (C15122007, TENN
063957). SWITZERLAND. Gr€aubunden: Rothenbrunnen,
edge of path, riparian Alnus incana forest, on black alluvial humus, 1 Sep 2006, leg. B. Senn-Irlet & P.-A. Moreau, herb. P.-A. Moreau n 06090110 (epitype LIP),
TENN 063587, TENN 063976.
Remarks
Our interpretation of Galerina clavus is based on the
detailed protologue of Romagnesi (1944 [1942]), which
matches collections from Belgium and Switzerland.
Unfortunately, no original material of Galerina clavus
exists. The herbarium packet corresponding to one of the
two collections cited by Romagnesi (1942: 145) Yerres,
bois de Cerçay, 18 Jun 1942, kept in herb. H. Romagnesi
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10
P. Brandon Matheny et al.
(PC) was empty. The other cannot be located. Because a
figure that depicts G. clavus exists in the protologue, this
must serve as the lectotype. Original drawings of G.
clavus made by Romagnesi also exist at PC. Thus, we designate PAM06090110 (LIP) as an epitype.
The species features some morphological and microscopic variation. The epitype (sequenced here), showed
purplish-grey tones on the stipe as well as the pileus, but
the protologue only mentions this colour on the stipe. Belgian collections described by de Haan & Walleyn (2009)
describe a more convex pileus, broadly adnate instead of
subdecurrent lamellae, a ‘weakly farinaceous’ taste, and
habitat in dry mineral spots in urban grasslands. However,
all collections show gradual variation in the pileus shape,
colour and lamellae attachment.
Coastal collections are probably better considered as variants of R. clavus. Despite some variation in spore dimensions we could not find any support for specific or
infraspecific distinctions. Detailed spore measurements
illustrate continuity in these variation patterns but apparent
differences (Appendix 2, see supplemental material online).
North African and Sardinian collections of Galerina
clavus, as described by Hausknecht & Zuccherelli (1993)
and Malençon (Moreau, 2009), differ from continental
collections by somewhat larger basidiospores [(7.0)
7.6 8.2 9.0 (10) £ (4.0) 4.9 5.2 5.5 (5.7) mm, Q D
1.48 1.59 1.70], slightly larger cystidia, and a filamentous pileipellis with a more or less continuous suprapellis
of slender cylindrical hyphae with sparse slightly upraised
terminal elements. It is possible that Mediterranean collections may represent distinct populations. One collection from Sardinia was sequenced (C15122007, leg. M.
Contu), in which the ITS1 region reveals nine site differences with R. clavus PAM6090110, four of which, however, are polymorphic in C15122007. Galerina clavus has
also been reported from the Netherlands (www.
verspreidingsatlas.nl/046620).
Romagnesiella clavus is probably often confused with
other naucorioid species frequent in the same environment,
uhner, Psilocybe
such as Galerina graminea (Velen.) K€
pratensis P.D. Orton, or Tubaria spp. The distant lamellae
and absence of a veil on the stipe are good distinctive field
characters. However, we discuss below three additional
species with which R. clavus could be confused.
Tubaria umbonata S. Lundell in Lundell & Nannfeldt
(1953: 23).
(Figs 9 13).
MYCOBANK No. 307168.
ISOTYPE: SWEDEN. Upland: Uppsala, Slottsbaken,
NW part below Gunillaklaken, 50 m from Stockholmsv€agen, 6 August 1944, leg. S. Lundell, ex Fungi exsiccati
Suecici 2041 (PC, about 20 well-preserved specimens).
Description
Exsiccata small to minute (2 6 mm), very slender,
entirely dark brown, without visible veil, with arcuate and
distant lamellae. Basidiospores (6.2) 6.5 7.2 8.0 (9.0) £
(3.0) 3.2 3.7 4.2 mm, Q D 1.70 1.96 2.24 (n D 23;
see also Appendix 2, see supplemental material online),
pale yellow, slightly thick-walled (<0.5 mm thick), ochraceous yellow in KOH, not collapsing, narrowly ovo-ellipsoidal to ellipsoidal, with slightly guttulate content, not
dextrinoid. Basidia 22 34 £ 6.5 7.5 mm, 4-spored
(occasionally 2-spored), clavate more or less capitate,
often strangulate under apex before maturity, hyaline;
subhymenium 12 15 mm thick, filamentous-ramose, with
hyphae 2 2.5 mm wide. Cheilocytidia 16 30 £
6 11.5 mm, often clustered ampullaceous, clavate, ellipsoidal, utriform, cylindrical, with a thin and smooth wall,
intermixed with some fertile basidia; lamella edge almost
sterile. Pleurocystidia if present, not studied. Hymenophoral trama regular, yellowish, made of slender hyphae
2 6 mm wide with thick encrusted wall. Pileipellis with a
discontinuous suprapellis made of C/¡ erected wide ellipsoidal to cylindrical catenulate elements, 18 30 £
6 13 mm, pale, slightly thick-walled, not or only locally
encrusted; subpellis made of slender hyphae 3.5 8 mm
wide, distinctly encrusted by granular pigment remaining
yellow in KOH. Stipitipellis with sparse traces of filamentous veil towards apex, composed of slender hyphae
3 5 mm wide; wall yellowish, up to 2 mm thick and
encrusted, terminal cells cylindrical with some vesicular
cells up to 20 mm wide; superficial hyphae slender, these
3 5 mm wide, with yellow walls up to 0.5 mm thick,
intermixed with large cylindrical hyphae 9 16 mm wide,
locally encrusted by gold-yellow pigment (KOH), especially at septa, and some sparse pale gleoplerous hyphae.
Clamp connections present at septa.
Remarks
Tubaria umbonata has not been revised nor documented
since its publication in Fungi Exsiccati Suecici (Lundell
& Nannfeldt 1953). We studied the isotype at PC (Fungi
Exsiccati Suecici fasc. 41 42). Based on our morphological analysis, we conclude that T. umbonata represents a
genuine species of Tubaria (W.G. Sm.) Gillet, but with
rather narrow spores. DNA extraction of the PC material
of T. umbonata yielded no PCR amplicons. Illustrations
of anatomical features (Fig. 5) and their description
(above) from the isotype are presented.
Two additional species could be confused with R.
clavus. The name Tubaria minima J.E. Lange (Lange,
1940) was misapplied by Moreau (in Matheny et al.,
2007a) to collections of R. clavus. Although Bon (1992)
maintains T. minima as an autonomous species, Romagnesi (1942) considers it to be a synonym of T. minutalis
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Two new genera of dark-spored Agaricales
11
Figs. 9 13. Anatomical features of Tubaria umbonata (Fungi Exsicatti Suecici fasc. 41 42, isotype). (Fig. 9) Spores. (Fig. 10) Basidia
and subhymenium. (Fig. 11) Cheilocystidia. (Fig. 12) pileipellis. (Fig. 13) Stipitipellis. Scale bars D 10 mm.
Romagn. (Romagnesi, 1937), a position followed by modern authors. This species (sensu Lange (1940), non Moreau) differs from G. clavus by its hygrophanous piles and
smaller spores (5.2 6.0 £ 3.2 3.8 mm), features that
reinforce its conspecificity with T. minutalis.
Tubaria embolus (Fr.) Sacc. is rather frequently cited
in the literature but has been interpreted several different
ways. Lange (1938: 655, pl. 127B) illustrates as
‘Tubaria embola’ a species with broadly adnexed lamellae and yellow tones especially in the context (conforming to Fries’ protologue, 1836 1838: 206), which seems
to represent Agrocybe pusiola (Fr.) R. Heim. Bon (1992)
cites the species in the genus Galerina, but examination
of his materials (LIP) showed that his concept was
unclear: coll. 741120 is Galerina uncialis (Britzelm.)
K€
uhner, and coll. 70624 (as ‘Galerina cf. embolus’) is a
species with pleurocystidia close to Galerina
vittaeformis (Fr.) K€uhner. Orton (1960: 176) mentions
five reports of T. embolus (as ‘Galerina embolus’) from
sand dunes, with comparable microscopical characters
(but with notably long spores, 9 11 £ 4.5 6 mm, compatible with our coastal collections of R. clavus), but
pleurocystidia and necrobasidia are not mentioned.
Moreover, yellow tones are described towards the pileus
margin when dry, incompatible with any species known
to us. In addition de Haan & Walleyn (2009) describe
without illustrations a collection of G. embolus (reported
here as R. clavus) and also found in fixed dunes in Belgium. Considering the ambiguities of the protologue
(Fries, 1836 1838), and the diversity of interpretations
proposed by various authors, we reject the name here.
Additional morphological and molecular study is
required to unravel the taxonomic relationships of these
variously interpreted collections to Romagnesiella.
12
P. Brandon Matheny et al.
Discussion
Downloaded by [University of Tennessee, Knoxville] at 13:56 31 October 2014
Polyphyly of Pachylepyrium and recognition
of Crassisporium and Romagnesiella as new
genera
Our results strongly support the polyphyletic status of
Pachylepyrium. The type species of the genus, P. fulvidula, lacks several of the features attributed to the residual
species. Originally described in Phaeomarasmius, P. fulvidula fruits on non-burnt woody debris and lacks the
broad germ pore observed in other species. Horak (1968)
reports seldom seeing any germ pore at all in the type of
P. fulvidula. Our examination of the isotype at MICH confirms this observation (a germ pore was not observed).
Thus, it is not surprising to see phylogenetic placement of
P. fulvidula apart from the residual Pachylepyrium species. Pachylepyrium fulvidula resides in the Tubariaceae
(Fig. 3) where it is closely related to other species of
Flammulaster, Phaeomyces, Phaeomarasmius and Tubaria (W.G. Sm.) Gillet, all of these also lacking a broad
germ pore (Horak, 2005).
We place three residual species of Pachylepyrium in the
new genus Crassisporium united by a combination of
basidiospore features (thick-walled spores with a broad
germ pore and rusty to reddish brown colouration in
KOH), anatomical features (non-gelatinous cutis, absence
of pleurocystidia and chrysocystidia, absence of a gelatinous subhymenial layer) and ecology (carbonicolous
habit). However, before our phylogenetic analysis based
on molecular data, we did not suspect that Galerina clavus
would be related to Crassisporium more so than to any
other group of Hymenogastraceae or Strophariaceae. In
order to point out differences between typical carbonicolous species with thick-walled pored spores (Crassisporium species) and non-carbonicolous species with thinnerwalled (<0.5 mm) spores such as G. clavus, we have proposed a new genus Romagnesiella to accommodate the
latter. No extra-European or North African species are
unequivocally attributable to Romagnesiella at present
without the addition of detailed morphological and molecular study. Galerina fuegiana Singer from Patagonia
(Smith & Singer, 1964) is a possible candidate.
The inclusion of sequences of Crassisporium and
Romagnesiella in a multigene phylogenetic analysis of
the Agaricoid clade shows these two taxa form a well-supported group (Fig. 3) sister to the Strophariaceae s.lat.
consortium (Gulden et al., 2005), including the families
Hymenogastraceae and Strophariaceae s. str. of Matheny
et al. (2006). Inclusion of Crassisporium and Romagnesiella in Strophariaceae s. str. would render the family
paraphyletic in this analysis. Consideration of a more
broadly conceived Hymenogastraceae, subsuming the
Strophariaceae, could be made since the name Hymenogastraceae Vitt. 1831 pre-dates that of the Strophariaceae
Singer & A.H. Sm. 1946. However, additional taxon and
gene sampling are needed to resolve the relationship
between these two families.
An alternative scenario to consider is inclusion of the
three species of Crassisporium into one genus with R.
clavus, thereby describing only a single genus as new.
Samples of each group form a clade with strong support, a
synapomorphy of which are the basidiospores that deepen
various shades of reddish brown in KOH. However, we
favour separate genera for the two lineages for several
reasons: (1) species of Crassisporium are carbonicolous,
whereas those of Romagnesiella are non-carbonicolous;
(2) the lamellae are adnate to subdecurrent in Romagnesiella but never subdecurrent in Crassisporium; (3) a veil
is absent in Romagnesiella but present in Crassisporium;
(4) the basidiospores of Crassisporium feature walls
>0.5 mm thick, a wide germ pore typically 1.0 1.5 mm
thick, and are subangular in face view. These features
may be correlated with the fire ecology in that heat may
be required to induce germination (Claridge, Trappe, &
Hansen, 2009). Basidiospores of Romagnesiella have
thinner walls (<0.5 mm thick), no germ pore and are not
subangular; (5) pleurocystidia are present in Romagnesiella but absent in Crassisporium; and (6) the relative
branch length differences between the two lineages correspond to branch length differences between other genera
of Strophariaceae and Hymenogastraceae. Therefore, we
prefer to recognize the two lineages as separate genera.
The monophyly of the Agaricoid clade is recovered here
for the first time with high bootstrap and significant Bayesian posterior probability. Most species in the Agaricoid
clade tend to have pigmented and thick-walled spores, perhaps indicative of adaptations to novel environments (e.g.
dung, burnt sites) (Garnica et al., 2007). Many species of
the Agaricoid clade also feature multiple nuclei per spore
and an open pore type of hilum (Matheny et al., 2006).
Acknowledgements
The authors are grateful to staff at herbaria G (P. Clerc),
IB (R. Kuhner), LIP (R. Courtecuisse and C. Lecuru),
MICH (R. Rabeler, P. Rogers), MPU (V. Bourgade, L.
Gomel and M.-J. Mauruc), PC (B. Buyck), PERTH (N.
Bougher), and the USDA Forest Service, Luqillo, Puerto
Rico (D. J. Lodge) for loan of collections. Egon Horak
(Innsbruck, Austria) is acknowledged for his study on Sardinian collections sent by M. Contu. Regis Courtecuisse
(Lille, France) is also acknowledged for his valuable suggestions and bibliographic expertise. Aaron Wolfenbarger, Emily Giles, Whitaker Hoskins, Sarah Sprague,
and Christine Braaten provided laboratory assistance at
the University of Tennessee. Three anonymous reviewers
and the Associate Editor, Karen Hansen, provided critical
feedback that helped improve this paper. Research was
conducted at the University of Tennessee, Universite Lille
Nord de France, and Universita di Torino.
Two new genera of dark-spored Agaricales
Funding
This work was supported by the U.S. National Science
Foundation under Grant DEB-0949517.
Supplemental data
Supplemental data for this article can be accessed here.
Downloaded by [University of Tennessee, Knoxville] at 13:56 31 October 2014
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