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ORIGINAL ARTICLE
Morphological characterization of clades of the Psathyrellaceae
(Agaricales) inferred from a multigene phylogeny
László G. Nagy &Csaba Vágvölgyi &Tamás Papp
Received: 20 May 2012 /Revised: 9 September 2012 / Accepted: 12 September 2012
#German Mycological Society and Springer-Verlag Berlin Heidelberg 2012
Abstract The phylogeny of the Psathyrellaceae has re-
ceived much attention recently. Despite repeated efforts for
inferring a stable phylogeny that can serve as a basis for
reclassification of the Psathyrellaceae, extensive taxonomic
rearrangements have been withheld by several factors;
among others, inadequate taxon sampling in several clades
and low support values for critical relationships. In this
paper, we present a well-resolved, robust phylogeny and
morphological circumscriptions for 14 clades of the Psathyr-
ellaceae. Sequence data from a matrix of four nuclear genes
(approximately 4,700 characters, including recoded indels)
and various phylogenetic methods were used to infer rela-
tionships. Unexpected relationships and other morphologi-
cally informed phylogenetic hypotheses have been tested by
constraint analyses. Nearly fully resolved consensus trees
have been obtained, with strong support for most of the
large clades and several early evolutionary events. We iden-
tified poorly sampled regions of the phylogeny and discuss
potential additions and extensions of the phylogeny for
future sampling. Prospects for and potential pitfalls of a
future reclassification are discussed in detail.
Keywords Basidiomycetes .Deliquescence .Coprinopsis .
Psathyrella .Taxonomy .Classification .Bayesian mixture
model
Introduction
The mushroom family Psathyrellaceae Vilgalys, Moncalvo
& Redhead contains small to large, dark-spored agarics,
which are generally considered difficult to identify to spe-
cies. Many of the taxa are deliquescent, well known for their
ability to digest themselves by means of autodigestive chi-
tinases (Kües 2000). These, known as "inky caps" or Copri-
nus s.l., include species used as model organisms in various
fields, spanning fungal ontogeny, breeding biology, devel-
opmental biology and genomics (Kemp 1975; Kües 2000;
Stajich et al. 2010). The type genus of the family, Psathyr-
ella (Fr.) Quél. by definition contains the non-autodigesting
species, mainly decomposers of leaf-litter or wood, more
rarely living on dung, or parasitizing other fungi (Kits van
Wav eren 1985;Singer1986;Smith1972). Some of the
species are well-known edible mushrooms, such as Ps. aff.
hymenocephala, which is used as a spice in Haiti (Nieves–
Rivera 2001), or C. micaceus and C. atramentarius, which
are sometimes collected for the table, although the latter is
also known to be toxic when consumed with alcohol.
Traditionally, the family (formerly treated under the name
Coprinaceae) included two large genera, Coprinus Pers.
(Fig. 1a–f)andPsathyrella (Fig. 1g–l), although several
other genera have also been included. For instance, Pseu-
docoprinus Kühner was introduced to accomodate partially
deliquescent species such as Coprinellus disseminatus
(Fig. 1e), which is now classified into Coprinellus P. Karst.
(Kühner 1928). The genera Ozonium Link and Hormogra-
phiella Guarro & Gené were established to accommodate
the conidial anamorphs of certain species now classified in
Coprinellus. These anamorph taxa, especially Hormogra-
phiella verticillata, have been reported as opportunistic
human and animal pathogens in several case reports of
severe or even fatal infections (Cáceres et al. 2006;
Rampazzo et al. 2009; Verweij et al. 1997). The name
Electronic supplementary material The online version of this article
(doi:10.1007/s11557-012-0857-3) contains supplementary material,
which is available to authorized users.
L. G. Nagy (*):C. Vágvölgyi :T. Papp
Department of Microbiology, Faculty of Science and Informatics,
University of Szeged,
Közép fasor 52,
6726 Szeged, Hungary
e-mail: cortinarius2000@yahoo.co.uk
Mycol Progress
DOI 10.1007/s11557-012-0857-3
Coprinaceae was used for many decades (Kühner and
Romagnesi 1953; Moser 1983; Singer 1986), but following
early phylogenetic studies (Hopple and Vilgalys 1994,
1999)Coprinus was split into four genera: Coprinus s.s.,
Coprinellus (Fig. 1c–e), Coprinopsis P. Karst. (Fig. 1a, f)
and Parasola Redhead, Vilgalys and Hopple (Fig. 1b), with
Coprinus s.s. belonging to the Agaricaceae (Redhead et al.
2001). A consequence of the transfer of Coprinus s.s. to the
Agaricaceae was that the name Coprinaceae became a
synonym of Agaricaceae, necessitating the establishment
of the new family name Psathyrellaceae.
The main morphologicalfeatures used in the reclassification
of coprinoid fungi were the structure of the pileipellis (cap
cuticle), the presence or absence of the universal veil and its
microstructure. Coprinopsis was defined as being deliques-
cent, having a cuticular pileipellis, always having a veil of
various structures (globose, hyphal, diverticulate, etc.) and
devoid of caulocystidia. This genus contains several well-
Fig. 1 Examples of different
groups of Psathyrellaceae. a
Coprinopsis strossmayeri,
showing rich fibrillose patchy
veil coverage on the cap, b
Parasola leiocephala,c
Coprinellus sp., a Coprinellus
species lacking all types of veil,
dCoprinellus micaceus, with a
thin layer of granular veil
remnants on the cap, e
Coprinellus disseminatus,f
Partially digested, mature pileus
of Coprinopsis stangliana.
Autodigestion starts from pileus
edge and progresses
concentrically in parallel with
spore maturation. gYoung
specimens of Psathyrella
gordonii with rich fibrillose veil
coverage, hPsathyrella
fibrillosa,iLacrymaria
vellutina,jPsathyrella gracilis,
the type species of the genus, k
Psathyrella ammophila,
lPsathyrella panaeoloides,a
species with very scanty veil, m
Spores of Coprinopsis lagopus,
nCheilocystidia of Psathyrella
prona,oCheilocystidia of
Parasola plicatilis,pSpores of
Coprinopsis calospora
(Holotype), qCheilocystidia of
Ps.lutensis,rLageniform cys-
tidia from the stipe surface of
Coprinellus pusillulus,sMi-
crostructure of veil elements of
Coprinopsis friesii wth thick
walls and diverticulate, coral-
loid appearance
Mycol Progress
known taxa, such as C. cinerea,C. lagopus or C. atramentaria.
Species of Coprinellus, on the other hand, possess hymeniform
(cellular) pileipellis, and veil, if present, consists of globose
cells. Several Coprinellus species have short, hair-like struc-
tures, pileocystidia on the cap surface and caulocystidia-like
structures on the stipes (Fig. 1r), whereas such structures are
never observed in Coprinopsis.Parasola is the only genus
consistently lacking all types of velar structures and having a
strongly grooved, parasol-like, membranous pileus. Deliques-
cence in this genus is incomplete or lacking. According to
Redhead et al. (2001), pleurocystidia are always present, but
this view has been challenged by subsequent publications
(Nagy et al. 2009;Padamseeetal.2008). An additional spe-
cies, Coprinus patuoillardii (0C. cordisporus), has not been
placed in any of the above-mentioned genera, since it was
grouped together with psathyrelloid species.
Although Psathyrella was also found to be paraphyletic, it
has not been reclassified, awaiting more species and more loci
to better understand their phylogeny and morphological evo-
lution. Sampling of additional Psathyrella taxa revealed that
the three coprinoid genera were not as uniform as formerly
thought; they include non-deliquescent psathyrelloid taxa as
well. Accordingly, Larsson and Örstadius (2008) placed Psa-
thyrella conopilus in Parasola,andPsathyrella marcescibilis
and P. p a n n u c i oi d e s in Coprinopsis. Several studies attempted
to decipher patterns of the phylogeny of Psathyrella taxa, but
the resolving power of the analyses has been limited by gene or
taxon sampling. Individual analyses uncovered single or a few
clades, but a well-supported multigene phylogeny of the Psa-
thyrellaceae phylogeny is still lacking.
In this study, we build on formerly published phylogenies
(Hopple and Vilgalys 1999; Larsson and Örstadius 2008;Nagy
et al. 2010,2011; Padamsee et al. 2008; Vasutova et al. 2008;
Wal the r et al. 2005), and extend those with additional taxa and
gene sequences (beta-tubulin) to achieve a comprehensive view
of Psathyrellaceae phylogeny. We conducted phylogenetic
analyses based on four nuclear genes, covering most of the
morphologically established groups of the family in order to
recover a robust, well-supported phylogeny of the family. We
analyzed 141 specimens using two different Bayesian
approaches and models, as well as ML and MP bootstrapping.
We also identified morphological characters that support the
phylogeny, with the aim to provide morphological key charac-
ters that correspond to recovered clade structure, and discuss
aspects of a future reclassification of the Psathyrellaceae.
Materials and methods
Taxon sampling
We extended previous sampling of taxa (98 specimens, Nagy
et al. 2011) by several species of the Psathyrellaceae from
Europe (134 ingroup taxa). Since the most severe drawback of
the current classification of the family is the paraphyly of the
genus Psathyrella and the ambiguous positions of psathyrel-
loid clades, our objective was to recover as many lineages of
the Psathyrellaceae as possible. To this end, the design of our
taxon sampling took advantage of formerlypublished phylog-
enies (Larsson and Örstadius 2008;Nagyetal.2010,2011;
Padamsee et al. 2008; Vasutova et al. 2008), extensive type
studies and a database of > 700 internal transcribed spacer
(ITS) sequences of Psathyrellaceae. Initially, we applied rough
morphological definitions for the larger clades recovered in
the above mentioned studies, and applied a denser sampling in
those clades. To identify hitherto unknown clades, we
searched the literature for species not fitting in any of the
formerly inferred larger clades, and inferred pilot phylogenies
based on only one or two genes (ITS, large subunit (LSU) or
both) with many hundred sequences. Taxa appearing as new
clades were included in the subsequent four-loci analyses.
Genbank sequences from our previous studies (Nagy et al.
2009,2010,2011)andofPs. pennata, Ps. spintrigeroides, Ps.
romagnesii, Ps. sphaerocystis, Ps. cotonea, Ps. stercoraria,
Ps. larga,andPs.gossypina (Accession numbers in order:
DQ389710, DQ389696, DQ389716, DQ389708,
AM712283, DQ389670, DQ389694, AM712294, Larsson
and Örstadius 2008; Vasutova et al. 2008)wereincludedin
the study. The complete list of specimens used in this study,
their origins, Accession Numbers and identifiers are listed in
Supplementary Table 1.
The Bolbitiaceae have been shown to be closely related
to the Psathyrellaceae (Matheny et al. 2006; Moncalvo et al.
2002), so we used representatives of this family plus Myth-
icomyces as outgroup in our analyses. GenBank sequences
of Cystoagaricus strobilomyces have been included in the
analyses, since evidence from former studies suggested that
this belongs to the Psathyrellaceae (Vellinga 2004). Singer
(1986) placed the genus Macrometrula in the Psathyrella-
ceae; therefore, we examined the type of this species, but
found it to be a very badly preserved representative of a
species closer to the Agaricaceae or the Pluteaceae.
DNA extraction, PCR and sequencing
Genomic DNA was extracted from 2–20 mg of dried herbar-
ium specimens by means of the DNeasy Plant Mini kit
(Qiagen), following the manufacturer’s instructions. PCR am-
plification targeted four loci: the nrLSU (ca. 1,500 bp), ITS
(ca. 700 bp), tef1 (ca. 1,200 bp) and btub (ca. 500 bp) genes,
and employed the primer combinations LROR/LR7, ITS1/
ITS4, 983 F/2218R and B12R_psa/B36F_psa for the nrLSU,
ITS, tef1 and btub genes, respectively (for primer sequences,
see Nagy et al 2011). Amplification followed standard proto-
cols (White et al. 1990). PCR clean-up and sequencing was
performed by LGC Genomics (Berlin) using the same primers
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as above, except for the tef1 gene, for which an internal
reverse primer (1567R) was used. Individual readings were
assembled to contigs using the PreGap and Gap4 programs of
the Staden package (Staden et al. 2000).
Alignments, congruence tests and modeling sequence evolution
Sequences of nrLSU, tef1 and btub were aligned by ClustalX
(Thompson et al. 2002), followed by minor manual adjust-
ments. ITS sequences were aligned by Probalign, a probabi-
listic alignment algorithm found to reduce the proportion of
incorrectly inferred homologies in sequences with numerous
indel events (Roshan and Livesay 2006). Probalign was
launched with default parameters. Indels in the ITS region
were recoded as a partition of binary characters by using the
simple indel coding algorithm (Simmons and Ochoterena
2000), as implemented in FastGap 1.2 (Borchsenius 2007).
This approach treats indels as one evolutionary event.
ITS alignments are frequently criticized for alignment
errors, especially in data sets spanning significant taxonomic
ranges. In order to examine the sensitivity of our conclusions
to potential phylogenetic noise originating from incorrectly
inferred homologies in the ITS1 and ITS2 regions, we per-
formed phylogenetic analyses with all the aligned regions, as
well as with ambiguously aligned regions excluded. To ex-
amine how different treatments of gapped regions affect the
topology and support values of trees, we identified ambigu-
ously aligned regions by GBlocks 1.0 by using a “less strin-
gent”set of parameters, allowing up to half of the sequences to
contain gaps in the final alignment (Castresana 2000). This
trimmed ITS alignment was subjected to the same phyloge-
netic analyses as the complete one.
To check for topological congruence between single-locus
alignments, we performed maximum likelihood (ML) boot-
strapping in 100 replicates using the parallel version of
RAxML 7.0.4 (Stamatakis 2006). Mutually exclusive, strong-
ly supported clades (MLBS≥70 %) were considered
indicative of significant topological incongruence. Based on
our former results obtained on a more restricted set of speci-
mens and formal model testing (Nagy et al. 2011), the GTR+
G model of sequence evolution have been used for all genes.
Because gamma approximation accounts for the same phe-
nomenon as P-invar and it has been raised that using both can
reduce the identifiability of parameters in the analyses
(Stamatakis 2006), we omitted the invariant sites model from
the analyses. For the concatenated data set, the following
partitioned model was used (Nagy et al. 2011): the ITS1,
5.8S, ITS2 and nrLSU regions were modeled by a GTR+G
model, while for the two protein coding genes, two site-
specific rate models were invoked by dividing the tef1 and
btub genes into 1st, 2nd, and 3rd codon positions, and esti-
mating a GTR+ G model for each codon position in each gene.
This resulted in ten partitions of nucleic acid supplemented
with recoded indel data as the 11th partition. Model parame-
ters were unlinked between partitions in the Bayesian analy-
ses. The concatenated alignment, as well as the Bayesian
consensus tree, are available in TreeBase (Accession Number:
11724).
Constraint analyses
Several morphologically informed phylogenetic hypotheses
were tested by constraint analyses (summarized in Table 1).
Constraint trees were designed either to test unexpected rela-
tionships, nodes contradicting previously published phyloge-
nies, or the affinities of morphologically similar taxa.
Mesquite 2.74 (Maddison and Maddison 2009) was used to
create constraint trees, choosing the most conservative way of
imposing the constraints. This often required several clades to
be collapsed to polytomy, which has later been resolved
according to the ML solution around the particular node.
RAxML was used to compute the unconstrained trees, to find
the ML solution for polytomous nodes of constrained trees
and to calculate single-site likelihoods. Trees were inferred in
Table 1 Relationships tested by constraint analyses. Constraints rejected at p< 0.05 are shown in bold
Constraint Approximately unbiased test P value
/cotonea and /Cystoagaricus monophyletic 0.003–0.015
/Coprinus patouillardii sister group to /Coprinellus 0.12–0.28
/Coprinus patouillardii and /Coprinellus sect Setulosi monophyletic 0.07–0.09
/Coprinus patouillardii and /Coprinellus sect Domestici and Micacei monophyletic 0.03–0.04
/gordonii clade and /Coprinopsis < 0.0001
Ps. melanthina, Ps. submicrospora, Ps. pannucioides and Ps. marcescibilis on one branch 0.019–0.021
/Coprinopsis sister group to /Parasola < 0.00001
/Lacrymaria clade in basalmost position 0.09
/Lacrymaria clade + /Cystoagaricus clade < 0.001
Ps. melanthina, belong to the /Cystoagaricus clade <0.00001
Ps. cotonea and Ps. caput–medusae as a separate lineage 0.22–0.62
Mycol Progress
ten replicates in all cases. Single-site likelihoods were then
imported into CONSEL (Shimodaira and Hasewaga 2001)to
calculate confidence intervals on the trees. CONSEL was run
with default settings, and priority was given to the results of
the approximately unbiased test (Shimodaira and Hasewaga
2001). P values ≤0.05 were considered significant.
MP and ML bootstrap analyses
We estimated nodal support for the trees by maximum
parsimony (MP) and ML bootstrapping. Parsimony anal-
yses have been performed using PAUP 4.0b10
(Swofford 2002). Initial, exploratory MP searches have
been performed in 1,000 replicates (TBR branch swap-
ping, MULTREES0YES), keeping only three of the
shortest trees per replicate. Subsequently, more thorough
branch swapping has been performed on the trees saved
during the first cycle. Bootstrapping was initiated in
1,000 replicates with TBR branch swapping, stepwise
sequence addition and ten replicates of branch swapping
per bootstrap replicate. A 50 % bootstrap consensus tree
has been computed in PAUP.
For ML bootstrapping, we used the parallel version of
PhyML 3.0 (Guindon and Gascuel 2003). One thousand
bootstrap replicates were analyzed under the GTR +G model
of evolution. Rate heterogeneity was modeled by a gamma
distribution discretized using four categories. Branch swap-
ping was set to SPR.
Bayesian analyses
We inferred treesby using two different Bayesian approaches,
under two different partitioned models found optimal for a
smaller version of this dataset in our former study (Nagy et al.
2011). The first, outlined above, was used in MrBayes 3.1.2
(Altekar et al. 2004), while a mixture model approach using
three GTR+G matrices for each of the sites of the partitions
described above was implemented by using BayesPhyloge-
nies 1.0 (Pagel and Meade 2006). The latter was found to
improve likelihood values significantly (based on Bayes Fac-
tors), and to converge to more realistic branch length estimates
than MrBayes (Nagy et al. 2011). Since topological differ-
ences between the two types of analyses have not yet been
examined in detail, we compare them in this paper. Sampling
frequency was set to 1,000 for both programs. The indel
matrix was appended to the end of the concatenated alignment
in both Bayesian analyses, and a correction for constant char-
acters not included in the matrix was imposed ("coding0
variable" in MrBayes and "cb0noconst" in BayesPhyloge-
nies). Indel data were modeled by a two-parameter Markov
model. MrBayes was launched with four incrementally heated
chains and two replicates, while BayesPhylogenies was run
with one chain, in three replicates.
Convergence of likelihood values was checked in Tracer
(Rambaut and Drummond 2008). Since topological conver-
gence was proved difficult to achieve and preliminary Bayesian
runs failed to converge on satisfyingly stable split posteriors [as
deduced from the average standard deviation of split frequen-
cies (in MrBayes) and the cumulative and compare functions of
AWTY (Wilgenbusch et al. 2004)], we performed long runs,
where the Markov chains were run for 100 million generations
inbothprograms.Burn-invaluesweredeterminedbyinspect-
ing likelihood and topological convergence.
Trees sampled after convergence were used to compute
50 % Majority Rule consensus trees. This was done by
combining trees sampled from paired or replicated MCMC
runs. Runs that failed to converge to the same posterior as
the others were excluded from consensus analyses. On the
basis of consensus trees, we compared Bayesian posterior
probabilities and branch length estimates obtained from the
MrBayes and the BayesPhylogenies runs. The notations
BPP
b
and BPP
m
hereafter refer to the BayesPhylogenies
and MrBayes posterior probabilities, respectively.
Results
Data sets and congruence tests
We sampled and analyzed four nuclear loci for 140 speci-
mens of the Psathyrellaceae and outgroup taxa. Of these,
Bolbitius vitellinus, Conocybe lactea, Agrocybe preacox, Ps.
melanthina, Ps. pennata, Ps. spintrigeroides, Ps. romagne-
sii, Ps. sphaerocystis, Ps. cotonea, Ps. stercoraria, Ps.
larga, Ps. gossypina and Cystoagaricus strobilomyces were
represented only by the ITS and LSU regions, while for
Coprinopsis friesii only LSU and btub fragments amplified
successfully. All other species were represented by all four,
or in the case of ten specimens, by three loci. The final
concatenated matrix contained 4,717 characters in total. Of
this, 3,952 were nucleic acid and 765 were binary indel
characters obtained by recoding gaps in the ITS alignment.
The length of the individual partitions was 730 for the ITS1,
172 for the 5.8S rRNA, 712 for the ITS2, 1,297 for the
nrLSU, 664 for the tef1 and 375 for the btub gene. Of these,
the number of variable positions was 528 for the ITS1, 27
for the 5.8S rRNA, 469 for the ITS2, 484 for the nrLSU, 435
for the tef1and 189 for beta-tubulin. There were two intron
regions in the tef1and the btub genes each, which were
deleted from the alignments, due to their excessive variabil-
ity. To examine the effects of excluding highly variable
regions of the ITS locus from the alignment, we identified
“ambiguously aligned”regions by GBlocks, which resulted
in a final alignment length of 318 bp.
We found that incongruence between the single locus
alignments was caused by two specimens, Coprinellus sp.
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(SZMC-NL-0978) and Psathyrella aff.lutensis (SZMC-NL-
4301). These were deleted from the alignments, since they
appeared in different positions in the single-gene ML trees
and caused major discrepancies both in combined Bayesian
analyses and congruence tests. The results of the constraint
analyses are summarized in Table 1.
Bayesian analysis
Likelihood values converged very rapidly to stationarity, usu-
ally within the first 2 million generations. The three replicated
runs of BayesPhylogenies terminated after 19 million gener-
ations, but converged to roughly the same likelihood values
(-lnL051431–51451). A Bayes Factor test indicated small
differences between the three runs (logBF02.13–8.44). On
the other hand, paired MrBayes runs converged to more
different likelihood values (-lnL052011–52049, logBF0
16.59), of which run 1 produced insufficient convergence,
so we excluded it from the calculation of consensus trees.
Consensus trees and support values
The different analyses yielded the same results with regard
to the gross topology of the trees. Based on analyses of
convergence, we established the burn-in as 70 million and
10 million generations in MrBayes and BayesPhylogenies,
respectively. The 50 % majority rule consensus trees have a
high resolution, with most larger clades receiving strong
support values (Fig. 2). Out of the four different analyses,
the two Bayesian trees are topologically closest to each
other, conflicting only in the placement of some minor
clades. The parsimony and ML trees also conflicted with
the Bayesian ones in deeper internal branches. For instance,
the Lacrymaria clade was placed in different positions in the
analyses. In the MP consensus and the ML trees, it was
placed sister to Coprinopsis (MPB: 93 %, MLB: 100 %),
whereas both Bayesian analyses placed it close to Coprinel-
lus (BPP
m
: 1.00, BPP
b
: 1.00) (see Fig. 2).
Trees inferred from the GBlocks-curated alignment, i.e.
from which gapped regions of the ITS locus were deleted
(Suppl. Fig. 1), were congruent with analyses from the
complete alignment, with the exception of some incongruent
nodes around Coprinellus. The candolleana clade was split
to two subclades, one (containing Ps. candolleana, Ps.
leucotephra, Ps. badiophylla) was nested within Coprinel-
lus, while the remaining species, Ps. typhae, together with
the calcarea clade, were inferred as a tritomy, sister to
Coprinellus. Further, as a result of GBlocks-curation, the
number of unresolved nodes in the clade of Coprinellus
hiascens—C. congregatus increased from zero to four.
Based on our trees and former studies, we distinguish 14
major clades within the Psathyrellaceae (Fig. 2): /spadiceogri-
sea, /fusca, /Coprinus patouillardii, /gordonii, /cotonea, /
calcarea, /Coprinellus, /candolleana, /gracilis, /Cystoagaricus,
/Lacrymaria, /Coprinopsis, /pseudonivea, and /Parasola.All
of these received significant support values from at least three
analyses, except for the fusca (−/−/.59/.80) and Coprinellus (−/
−/.91/1.00) clades. These are discussed in greater detail in the
next sections.
Discussion
Relationships between early branching clades
There is persistent contradiction between different published
papers and different analyses with regard to the topology at
the basal nodes of the phylogeny. This most severely affects
the position of the Lacrymaria clade, the genera Coprinopsis
and Parasola, but also affects the gracilis clade and Copri-
nellus. Paradoxically, posterior probabilities can be high for
alternative placements of a clade, even with extensive data
sets, as seen in the Lacrymaria clade. Studies utilizing only
one to two genes usually failed to find significant support
for the placement of this clade, whereas multilocus analyses
provided contradicting (Larsson and Örstadius 2008;
Padamsee et al. 2008; Vasutova et al. 2008), often strongly
supported, results (Nagy et al. 2009,2010). Its position
varied from being the sister group to the rest of the Psathyr-
ellaceae, to the sister group of Coprinopsis or Coprinellus
plus the other psathyrelloid clades. Based on three genes, we
formerly sustained the latter placement, although with mod-
erate support (Nagy et al. 2010). Sensitivity of topologies to
model selection has also been examined on the basis of a
more restricted taxon sampling with four genes (Nagy et al.
2011). Inference under some of the models of sequence
evolution (JC69 with and without partitioning, GTR+G
without partitioning, Site-Specific Rates /SSR/ model,
codon-based and mixture models) placed the Lacrymaria
clade sister to all other Psathyrellaceae with significant
support (BPP: 1.00 in all cases), whereas the minority of
tested models (JC69+G without partitions, GTR+ G parti-
tioned) produced a relationship congruent with the tree
Fig. 2 50 % Bayesian Majority Rule consensus tree. Support values
above branches are given as: Maximum Parsimony bootstrap / Maxi-
mum Likelihood bootstrap / Bayesian Posterior probability from
MrBayes / Bayesian Posterior probability from BayesPhylogenies. A
dash (−) represents that the clade was either not present on the tree, or
the support values were < 50 %/0.50. An asterisk (*) denoted 100 % or
1.00 support for bootstrap and Bayesian analyses, respectively. Support
values for specimens of the same species have been removed. Copri-
noid species, as defined by Nagy et al. (2011) (complete or partial
deliquescence, bimorphic basidia, voluminous cystidia, pseudoparaph-
yses and plicate pileus), are denoted by checked circles, while non-
deliquescent psathyrelloid taxa are denoted by striped circles. Out-
group taxa are identified by the Accession numbers of their ITS
sequences
b
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Psathyrella sylvestris NL-0640
Psathyrella sylvestris NL-3055
*/*/*/*
Psathyrella larga LÖ 223-90
62/68/*/.98
Psathyrella gossypina WU 25069
65/69/*/*
Cystoagaricus strobilomyces Nagasawa 9740
/Cystoagaricus
Psathyrella pseudogracilis NL-2142
Psathyrella polycystis NL-1951
*/*/*/*
Psathyrella corrugis NL-3395
-/-/*/.99
Psathyrella bipellis NL-2535
Psathyrella bipellis NL-0638
Psathyrella romelli NL-3526
Psathyrella sp. NL-2349
-/72/.99/.97
-/69/.98/.86
-/73/*/*
-/-/*/*
/Coprinus patouillardii
/gracilis
Psathyrella pennata LÖ 206-03
Psathyrella fibrillosa NL-0201
Psathyrella spintrigeroides LÖ 127-86
56/62/.98/.98
Psathyrella umbrina NL-1949
Psathyrella suavissima NL-3995
*/95/*/*
51/57/*/*
Psathyrella fagetophila NL-2530
Psathyrella romagnesii LÖ 267-04
*/*/*/*
Psathyrella sphaerocystis LÖ 126-99
-/-/.71/.65
Psathyrella piluliformis NL-3923
Psathyrella piluliformis NL-2936
Psathyrella pertinax NL-2350
Psathyrella sp. CBS206.33
*/*/*/*
-/-/.75/.64
Psathyrella pygmaea NL-2139
Psathyrella pygmaea NL-2325
Psathyrella olympiana NL-2935
93/97/*/*
Psathyrella reticulata NL-0441
Psathyrella noli-tangere NL-3403
*/-/.95/-
Psathyrella panaeoloides NL-2537
93/-/.60/.65
Psathyrella fusca NL-0630
51/56/*/.99
99/*/*/*
-/-/.59/.80
Psathyrella vestita NL-2346
Psathyrella gordonii NL-1950
*/*/*/*
Coprinus patouillardii NL-1684
Coprinus patouillardii NL-1687
Coprinus patouillardii NL-1685
Coprinus patouillardii NL-1695
*/*/*/*
-/59/*/*
Psathyrella caput-medusae WU 9982
Psathyrella cotonea BRNM 705616
95/95/*/*
Psathyrella prona NL-0398
Psathyrella senex NL-0199
Psathyrella microrhiza NL-3059
Psathyrella ammophila NL-1450
Psathyrella ammophila NL-3398
Psathyrella magnispora NL-1954
Psathyrella ammophila NL-2151
50/72/*/*
*/*/*/*
Psathyrella fatua NL-0603
89/99/*/*
Psathyrella spadiceogrisea NL-0440
96/98/*/*
Psathyrella phegophila NL-3524
Psathyrella clivensis NL-1952
94/97/*/* */*/*/*
-/-/*/-
-/-/.99/.92
Psathyrella stercoraria LÖ 460-05
Psathyrella calcarea NL-2434
70/67/*/*
Coprinellus congregatus NL-2138
Coprinellus bisporus NL-2512
51/52/.97/.95
Coprinellus callinus NL-1931
54/67/*/.98
Coprinellus sassi NL-1495
Coprinellus impatiens NL-1164
-/-/.88/-
Coprinellus brevisetulosus NL-1956
Coprinellus pellucidus NL-2344
*/*/*/*
-/55/.96/-
Coprinellus subpurpureus NL-2152
-/57/.78/-
Coprinellus hiascens NL-2536
*/*/*/*
Coprinellus pusillulus NL-2144
Coprinellus curtus NL-2339
99/*/*/*
Coprinellus flocculosus NL-1661
Coprinellus flocculosus NL-1567
-/-/.91/.62
Coprinellus xanthothrix NL-1292
Coprinellus disseminatus NL-2337
Coprinellus truncorum NL-1101
Coprinellus truncorum NL-1294
Coprinellus rufopruinatus NL-1959
-/-/*/*
79/95/*/*
Coprinellus aureogranulatus CBS973.95 TYPE
-/55/*/.97
-/-/.97/.98
88/95/*/*
Coprinellus verrucispermus NL-2146
*/*/*/*
-/-/.91/*
Psathyrella multipedata CBS1211.89
Psathyrella candolleana NL-2937
Psathyrella badiophylla NL-2347
Psathyrella leucotephra NL-1953
*/*/*/*
Psathyrella typhae LÖ 21-04
*/*/*/*
-/-/.86/.79
59/77/*/*
92/97/*/*
/spadiceogrisea
/cotonea
/fusca
/candolleana
/Coprinellus
/calcarea
/gordonii
Mycol Progress
shown in this study (BPP: 0.64–0.82). We observed no
correlation between taxon sampling and the position of this
clade. The prevalence of trees with the Lacrymaria clade as
the most basal one prompts us to regard this relationship as
more probable. Consistent with this, constraint analysis
cannot reject the possibility that /Lacrymaria is sister to the
rest of the Psathyrellaceae (P00.09, Table 1).
A counterexample of the Lacrymaria clade is the Cystoagar-
icus clade, which seems to have a very stable position in the tree.
It has been inferred in its current placement in several former
Psathyrella spadicea NL-3996
Psathyrella spadicea NL-3450
*/*/*/*
Lacrymaria glareosa WU 16293
Lacrymaria velutina NL-2150
*/*/*/*
*/*/*/* Lacrymaria pyrotricha CBS573.79 /Lacrymaria
Coprinopsis calospora CBS612.91 TYPE
Coprinopsis mitraespora NL-3031
Coprinopsis laanii CBS476.70
Coprinopsis tuberosa CBS596.80
61/62/.99/*
Coprinopsis narcotica NL-2342
Coprinopsis semitalis CBS291.77
*/*/*/*
81/84/*/*
Coprinopsis stercorea NL-2343
-/92/.91/.88
-/-/.97/.85
"Coprinus" poliomallus NL-2336
-/-/.79/-
-/57/.97/-
Coprinopsis vermiculifer CBS132.46
Coprinopsis gonophylla NL-0378
Coprinopsis friesii NL-0565
66/53/*/.97
Coprinopsis episcopalis NL-3032
*/*/*/*
-/-/*/.98
-/-/.98/-
Coprinopsis cinerea NL-1054
Coprinopsis erythrocephala NL-4153
Coprinopsis bresadoliana NL-2148
Coprinopsis atramentaria NL-4245
*/*/*/*
-/-/.92/-
Coprinopsis krieglsteineri NL-2345
-/63/.99/-
-/-/.88/*
Coprinopsis lagopus var. vacillans NL-2432
Coprinopsis lagopus NL-2143
*/*/*/*
Coprinopsis lagopus NL-1558
Coprinopsis lagopus NL-2493
Coprinopsis jonesi NL-0777
90/63/*/*
92/94/*/*
Coprinopsis macrocephala NL-1376
Coprinopsis goudensis NL-4139
Coprinopsis candidolanata NL-2338
76/-/.90/-
-/-/.90/-
80/94/*/*
-/-/.71/-
Coprinopsis insignis NL-4244
-/-/.86/.97
-/-/.51/*
-/-/.54/-
91/98/*/-
93/*/*/*
Psathyrella melanthina NL- 6410
/Coprinopsis p.p.
Agrocybe praecox AY194531
Agrocybe pediades DQ484057
Conocybe lactea DQ486693
Bolbitius vitellinus AY194519
Bolbitius vitellinus DQ200920
Mythicomyces corneipes DQ404393
Parasola sp. NL-4185
Parasola sp. NL-0295
Parasola sp. NL-0284
*/*/*/*
*/*/*/*
Parasola plicatilis NL-0287
*/97/*/*
Parasola megasperma NL-1924
Parasola megasperma AH 13089
56/61/*/.97
Parasola leiocephala NL-0283
Parasola_leiocephala 57_122
Parasola leiocephala NL-0288
-/61/*/.94
Parasola lilatincta NL-0660
Parasola misera NL-0667
-/70/.99/.99
*/99/*/*
Parasola auricoma NL-0087
Parasola conopilea NL-0285
Coprinopsis stangliana NL-2153
Coprinopsis picacea NL-0174
Coprinopsis picacea NL-3033
*/*/*/*
"Coprinus" cortinatus NL-1621
"Coprinus" bellulus NL-2341
"Coprinus" coniophorus NL-3414
98/78/.79/.53
"Coprinus" utrifer NL-0591
*/*/*/*
Coprinopsis pseudonivea NL-2340
81/83/*/*
Psathyrella submicrospora NL-0635
Coprinopsis pannucioides NL-3528
*/*/*/*
-/-/.95/*
Coprinopsis marcescibilis NL-2140
Coprinopsis marcescibilis NL-0629
*/*/*/*
97/99/*/*
*/*/*/*
57/*/0.99/*
0.01
/Parasola
/pseudonivea
*/*/*/*
Fig. 2 (continued)
Mycol Progress
studies as well (Larsson and Örstadius 2008;Nagyetal.2011;
Padamsee et al. 2008; Vasutova et al. 2008), where it has been
called Ps. larga (Larsson and Örstadius 2008; Padamsee et al.
2008), Ps. delineata, Cystoagaricus, (Padamsee et al. 2008)or
Ps. gossypina+Ps. delineata (Vasutova et al. 2008) clade. In all
previous studies (except for Nagy et al. 2011), the /Cystoagaricus
clade was split to smaller, non-monophyletic groups.
The parasola clade has been inferred as basal in the Psa-
thyrellaceae, although in previous studies, it was repeatedly
grouped together with Coprinopsis (e.g. Hopple and Vilgalys
1999;Nagyetal.2010,2011;Padamseeetal.2008). In fact,
this relationship would seem probable, since both clades con-
tain primarily deliquescent species, but constraint analyses
rejected such a relationship significantly at p≤0.00001.
There are two major clades in which the branching order
has been somewhat uncertain: Coprinellus and the fusca—
Coprinus patouillardii clades. The monophyly of Coprinellus
has been questioned in a former study (Nagy et al. 2010),
where the two large clades therein have been inferred as non-
monophyletic, placed along a completely unbalanced back-
bone of the tree. However, constraint analyses showed that
their monophyly is plausible on the basis of the approximately
unbiased test. The other complex in which it has been difficult
to resolve the internal branching order with confidence is the
clade formed by the /Coprinus patouillardii,/cotoneaand/
fusca. In addition, finding morphological definitions for these
clades is difficult, which suggests that these taxa might have
experienced rapid morphological diversification that erased
the historical signature of their relationships.
Phylogenetic relationships and morphological traits
In this study, we extended sampling of psathyrelloid taxa
substantially, with the aim to recover all lineages of the
family. This untangled several clades that formerly proved
hard to define, because of sparse sampling and the lack of
key species from the phylogeny. However, it is possible that
certain lineages are still missing from the phylogeny, which
should be examined by even more sampling of taxa from
morphologically heterogeneous and/or tropical groups. In
the following paragraphs, we discuss the 14 major clades
and their morphological characteristics.
In the spadiceogrisea clade (MPB: 50 %, MLB: 72 %, BPP
m
:
1.00, BPP
b
: 1.00, Fig. 1k) the basidiomes are fairly large (>
3 cm), non-deliquescent with medium-sized, ellipsoid-
subphaseoliform spores (7–9μm), and fibrillose, scanty veil that
is visible only on young specimens. The gill edge is lined mainly
with poorly developed globose-sphaeropedunculate cells
(paracystidia), whereas true, utriform cheilocystidia are very
scarce. The presence of this monophyletic assemblage,
corresponding to subsection Spadiceogriseae of Kits van Wav-
eren (1985) has already been noted by Vasutová et al. (2008)and
Larsson and Örstadius (2008), independently of each other.
The calcarea clade (MPB: 70 %, MLB: 67 %, BPP
m
:
1.00, BPP
b
: 1.00): the ML tree and the MP consensus tree
supports the placement of this clade sister to the /candol-
leana, although with very weak support. Although these
species are morphologically most similar to Ps. prona,so
that Ps. calcarea has been treated as a variety of the latter (as
Ps. prona var utriformis), trees with these three species on
one clade were significantly rejected by the approximately
unbiased test (Table 1). Traditional taxonomic treatments
considered these species closely related to representatives
of the gracilis clade (Kits van Waveren 1985; Larsson and
Örstadius 2008;Smith1972). Although not forming a
monophyletic assemblage, these two clades also fall in the
vicinity of each other in the Bayesian analyses. In support of
their relatedness are several former studies (Larsson and
Örstadius 2008; Nagy et al 2010; Padamsee et al. 2008),
where the calcarea clade (≈section Atomatae), the gracilis
clade (≈section Psathyrella) and Ps. bipellis (0Ps.odorata,
section Bipelles)showedhighaffinitytoeachotheror
formed a monophyletic group, although support for this
relationship was lacking in all cases. However, those studies
sampled more species, such as Ps. potteri, Ps. prona, Ps.
effibulata, Ps. orbitarum etc. Representatives of these
clades/sections share a similar habit, ellipsoid spores longer
than 10 μm and scanty veil. Therefore, we raise the possi-
bility that they actually are monophyletic, but more data and
denser sampling is necessary to substantiate that.
The fusca clade (MPB: -, MLB: -,BPP
m
: 0.59, BPP
b
:0.80,
Fig. 1h, l) constitutes a huge heterogeneous assemblage, re-
garding both morphology and traditional taxonomy. There-
fore, it is difficult to provide an unequivocal morphological
definition. The spores are shorter than 10 μminmostspecies,
and the cheilocystidia are mostly abundant along the gill edge.
All sampled Psathyrella species with lageniform and/or acute
fusiform cystidia and spores shorter than 10 μm (except for
Ps. fagetophila) are nested within this clade. These traits
discriminate this clade from the spadiceogriseae clade, in
which the spores are also shorter than 10 μm, but the cheilo-
cystidia of those species are very scarce and utriform when
present. Species of other clades have longer spores or they
lack pleurocystidia, or the cystidia are distinctly thick-walled
(> 1 μm). Species of subsection Lutenses and section Penna-
tae of Kits van Waveren (1985) are included in this clade, as
well as Ps. sphaerocystis, which is characterized by a granular
veil. Kits van Waveren (1985) placed this species in section
Cystopsathyra of subgenus Psathyra. It seems that psathyr-
elloid species with granular veil do not form a monophyletic
group, since Ps. sp. (NL-2349), an undescribed species with a
granular veil, fell within the gracilis clade. Further, it is ques-
tionable whether the granular veil in psathyrelloid taxa is of
the same origin as that of Coprinellus or Coprinopsis.
The Coprinus patouillardii clade (MPB: 100 %, MLB:
100 %, BPP
m
:1.00,BPP
b
: 1.00, also known as C.
Mycol Progress
cordisporus) contains small species with deliquescent basi-
diomes, granular veil and more or less hexagonal, lentiform
spores. This group consists of two species on our trees out of
a total of three (see Keirle et al. 2004). Earlier studies have
also proved its affinities to the fusca clade (Hopple and
Vilgalys 1999; Larsson and Örstadius 2008;Nagyetal.
2010,2011; Padamsee et al. 2008; Vasutová et al. 2008;
Walther et al. 2005), and constraint analyses reinforce its
position there (Padamsee et al. 2008). However, its exact
placement within/near this clade is uncertain, as shown by
the results of the constraint analyses. Trees with C. patouil-
lardii placed as a sister group of all other species of the fusca
clade cannot be rejected; moreover, when forced to mono-
phyly with Coprinellus (as a sister group of that), the like-
lihoods decreased only slightly (trees could not be rejected
at p≤0.05). This implies that—given our data—C. patouil-
lardii may occupy several places on the tree with nearly
equal probability. In support of the above finding, develop-
ment of the veil in early-median phases of the ontogeny
sheds more light on the relationship of this clade to /Copri-
nellus. The velar structures of C. patouillardii are not sharp-
ly delimited from cell layers of the pileipellis, whereas it is
clearly demarcated from the underneath layers of palisade in
Coprinellus (Reijnders 1979,asC. cordisporus). In this
respect, C. patouillardii is similar to the C. cortinatus group,
which belongs to the pseudonivea clade.
The gordonii clade (MPB: 100 %, MLB: 100 %, BPP
m
:
1.00, BPP
b
: 1.00, Fig. 1g) contains species with whitish or
pale grayish brown, medium-sized basidiomes, with thick
fibrillose, white veil when young, cuticular pileipellis, and
almost opaque spores, which are about 10 μm. We exam-
ined whether these species can be placed in Coprinopsis,
with which it shares the cuticular pileipellis and spore shape,
but it seems that such a relationship is improbable (au test
pvalues: 10
−4
–6×10
−35
). This, interestingly, at the same
time entails multiple independent origins and/or losses of
cuticular pileipellis in the Psathyrellaceae.
The cotonea clade (MPB: 95 %, MLB: 95 %, BPP
m
:
1.00, BPP
b
: 1.00) is recognized here as a monophyletic
group. Both species are morphologically peculiar in that
their basidiomes are large, non-deliquescent, and the veil
is whitish, abundant, cottony, blackening with age. The two
species here come in a rather isolated position and their
placement is somewhat uncertain. On preliminary phyloge-
nies, their position varied and ML analyses placed them
within the fusca clade. However, trees where they are forced
outside the fusca clade could not be rejected by the au test
(0.22<p<0.46). Because of their superficial similarity to
species of the Cystoagaricus clade, primarily the size of
the basidiomes, and the darkening of veil on ageing, we
tested the monophyly of these two groups. However, the p
values were close to 0.01, thereby rejecting the monophyly
of the cotonea and Cystoagaricus clades. Because the
constraint analyses provide evidence for an unstable posi-
tion for this clade on the tree, we raise the possibility that
they represent an independent, enigmatic lineage of the
Psathyrellaceae similar to the C. patouillardi clade.
/Coprinellus (MPB: -, MLB: -, BPP
m
: 0.91, BPP
b
: 1.00
(Fig. 1c,d,e,r)) is characterized by hymeniform pileipellis,
presence or absence of a granular veil, and deliquescent or
collapsing basidiomes. When the veil is lacking, the cap is
covered by lageniform, hair-like pileocystidia. The two sub-
clades therein correspond to subsection Setulosi p.p. (C. hia-
scens–C. congregatus) and subsections Domestici and
Micacei (C. verrucispermus–C. truncorum). The former is
characterized primarily by the lack of veil, while the latter
generally has rich granular veil on the cap. Exceptions from
this are the taxa originally placed in subsection Setulosi,such
as C. disseminatus, C. verrucispermus, C. curtus etc., which
are nested in the clade of veiled species. These species, how-
ever, have both veil and pilocystidia, and their spores are often
mitriform, very similar to that of C. micaceus.
The candolleana clade (MPB: 100 %, MLB: 100 %, BPP
m
:
1.00, BPP
b
: 1.00) contains species with rather pale basidiomes
lacking pleurocystidia, but having a fibrillose, scanty veil
which is visible only on young specimens. The affinities of
this clade to Coprinellus have already been noted by Hopple
and Vilgalys (1999) and many subsequent studies (Walther et
al. 2005; Padamsee et al. 2008;LarssonandÖrstadius2008;
Vasutová et al. 2008;Nagyetal.2010;2011).
The gracilis clade (MPB: -, MLB: 73 %, BPP
m
: 1.00,
BPP
b
: 1.00, Fig. 1j) is named after Ps. gracilis, the type
species of Psathyrella. Although we did not include it in the
present data set, two very closely related species, Ps. corrugis
(also known as Ps. gracilis f. corrugis)andPs. pseudogracilis
are included, and there is phylogenetic evidence for these
species belonging to the same clade (Larsson and Örstadius
2008; Vasutová et al. 2008). These species are characterized
by slender, non-deliquescent basidiomes, often rooting in the
substrate. Lageniform pleurocystidia and cheilocystidia are
always present, spores on average longer than 10 μm, regular
ellipsoid, and veil fibrillose, scanty and visible only on young
specimens. Species of this clade have been placed in section
Psathyrella of Kits van Waveren (1985) and section Psathyr-
ella, subsection Psathyrellae of Smith (1972). On the basis of
morphology, Ps. microrhiza also falls in the vicinity of Ps.
gracilis and Ps.pseudogracilis, which is in contradiction with
its phylogenetic position in the spadiceogrisea clade (MPB:
50 %, MLB: 72 %, BPP
m
,BPP
b
: -) on our phylogeny. Sim-
ilarly, Larsson and Örstadius (2008)inferredanambiguous
position for this taxon.
The Cystoagaricus clade (MPB: 65 %, MLB: 69 %,
BPP
m
: 1.00, BPP
b
: 1.00) has already appeared on the tree
published by Padamsee et al. (2008), although Ps. delineata
(0Ps. gossypina) was excluded. It contains large species
with mostly firm stipe and cap, where the cap surface is
Mycol Progress
often covered by appressed scales, which are darker than the
background, sometimes vanishing somewhat and then
remaining only in the center of pileus. Exceptions are Ps.
gossypina and Ps. larga, which have smooth pileus surface.
The spores are small (6–8μm), and smooth or subangular
(in Cy. strobilomyces). However, this clade is very hetero-
geneous and more research is needed to find better morpho-
logical characters to circumscribe it.
Species of the Lacrymaria clade (MPB: 100 %, MLB:
100 %, BPP
m
: 1.00, BPP
b
: 1.00) have been placed in section
Spadiceae and the genus/section Lacrymaria by traditional
taxonomists (Kits van Waveren 1985; Smith 1972). Two sub-
clades corresponding to taxonomic grouping can be recog-
nized, of which the Lacrymaria clade contains species with
large, ornamented, almost opaque spores, a thick, fibrillose
veil composed of pigmented hyphae, and large basidiomes
(Fig. 1i). The Psathyrella spadicea clade (here missing species
like Ps. cernua, Ps. conissans etc. of former studies) is char-
acterized by thick-walled cystidia, small (6–9μm) pale spores
and lacking a veil. It seems difficult to establish a uniform
morphological definition for the whole Lacrymaria clade, due
to the extraordinary morphological disparity exhibited by the
above mentioned subclades. However, to date, no published
phylogenetic analyses question their monophyly. Lacrymaria
lacrymabunda and its close allies have been treated a section
of Psathyrella by certain authors (e.g. Smith 1972), but that
classification does not provide any specific morphological trait
for the two groups concerned here.
The Coprinopsis clade (MPB: 91 %, MLB: 98 %, BPP
m
:
1.00, BPP
b
:1.00,Fig.1a, f,p,s)containsbothdeliquescent
and non-deliquescent taxa. The pileipellis is cuticular, or in a
few taxa, hymenidermal (C. poliomallus), the veil is well-
developed, composed of globose, filamentous or diverticulate
elements. The veil on the margin of the pileus is powdery, as
opposed to the pseudonivea clade (see below); when partially
"beard-like", then the fruiting body is grayish and the spores
are shorter than 9 μm. The psathyrelloid species, Ps. melan-
thina is also nested in the Coprinopsis clade, and should be
recombined in this genus. These have cuticular pileipellis, like
other Coprinopsis species, grayish non-deliquescent basi-
diomes and grayish, fibrillose scaly veil on the cap.
The pseudonivea clade (MPB: 100 %, MLB: 100 %,
BPP
m
: 1.00, BPP
b
: 1.00) takes an isolated position basal to
all other Coprinopsis species. Characteristic for this clade is
that the veil forms a special “beard-like”structure at the cap
margin of young basidiomes, composed of long white fibrils
connecting cap margin to the stipe and a few globose cells in
between. The basidiomes are usually pure white or whitish,
sometimes brown (Ps. submicrospora). The pileipellis is
cuticular when the fruiting body is deliquescent, and cutic-
ular or hymenidermal when non-deliquescent. Psathyrelloid
members of this clade have scanty veil on the cap surface,
but beard-like fibrils on the margin. Separation of this clade
from the rest of the Coprinopsis clade is proposed for the
first time in this study. Its morphological uniformity, basal
relationship and the strong support values raise it as an
enigmatic lineage of Psathyrellaceae, deserving more atten-
tion in the future. The position of non-deliquescent members
is noteworthy. In contrast to our previous thoughts (Nagy et
al. 2010), they split into several smaller clades and are not
inferred as the basal ones in Coprinopsis. This implies more
shifts between fruiting body types (i.e. between non-
deliquescent and deliquescent) than inferred without the
inclusion of Ps. pannucioides, Ps. submicrospora, and Ps.
melanthina (Nagy et al. 2010). Our study is the first to
include multiple representatives of the “C. cortinatus com-
plex”in phylogenetic studies, such as C. coniophorus, C.
bellulus,orC. cortinatus. Of these tiny species, the two
latter have not been classified in Coprinopsis by Redhead et
al. (2001), probably due to their superficial similarity to
certain Coprinellus species. Our study, however, provides
unequivocal evidence for their placement close to Copri-
nopsis pseudonivea, with which they share the characteristic
beard-like veil on the cap margin.
The Parasola clade (MPB: 100 %, MLB: 100 %, BPP
m
:
1.00, BPP
b
: 1.00, Fig. 1b, o) has been recovered in almost all
phylogenetic studies of Psathyrellaceae as monophyletic
(Hopple and Vilgalys 1999; Larsson and Örstadius 2008;
Nagy et al. 2010,2011;Padamseeetal.2008; Vasutova et
al. 2008; Walther et al. 2005), including a detailed study of
species limits (Nagy et al. 2009). The lack of any velar
structures and caulocystidia uniquely defines this genus. In-
terestingly, the basidiomes are collapsing (incompletely deli-
quescent); if not collapsing (psathyrelloid), then there are
thick-walled, brown setulae on the pileus. The spores are
usually rounded-triangular, flattened, which is rather peculiar
for the Psathyrellaceae. Only the pseudonivea clade and the
Coprinus patouillardii clade contain species with strongly
flattened, often angular spores. The position of P. conopilea
varied somewhat across different phylogenies, and appeared
either as a sister to all other Parasola taxa, or on a weakly
supported clade together with P. auricoma.Constraintanaly-
ses confirm the plausibility of Pconopileabeing basal to all
other Parasola taxa. This basal relationship would imply
gradual loss of pilocystidia, the emergence of partial deliques-
cence, as well as gradual flattening and expansion of the
spores within the genus Parasola (Nagy et al. 2009).
Classification of Psathyrellaceae
Of the several taxonomic difficulties in fungal classification,
that of the Psathyrellaceae is an exemplary case. Early
phylogenetic studies and subsequent reclassification of
coprinoid lineages (Coprinellus,Coprinopsis and Parasola)
left the genus Psathyrella para/polyphyletic (Redhead et al.
2001). In the following years, several studies tried to
Mycol Progress
decipher phylogenetic relationships within the family
(Larsson and Örstadius 2008;Nagyetal.2009,2010,
2011; Padamsee et al. 2008; Vasutová et al. 2008; Walther
et al. 2005), which added valuable information used here as
a basis for designing a balanced taxon sampling. One of the
major objectives during this study—which we consider an
absolute prerequisite for a classification—was to uncover all
major lineages within the family, and this has also strongly
relied on previous studies. The available published sequence
data allows us to conclude that most, if not all, major clades
have been uncovered. Fortunately, there is very little phylo-
genetic uncertainty about the major clades within the family,
and uncertainty at early splits of the phylogeny has no or
little impact on a reclassification in the future.
In conjunction with former studies (Hopple and Vilgalys
1999; Larsson and Örstadius 2008; Nagy et al. 2010,2011;
Padamsee et al. 2008; Vasutová et al. 2008; Walther et al.
2005), our results reinforce the para/polyphyly of Psathyr-
ella, and warrant extensive rearrangement of the genus into
new genera. The clade containing Ps. gracilis,thetype
species of Psathyrella, is sister to Coprinellus and /Candol-
leana, and forms a clade of its own with strong support. In
our opinion, the current topology and the uncertainties in the
placement of some clades would make it very impractical to
consider a large psathyrelloid "supergenus", i.e. treating all
the clades as one genus. This would save the generic name
Psathyrella for most of the species, but in that case, species
of Coprinellus and the Coprinus patouillardii complex
would have to be merged with the psathyrelloid species (if
the Lacrymaria clade is excluded, and in that case even
Coprinopsis,Parasola and the Cystoagaricus clade would
have to be included). We think the most reasonable solution
for the paraphyly of Psathyrella requires splitting it as
currently circumscribed into several smaller genera. If rec-
ognized as a separate genus, the gracilis clade should evi-
dently retain the name Psathyrella (Fr.) Quél., because the
type species belongs to this clade. For other clades, other
generic names should be sought, although their arrangement
should not necessarily follow the clade structure presented
here. Also, whether deliquescent taxa should be merged
with non-deliquescent ones, as would be the case with
Coprinus patouillardii or when merging the candolleana
clade with Coprinellus, should be considered carefully, even
if some non-deliquescent species have already been recom-
bined in deliquescent genera (e.g. Coprinopsis marcescibilis
or Parasola conopila; Larsson and Örstadius 2008). It
seems that such mixed genera cannot be avoided in a natural
classification, although this makes any future identification
keys either highly unnatural or very hard to write and use.
The placement of the “Coprinus cortinatus complex”in
Coprinopsis entails reconsideration of the generic definition
of Coprinopsis as given by Redhead et al. (2001). The
pileipellis of these species plus C. poliomallus (shown to
belong to Coprinopsis by Nagy et al. 2010) is hymenider-
mal, whereas that of other Coprinopsis species, including
non-deliquescent ones, is cuticular. However, the combina-
tion of hyphal veil cells and sphaerocysts, and the whitish
colours support their inclusion in Coprinopsis.
Deeper branches of the phylogeny presented here are not
robust; more data, or even phylogenomic approaches, may be
necessary to resolve the earliest evolutionary events. Similar-
ly, morphology provides no stronger evidence for any group-
ing of the larger clades, due to extensive convergence and
difficulties in recognizing homologous traits. However, the
larger clades can be defined by morphological traits unambig-
uously. The generality of these clade definitions should be
tested by the inclusion of more taxa in the phylogeny, espe-
cially from tropical regions or the southern hemisphere. Al-
though other traits may also prove helpful in circumscribing
certain clades, the morphological definitions presented here
represent a step forward in formulating taxonomic descrip-
tions of clades when a natural classification is to be designed.
Acknowledgements The authors would like to express their grati-
tude to Ellen Larsson and Leif Örstadius for valuable discussions and
exchange of specimens and voucher information. Sándor Kocsubé is
thanked for his generous help in designing the figures. This study has
been supported via a grant to LGN from the Fungal Research Trust.
References
Altekar G, Dwarkadas S, Huelsenbeck JP, Ronquist F (2004) Parallel
Metropolis coupled Markov chain Monte Carlo for Bayesian
phylogenetic inference. Bioinformatics 20:407–415
Borchsenius F (2007) FastGap 1.0.8. Software distributed by the
authors at (http://192.38.46.42/aubot/fb/FastGap_home.htm).
Cáceres O, Kirschner R, Piepenbring M, Schofer H, Gene J (2006)
Hormographiella verticillata and an Ozonium stage as anamorphs
of Coprinellus domesticus. Antonie Van Leeuwenhoek 89:79–90
Castresana J (2000) Selection of conserved blocks from multiple align-
ments for their use in phylogenetic analysis. Mol Biol Evol
17:540–552
Guindon S, Gascuel O (2003) PhyML—A simple, fast, and accurate
algorithm to estimate large phylogenies by maximum likelihood.
Syst Biol 52:696–704
Hopple JS, Vilgalys R (1994) Phylogenetic relationships among copri-
noid taxa and allies based on data from restriction site mapping of
nuclear rDNA. Mycologia 86:96–107
Hopple JS, Vilgalys R (1999) Phylogenetic relationships in the mush-
room genus Coprinus and dark-spored allies based on sequence
data from the nuclear gene coding for the large ribosomal subunit
RNA: divergent domains, outgroups, and monophyly. Mol Phy-
logenet Evol 13:1–19
Keirle MR, Hemmes DE, Desjardin DE (2004) Agaricales of the
Hawaiian Islands 8: Agaricaceae. Coprinus and Podaxis, Psathyr-
ellaceae: Coprinellus,Coprinopsis and Parasola. Fungal Divers
15:33–124
Kemp RFO (1975) Breeding biology of Coprinus speciesin the section
Lanatuli. Trans Br Mycol Soc 65:375–388
Kits van Waveren E (1985) The Dutch, French and British species of
Psathyrella. Persoonia Suppl. 2:1–300
Mycol Progress
Kües U (2000) Life history and developmental processes in the basid-
iomycete Coprinus cinereus. Microb Mol Biol Rev 64:316–353
Kühner R (1928) Le développement et la position taxonomique de
l’Agaricus disseminatus Pers. Le Botaniste 20:147–195
Kühner R, Romagnesi H (1953) Flore Analytique des Champignons
Supérieurs. Paris, pp 556
Larsson E, Örstadius L (2008) Fourteen coprophilous species of Psa-
thyrella identified in the Nordic countries using morphology and
nuclear rDNA sequence data. Mycol Res 112:1165–1185
Maddison WP, Maddison DR (2009) Mesquite: a modular system for
evolutionary analysis. Version 2.6. http://mesquiteproject.org
Matheny PB, Curtis JM, Hofstetter V, Aime MC, Moncalvo JM, Ge ZW,
Slot JC, Ammirati JF, Baroni TJ, Bougher NL, Hughes KW, Lodge
DJ, Kerrigan RW, Seidl MT, Aanen DK, DeNitis M, Daniele GM,
Desjardin DE, Kropp BR, Norvell LL, Parker A, Vellinga EC,
Vilgalys R, Hibbett DS (2006) Major clades of Agaricales: a multi-
locus phylogenetic overview. Mycologia 98:982–995
Moncalvo JM, Vilgalys R, Redhead SA, Johnson JE, James TY, Aime
CM, Hofstetter V, Verduin SJ, Larsson E, Baroni TJ, Greg Thorn R,
Jacobsson S, Clémençon H, Miller OK Jr (2002) One hundred and
seventeen clades of euagarics. Mol Phylogenet Evol 23:357–400
Moser M (1983) Die Röhrlinge und Blätterpilze. in GAMS H.: Kleine
Kryptogamenflora IIb/2. Gustav-Fischer-Verlag, Jena, p 532
Nagy GL, Kocsubé S, Papp T, Vágvölgyi C (2009) Phylogeny and char-
acter evolution of the coprinoid mushroom genus Parasola as inferred
from LSU and ITS nrDNA sequence data. Persoonia 22:28–37
Nagy GL, Urban A, Örstadius L, Papp T, Larsson E, Vágvölgyi C
(2010) The evolution of autodigestion in the mushroom family
Psathyrellaceae (Agaricales) inferred from Maximum Likelihood
and Bayesian methods. Mol Phylogenet Evol 57:1037–1048
Nagy GL, Walther G, Házi J, Vágvölgyi C, Papp T (2011) Under-
standing the evolutionary processes of fungal fruiting bodies:
correlated evolution and divergence times in the Psathyrellaceae.
Syst Biol 60:303–317
Nieves–Rivera AM (2001) The edible Psathyrellas of Haiti. Inoculum
52:1–3
Padamsee M, Matheny PB, Dentinger BT, McLaughlin DJ (2008) The
mushroom family Psathyrellaceae: evidence for large-scale poly-
phyly of the genus Psathyrella. Mol Phylogenet Evol 46:415–429
Pagel M, Meade A (2006) BayesPhylogenies 1.0. Software distributed
by the authors
Rambaut A, Drummond A (2008) Tracer v 1.4.1. Soeftware distrib-
utedby the authors at http://beast.bio.ed.ac.uk/
Rampazzo A, Kuhnert P, Howard J, Bornand V (2009) Hormogra-
phiella aspergillata keratomycosis in a dog. Vet Ophthalmol
12:43–47
Redhead SA, Vilgalys R, Moncalvo JM, Johnson J, Hopple JS (2001)
Coprinus Persoon and the disposition of Coprinus species sensu
lato. Taxon 50:203–241
Reijnders AFM (1979) Developmental anatomy of Coprinus. Persoonia
10:383–424
Roshan U, Livesay DR (2006) Probalign: multiple sequence alignment
using partition function posterior probabilities. Bioinformatics
22:2715–2721
Shimodaira H, Hasewaga M (2001) CONSEL: for assessing the con-
fidence of phylogenetic tree selection. Bioinformatics 17:1246–
1247
Simmons MP, Ochoterena H (2000) Gaps as characters in sequence-
based phylogenetic analyses. Syst Biol 49:369–381
Singer R (1986) The Agaricales in modern taxonomy, 4th edn. Koeltz
Scientific Books, Koenigstein, pp 912
Smith AH (1972) The North American species of Psathyrella. Mem
NY Bot Gard 24:1–633
Staden R, Beal KF, Bonfield JK (2000) The staden package. Methods
Mol Biol 132:115–130
Stajich JE, Wilke SK, Ahren D, Au CH, Birren BW, Borodovsky M,
Burns C, Canback B, Casselton LA, Cheng CK, Deng J, Dietrich
FS, Fargo DC, Farman ML, Gathman AC, Goldberg J, Guigo R,
Hoegger PJ, Hooker JB, Huggins A, James TY, Kamada T, Kilaru
S, Kodira C, Kues U, Kupfer D, Kwan HS, Lomsadze A, Li W,
Lilly WW, Ma LJ, Mackey AJ, Manning G, Martin F, Muraguchi
H, Natvig DO, Palmerini H, Ramesh MA, Rehmeyer CJ, Roe BA,
Shenoy N, Stanke M, Ter-Hovhannisyan V, Tunlid A, Velagapudi
R, Vision TJ, Zeng Q, Zolan ME, Pukkila PJ (2010) Insights into
evolution of multicellular fungi from the assembled chromosomes
of the mushroom Coprinopsis cinerea (Coprinus cinereus). Proc
Natl Acad Sci USA 107:11889–11894
Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based
phylogenetic analyses with thousands of taxa and mixed models.
Bioinformatics 22:2688–2690
Swofford DL (2002) PAUP: phylogenetic analysis using parsimony
(and other methods) version 4.0b10. Sinauer Associates,
Sunderland
Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence
alignment using ClustalW and ClustalX. Curr Protoc Bioinfor-
matics Chapter 2, Unit 2 3
Vasutova M, Antonin V, Urban A (2008) Phylogenetic studies in Psathyrella
focusing on sections Pennatae and Spadiceae–new evidence for the
paraphyly of the genus. Mycol Res 112:1153–1164
Vellinga EC (2004) Genera in the family Agaricaceae—Evidence from
nrITS and nrLSU sequences. Mycol Res 108:354–377
Verweij PE, van Kasteren M, van de Nes J, de Hoog GS, de Pauw BE,
Meis JF (1997) Fatal pulmonary infection caused by the basidio-
mycete Hormographiella aspergillata. J Clin Microbiol 35:2675–
2678
Walther G, Garnica S, Weiss M (2005) The systematic relevance of
conidiogenesis modes in the gilled Agaricales. Mycol Res
109:525–544
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Proto-
cols: a guide to methods and applications pp 315–322
Mycol Progress
