Fourteen coprophilous species of
Psathyrella
identified inthe Nordic countries using morphology and nuclearrDNA sequence data
Ellen LARSSON
a,
a
Deptartment of Plant and Environmental Sciences, University of Gothenburg, Box 461, SE-405 30 Go¨ teborg, Sweden
b
Lyckans va¨ g 39A, SE 291 43 Kristianstad, Sweden
a r t i c l e i n f o
Article history:
Received 31 October 2007Received in revised form19 March 2008Accepted 1 April 2008
Corresponding Editor
:David L. Hawksworth
Keywords:AgaricalesBasidiomycota
Molecular systematicsPhylogenyTaxonomy
a b s t r a c t
Psathyrella
species growing on dung or occasionally on dung in the Nordic countries werestudied using morphological characters and nu-rDNA sequence data and type collectionswere examined when available. Fourteen species capable of growing on dung were identi-fied. Descriptions are given of all dung-inhabiting species and to a lesser extent of the spe-cies occasionally growing on dung. Three new species are described:
Psathyrella fimiseda,P. merdicola
, and
P. scatophila
.
P. stercoraria
is described as a new species in order to validatethe name. A key to the coprophilous species in Europe including the species described byPeck & Smith from North America is provided. The phylogenetic analyses recovered fourmajor supported clades within
Psathyrellaceae
corresponding to
Parasola, Coprinopsis, Lacry-maria/Spadiceae pro parte,
and
Psathyrella
. The status of
Coprinellus
was ambiguous. Thecurrent morphology-based infrageneric classification of
Psathyrella
was not supported bythe phylogenetic analyses and a coprophilous habit has apparently evolved on multipleoccasions. Three new combinations are proposed:
Parasola conopilus
,
Coprinopsis marcescibi-lis,
and
Coprinopsis pannucioides
.
ª
2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction
The genus
Psathyrella
was traditionally placed in the family
Coprinaceae
. Molecular phylogenetic studies based on nu-rDNA sequences have considerably altered the understanding of the relationships within coprinoid fungi (Hopple & Vilgalys1999; Park
Coprinus
is non-monophyletic. Although themajority of species form a large clade (/psathyrellaceace inMoncalvo
2002), the type species,
C. comatus,
belongs tothe lepiotoid fungi in
Agaricaceae
Psathyrellaceae
Psathyrellaceae
comprises six genera inEurope,
viz
.
Psathyrella
(Fr.) Que´l. 1872,
Lacrymaria
Pat. 1887,
Coprinellus
P. Karst 1879,
Parasola
Redhead, Vilgalys & Hopple2001,
Coprinopsis
P. Karst. 1881, and the small genus
Macrome-trula
Donk & Singer 1948. However, some limits and subdivi-sion of genera are still unresolved, especially around
Psathyrella
,
Coprinopsis,
and
Coprinellus
. Several recent molecu-lar studies on these genera have indicated that
Psathyrella
2008). The species in
Psa-thyrella
have a saprotrophic habit and most species occur onsoil or on wood. However, a number of them grow on dung and a few occur on old bonfires or in swamps. The ecologicalspecialization to dung of various animals, such as cow, horse,
* Corresponding author. Tel.:
þ
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mycological research 112 (2008) 1165–1185
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ª
2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.mycres.2008.04.003
and wild boar, is widespread among species in
Coprinopsis
,
Coprinellus
, and
Psathyrella
.
Psathyrella
was first introduced as a tribus, within
Agaricus
(Fries 1838) and a few years later reclassified as a subgenus(Fries 1849). Fries included ten species, but only four of themare currently recognized in
Psathyrella
. The sole species forwhich Fries mentions dung as a substrate is
Agaricus subtilis
(Fries 1821), a name adopted by few later authors. Kirchner
& Eichler (1894) mentioned it from Germany and also Massee(1902) used the name and gave the spore measurement 12–16
5–8
m
Drosophila subtilis
as‘assez rare’ from France, but did not include it in his laterworks. The interpretation of the name is unclear, but the de-scription recalls a tiny
Psathyrella
growing on dung in moistforest (‘
in fimo locis udis silvaticis
Drosophila
(syn.
Psathyrella
),
viz
.
D. coprobia
(syn.
P.hirta
) and
D. stercoraria,
both referred to subgenus
Psathyrella
,but within the subgenus placed in sections
Atomatae
and
Pro-nae,
P. galericolor, P. minima, P. argentata, P.equina
, and
P. sphaerocystis)
and nine species associated withdung or manured, fertilized soil (
P. pseudolimicola, P. pratensis,P. odorata, P. nezpercii, P. potteri, P. pruinosipes, P. prona, P. hirta
,and
P.conopilus
).Withtheexceptionof
P.sphaerocystis
and
P.con-opilus,
allspecieswereclassifiedinthesubgenus
Psathyrella
sect.
Atomatae
,asectioncontainingcoprophilousspeciesandspeciesgrowing on well-fertilized soils.
Psathyrella conopilus
was placedin subgenus
Psathyrella
section
Subatratae
, a sect. distinguishedby the absence of pleurocystidia.
P. sphaerocystis
was placed insubgenus
Cystopsathyra
on the strength of the powdery veilwith cells mostly in the form of sphaerocysts (Smith 1972).In the systematic arrangement of
Psathyrella
Atomatae
and
Pronae
were united to sect.
Atom-atae
.
P.cobrobia
wasplacedinsubgenus
Psathyrella
sect.
Bipelles
together with the non-coprophilous
P. bipellis
(syn.
P. odorata
).Kits van Waveren (1985) listed five dung-inhabiting speciesfrom France, the UK, and The Netherlands,
viz
.
P. waverenii
,
P.hirta
,
P.stercoraria
,
P.coprophila
,and
P.sphaerocystis
.Inaddition,
P. prona
var.
prona
f.
cana,
normally a non-coprophilous taxon,wasrecordedfromhorsedung.Inthesystematicarrangement
P.sphaerocystis
wasplacedinsubgenus
Psathyra
section
Cystop-sathyra,
whereas all other species,including
P. prona
var.
prona
f.
cana,
were placed in subgenus
Psathyrella
section
Atomatae
P. coprophila
and
P. hirta
Psathyra gordonii
f.
minor
with pale cap colour, growing ongrass, with a spore size of 10.5–12.5
5.5–6
m
m and obtuselylageniform cystidia, characters reminiscent of
Psathyrella mar-cescibilis
. Besides growing on grass, Lange also remarked inbrackets ‘with remnants of horse-dung’, a habitat not knownfor
P. marcescibilis
Psathyra cobrobia
(syn.
Psathyrella hirta
) from dung.A species of uncertain identity is
Psathyrella polaris
col-lected by H. G. Simmons on dung of muskox (
Bovis maschatis
)innorthernCanadaduringtheSecondNorwegianArcticExpe-dition on the Fram 1898–1902 (Rostrup 1906). Neither the typenor any other collections of the species have been located sofar. Based upon the short original decription, it is not clear if the species belongs in
Psathyrella
Psathyrella ascarioides
based on a single basidiome col-lected on horse dung in Spain. The cap is described aslemon yellow, viscid, and 10.5 mm broad. Other charactersare the viscid upper part of the stem, the spore size18
9.5
m
m, and the absence of pleurocystidia. Some of thesecharacters deviate from the concept of
Psathyrella
and thespecies may belong to another genus. According to Esteve-Ravento´s & Barrasa (1989), it could be the same as
Psilocybeluteonitens
(syn.
Stropharia luteonitens
).In current classifications of
Psathyrella
P. sphaerocystis
and
P. conopilus,
are placed in sect.
Atomatae
. However, whencarefully studied the dung-inhabiting species show consider-able variation both in macro- (veil characteristics) and micro-morphology. These observations suggested to us that dung-growth might not reflect common ancestry and that section
Atomatae
could be non-monophyletic.Theprincipalaimsofthepresentstudyweretoidentifythenumber of coprophilous species occuring in the Nordic region(Denmark, the Faroe Islands, Finland, Iceland, Norway, andSweden), to identify and describe new taxa, to infer the phylo-genetic relationships of coprophilous species within
Psathyr-ella
, and contribute to a more fully resolved phylogeny of
Psathyrella
and related taxa.
Materials and methods
Morphological studies
Many species of
Psathyrella
are fragile and important charac-ters are easily destroyed during handling. Therefore, all basi-diomata were photographed before being collected. The aimwas to describe the collection complete with notes on ecologyalready in the field. On dung or on heaps of manure basidio-mata sometimes grow in smaller or larger groups. The fea-tures of each basidiome were then carefully compared toprevent a mixture of species being collected, and the speci-menswere put into separateboxes.The presence ofa pseudo-rhiza was checked. As most species are hygrophanous, it isnecessary to note the colours of moist and striate caps beforethey are dried. Colour names follow the Munsell soil colourcharts (Munsell 1975), cited as Mu in the text. If present, theevanescent veil on both cap and stem was described. Some-times the veil is present only in young basidiomata and seenas fibrils close to the cap margin or as dispersed fibrils onthe stem surface. When well-developed, the veil can be ob-served as flocci, scales, or patches on the cap surface. Inmost species, the stem is pulverulent or pruinose at apexwith more or less evident veil remnants below. The upperpart of the veil sometimes leaves an annulus or fugaciousring-zone that must be noted. This zone is rarely present atthe lowerpartofthestem.If possibleall stagesofbasidiomatadevelopment were collected in order to cover changes in capcolours and veil features. Moreover, the cap and stem surfacewas examined for projecting hairs that are present in a fewspecies (e.g.
P. tenuicula
). Before drying the material spore1166 E. Larsson, L. O ¨ rstadius
Table 1 – Data of specimens sequenced in this study
Species Coll. ID./Origin Ecology, substrate GenBank no.
Psathyrella
sp. LO ¨ 382-89/Sweden In a moist, rich forest DQ389667
P. albofloccosa
Sivertsen 65-89 (TROM)/Norway On grass remnants DQ389708
P. berolinense
LO ¨ 37-04/Sweden Wild boar dung DQ389704
P. berolinense
LO ¨ 148-91/Sweden Wild boar dung DQ289705
P. calcarea
LO ¨ 211-03/Sweden On dry, calcareous soil DQ389671
P. candolleana
LO ¨ 38-00/Sweden In a rich deciduous forest DQ389720
P. cernua
LO ¨ 134-98/Sweden Caespitose on a stump DQ389726
P. clivensis
LO ¨ 182-03/Sweden On the great alvar DQ389683
P. conopilus
LO ¨ 186-02/Sweden On buried wood DQ389725
P. corrugis
LO ¨ 171-01/Sweden Attached to buried wood DQ389674
P. dicrani
LO ¨ 270-04/Sweden On dry, sandy soil DQ389698
P. effibulata
LO ¨ 37-96 type/Sweden In a field margin DQ389672
P. fatua
LO ¨ 132-97/Sweden On nitrophilous soil DQ389681
P. fibrillosa
LO ¨ 138-00/Sweden Among leaves of
Fagus
DQ389686
P. fimiseda
LO ¨ 56-96 type/Sweden Cow dung DQ389690
P. hirta
LO ¨ 142-00/Sweden Cow dung DQ389702
P. kitsiana
LO ¨ 217-85 type/Sweden On or near a stump DQ389689
P. larga
LO ¨ 223-90/Sweden In a rich deciduous forest DQ389694
P. larga
LAS97-054/Sweden Well decayed stump DQ389695
P. longicauda
LO ¨ 201-02/Sweden In a copse DQ389676
P. longicauda
Kyto ¨ vuori 94-009 (H)/Finland Between compost heaps DQ389677
P. lutensis
LO ¨ 98-03/Sweden On soil attached to sticks DQ389685
P. marcescibilis
LO ¨ 31-03/Sweden In a park with
Urtica dioica
DQ389728
P. merdicola
LO ¨ 45-02 type/Sweden Cow dung DQ389688
P. microrhiza
LO ¨ 185-02/Sweden In a rich deciduous forest DQ389684
P. mucrocystis
LO ¨ 103-98/Sweden On a stump DQ389700
P. noli-tangere
LO ¨ 83-03/Sweden Among leaves in a forest DQ389713
P. obtusata
LO ¨ 88-01/Sweden On moist soil DQ389711
P. odorata
LO ¨ 207-96/Sweden On soil in a pasture DQ389679
P. odorata
LO ¨ 50-04/Sweden In a rich deciduous forest DQ389680
P. olympiana
LO ¨ 32-02/Sweden On soil attached to wood DQ389722
P. orbicularis
LO ¨ 211-04/Sweden On dry, sandy soil DQ389692
P. orbitarum
LO ¨ 257-90/Sweden In a rich deciduous forest DQ389673
P. panaeoloides
LO ¨ 44-03/Sweden In mud of a fen DQ389719
P. pannucioides
LO ¨ 143-03/Sweden On clayey soil,
Fagus
forest DQ389727
P. pennata
LO ¨ 206-03/Sweden On burnt soil DQ389710
P. pertinax
LO ¨ 259-91/Sweden On mossy twigs of
Picea
DQ389701
P. piluliformis
LO ¨ 162-02/Germany On stump of
Fagus
DQ389699
P. potteri
LO ¨ 271-01/Sweden On nitrophilous soil DQ389665
P. prona
LO ¨ 91-99/Sweden In a rich deciduous forest DQ389666
P. pseudocasca
LO ¨ 17-04/Sweden In a rich deciduous forest DQ389691
P. pseudogracilis
LO ¨ 172-02/Sweden In a shrubbery DQ389675
P. purpureobadia
LO ¨ 23-94/Sweden Gregarious on cow dung DQ389678
P. purpureobadia
9956 (L) type/The Netherlands In a grazed grassland EU126026
P. pygmaea
LO ¨ 97-04/Sweden On wood of
Salix
DQ389718
P. romagnesii
LO ¨ 213-96/Sweden Horse dung DQ389714
P. romagnesii
LO ¨ 85-98/Sweden Horse dung DQ389716
P. romagnesii
LO ¨ 267-04/Sweden Horse dung DQ389715
P. rostellata
LO ¨ 228-85 type/Sweden Deciduous wood DQ389693
P. saponacea
LO ¨ 204-96/Sweden Horse dung DQ389717
P. scatophila
LO ¨ 64-95 type/Sweden Horse dung DQ389703
P. senex
LO ¨ 115-02/Germany In a park, among leaves DQ389712
P. spadicea
Enderle epitype/Germany Caespitose on a tree base DQ389729
P. spadiceogrisea
LO ¨ 92-01/Sweden In a deciduous forest DQ389682
P. sphaerocystis
LO ¨ 126-99/Sweden Horse dung DQ389709
P. spintrigeroides
LO ¨ 122-86/Sweden On a stump DQ389696
P. squamosa
LO ¨ 104-95/Sweden In a gravel at a roadside DQ389687
P. stercoraria
LAS80-94/Sweden Horse dung DQ389668
P. stercoraria
Kyto ¨ vuori Virrat 1991 (H)/Finland In a pasture DQ389670
P. stercoraria
LO ¨ 460-05 type/Sweden Cow dung DQ389669
P. tenuicula
LO ¨ 58-03/Sweden On soil rich in humus DQ389706
P. tenuicula
Brown (K, 49734)/England Horse dung DQ389707
P. typhae
LO ¨ 21-04/Sweden On remnants of
Typha
DQ389721(
continued on next page
)
Coprophilous species of
Psathyrella
1167
prints were taken and any green reaction of the gill edge in10 % NH
4
OH was observed in a microscope.Micromorphological characters were observed using a Beck Zenith V microscope equipped with phase contrast.For each collection ten to 20 mature spores were measuredin water at
1250 magnification. Unusually large or smallspores were not considered. Other morphological characterswere studied in a 10 % NH
4
OH solution and measured tonearest micron. To observe the hymenial cystidia a completelamella was cut off with a razor blade and soaked for a while.The gill edge was removed in order to check the cheilocysti-dia. The middle portion of the gill was cut out, crush-mounted, and pleurocystidia, basidia, subhymenium, andhymenophoral trama studied. The layers of the pileus wereobserved half-way from the margin by cutting tangential tothe pileus, a ‘scalp’. The cells of the pileipellis and the hyphaeof the pileitrama can then be observed. If the material admit-ted, a radial cut to the pileus was done instead, resulting ina better picture of the different layers. In addition, if a veilwas present above the pileipellis it could be more satisfactorylocated. Finally, the veil tissue from cap margin and the pres-ence of clamps were checked. As for the shape of spores andcystidia the terminology of Vellinga (1988) was followed.Drawings were made with the aid of a drawing tube attachedto the microscope.
Material studied
Collections are deposited in the herbarium at Plant and Envi-ronmental Sciences, Go ¨ teborg University (GB) if not otherwiseindicated. Numerous collections from Nordic herbaria havebeen examined. Many types and other collections fromEurope and North America were studied as loans, among these the Charles Peck collections from NYS and collectionsfrom AMNH, B, C, E, G, GB, H, K, L, LD, MICH, NYS, O, S,TROM, TURA, UPS, and WBS. Material was also received asgifts or loans from several private herbaria.Data on sequenced specimens are provided in Table 1.Additional specimens of the four new species,
Psathyrella fimiseda
,
P. merdicola
,
P. scatophila
, and
P. stercoraria
are listedunder each species description. Additional specimens studiedare provided as a supplementary data file.
Taxon sampling
Sequences of the completeITS regionand 1200 bp of the 5
0
endof nu-rDNA from 65 specimens of
Psathyrella
were generatedfor the study. The specimens represent almost half of the spe-cies of
Psathyrella
that occur in North Europe. Specimens wereselectedtorepresentabroadspectrumofmorphologicalchar-acters and infrageneric classification groups but with an em-phasis on coprophilous species. Availability of type material,the state of the material, and the possibility to make a com-plete description of the collection was also considered. Typecollections were sequenced only when deemed unharmfulandpermissionreceived.FifteenLSUsequencesof
Coprinopsis
,
Coprinellus
,
Parasola
, and
Psathyrella
were taken from GenBank(AF261489, AF041488, AF041489, AF041503, AF041507,AF041510, AF041517, AF041518, AF041519, AF041527,AF041525, AF041515, AF041523, AF041520, AF041511).Based on results of earlier molecular phylogenetic studiesof
Agaricales
Agro-cybe pusiola
,
Conocybe siliginea
,
Psilocybesemilanceata
, and
Galer-ina marginata
were selected as out-group.
DNA extraction, PCR, and sequencing
Sequence data were obtained from herbarium specimens(Table 1). Total DNA was isolated using DNeasy plant minikit (QIAGEN, Valencia), following the manufacturers recom-mendations. PCR reactions were carried out using Ready-To-Go
PCR beads (Amersham Biosciences, Uppsala). Primers usedto amplify the complete ITS region and the 5
0
end of the LSUregion were ITS1F (Gardes & Bruns 1993) and LR21, LR0R andLR7 (Hopple &Vilgalys1999).Amplifiedproductswerepurifiedusing Qiaquick spin columns (QIAGEN, Hilden). Primers usedfor sequencing were ITS1, ITS3, ITS4 (White
bruns/), Lr5 and LR3R (Hopple& Vilgalys 1999). Fifty to 75 ng of PCR products were used ineach sequencing reaction using DTCS Quick Start Kit (Beck-man Coulter, Fullerton). Sequences were obtained using CEQ 8000 DNA analysis system (Beckman Coulter).
Phylogenetic analyses
Sequences were edited and assembled using Sequencher 3.1(Gene Codes, Ann Arbor). Sequences were aligned automati-callyusingthesoftwareMAFFT(Katoh
2002)andadjustedmanually using the data editor in PAUP (Swofford 2003).Sequences have been deposited in GenBank and accessionnumbers are listed in Table 1.Heuristic searches for most parsimonious trees wereperformed using PAUP. All transformations were consideredunordered and equally weighted. Variable regions with am-biguous alignment were excluded and gaps were treated asmissing data. Heuristic searches with 1 K random-additionsequence replicates and tree bisection–reconnection (TBR)branch swapping were performed. Relative robustness of
Table 1 – (
continued
)
Species Coll. ID./Origin Ecology, substrate GenBank no.
P. umbrina
LO ¨ 235-04/Sweden In a rich deciduous forest DQ389697
Agrocybe pusiola
LO ¨ 304-05/Sweden On calcareous soil DQ389732
Conocybe siliginea
LO ¨ 93-04/Sweden In a pasture DQ389731
Coprinus cordisporus
LO ¨ 41-01/Sweden Cow dung DQ389723
Galerina marginata
RM3225/Sweden On wood AF195590
Lacrymaria lacrymabunda
EL70-03/Sweden In a pasture DQ389724
Psilocybe semilanceata
Holst84/Sweden In grass EU029945
1168 E. Larsson, L. O ¨ rstadius