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Molecular phylogenetic studies show Omphalina giovanellaerepresents a new section of Clitopilus (Agaricomycetes)

Gabriel MORENOa,*, Marco CONTUb, Antonio ORTEGAc,Gonzalo PLATASd, Fernando PELAEZd

aDepartamento de Biologıa Vegetal, Facultad de Biologıa, Universidad de Alcala, Alcala de Henares, ES-28871 Madrid, SpainbVia Traversa via Roma 12, I-07026 Olbia (SS), ItalycDepartamento de Botanica, Facultad de Ciencias, Universidad de Granada, ES-18071 Granada, SpaindCentro de Investigacion Basica, Merck Sharp & Dohme de Espana, Josefa Valcarcel 38, ES-28027 Madrid, Spain

a r t i c l e i n f o

Article history:

Received 13 April 2007

Accepted 27 September 2007

Published online 9 October 2007

Corresponding Editor:

David L. Hawksworth

Keywords:

Agaricales

Basidiomycota

Italy

Molecular phylogeny

Spain

a b s t r a c t

After reconsidering the specific characters of Omphalia giovanellae and applying data from

the sequences of the D1–D2 domain of the 28S rRNA gene and the ITS region of material

collected in Italy and Spain, the species is ascribed to the genus Clitopilus. Considering

the rather peculiar characters, especially the smooth spores, C. giovanellae is ascribed to

the new section Omphaloidei sect. nov. The species is illustrated with drawings of fresh

basidiomata observed in situ, as well as with SEM micrographs of the spores. Also based

on DNA sequence data, O. farinolens, recently described from Spain, is shown to be a syno-

nym of C. giovanellae.

ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

.

Introduction

Bresadola (1881) described a small greyish agaric from Trento,

Italy, which he named Omphalia giovanellae, providing a very

representative colour plate. After studying Bresadola’s collec-

tion preserved in the New York Botanic Garden, New York

(NY) dated 1899 (‘Gocciadoro.locis herbidis’), Singer (1942)

noted that the spores of the fungus had very slight longitudi-

nal ridges. Therefore, he decided to transfer the species to Cli-

topilus, and later (Singer 1946) published a detailed description

based either on the authentic material from Bresadola or from

two collections from the USA (Florida) in 1943, one made by

himself and another by Murrill. In the latter description, using

the name C. giovanellae, Singer described a small greyish

fungus with a cyathiform cap and smooth surface, shining

and sericeous, with thin and rather decurrent gills, the flesh

smelling and tasting of flour, a pink spore mass, and ovoid

to subfusiform spores ornamented with hardly visible longi-

tudinal ridges. The lamellar trama was described as ‘partly

intermixed’. The American specimens were from sandy soil

in northern Florida.

Subsequently, several authors have reported macro- and

microscopic descriptions of C. giovanellae. Those of Josserand

(1955), Contu (1992), and Cetto (1993) deserve special mention

as they were based on fresh material. Cetto (1993) published

a colour plate of the specimens studied, which is particularly

significant as he usually collected in the same places as Bresa-

dola (Contu, pers. comm.). The material used by Singer (1946)

* Corresponding author.E-mail address: gabriel.moreno@uah.es

0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.mycres.2007.09.009

1400 G. Moreno et al.

was recently examined by Baroni (1995), who compared the

data collected with those from an original collection of O. gio-

vanellae preserved in the Rijksmuseum, Stockholm (S) that

was made by Bresadola in Gocciadoro on 25 June 1888.

Although this is not the same collection used by Singer, we

consider it relevant for the purpose of the assessment of the

generic adscription of O. giovanellae, as Baroni showed that,

in both cases, the spores of the material were actually smooth,

even using SEM, and therefore, that Bresadola had correctly

understood this to be a species with non-ornamented spores.

Likewise, Baroni, having also studied the collection cited by

Singer (1946), established that that collection belonged to

a species with ornamented spores that could easily be in-

cluded within Clitopilus, describing it as the new species Clito-

pilus bigelowii (Baroni 1995). The reports by Josserand (1955)

and Contu (1992) certainly belong to C. bigelowii, and the de-

scriptions of C. giovanellae published by Moser (1967), Cetto

(1993), Noordeloos (1993), and Horak (2005) also somewhat fit

into the concept of C. bigelowii.

The taxonomic placement of Bresadola’s species is very

difficult. For this reason, mycologists have included Omphalia

giovanellae either in Clitopilus (Singer 1946) or Clitocybe (sensu

Bigelow 1982; cfr Baroni 1995). However, based on the above

observations, it is clear that this species has been misinter-

preted by most mycologists for over 110 years, and that it

has been confused with the macroscopically similar fungi

currently known as Clitopilus bigelowii or Clitopilus scyphoides

(syn. Omphalia scyphoides). In order to resolve the question

of the identity and placement of Bresadola’s taxon, we con-

ducted molecular phylogenetic studies and SEM of the basid-

iospores, which showed it is best placed in a new section of

Clitopilus.

Materials and methods

The material studied is preserved in the following dried refer-

ence collections: AH (Universidad de Alcala de Henares), GDA

(Universidad de Granada), S (Rijksmuseum, Stockholm), and

NY (New York Botanic Garden, New York).

The description of the macro- and micromorphological

characteristics was based on fresh material; the micromor-

phological characters were also studied from dried reference

material, using 5 and 10 % potassium hydroxide, ammoniacal

Congo red, and distilled water. Phoxin B and Cotton blue were

used to study spore ornamentation. Spore morphology was

further observed under field-emission scanning electron mi-

croscope (FESEM) Leo model 1539 Gemini (Zeiss, Jena) follow-

ing critical point drying, as described in Moreno et al. (1995).

DNA extraction, PCR amplification, and DNA sequencing

DNA was extracted from small fragments of the basidiomata

following previously described methods (Pelaez et al. 1996).

Amplification of the D1–D2 region of the 28S rDNA was carried

out using primers LR0R (Hopple & Vilgalys 1994) and NL4

(O’Donnell 1993). Amplification of the ITS1–5.8S–ITS2 frag-

ment was performed using primers ITS5 (White et al. 1990)

and ITS4b (Gardes & Burns 1993) in a first attempt, and then

primer pair OMP-1 (50-GGC AGC CAG AGA CTA CCA GAT

TTT-30) and 18S-3 (50-TTA CGT CCC TGC CCT TTG TAC A-30)

for the specific amplification of the ITS1–5.8S–ITS2 region.

PCR reactions were performed following standard proce-

dures (5 min at 93 �C, followed by 40 cycles of 30 s at 93 �C,

30 s at 53 �C, and 2 min at 72 �C) with Taq DNA polymerase

(QBiogene, Carlsbad, CA) following the procedures recom-

mended by the manufacturer. The amplification products

(0.1 mg ml�1) were sequenced using the Big Dye Terminator

Cycle Sequencing Kit (Applied Biosystems, Foster City, CA)

following the manufacturer’s recommendations. Each strand

of all the amplification products was sequenced with the

same primers used for the initial amplification. Partial

sequences obtained in sequencing reactions were assembled

using Bioedit 7.0.5.3 (Hall 1999).

Consensus sequences were aligned using CLUSTAL X

(Thompson et al. 1997) and the program-generated multiple

alignments were visually adjusted with GeneDoc 2.5 software

(Nicholas et al. 1997). Each sequence was compared to both

GenBank and our own internal database of local accessions

using FastA applications (GCG Wisconsin Package, Version

10.3-UNIX, Accelrys, San Diego, CA).

Phylogenetic analysis

Bayesian analysis based on the MCMC approach was run as

implemented in the computer program MrBayes 3.01 (Huel-

senbeck et al. 2002). To improve mixing of the chain, four

incrementally heated simultaneous MCMCs were run over

2 M generations. MrModeltest 2.2 (Nylander 2004) was

used for hierarchical likelihood ratio tests to calculate the

Akaike information criterion (AIC) values of the nucleotide

substitution models. The model selected by AIC for the

alignment of the 28S rRNA gene fragment was the

GTRþ IþG model of DNA substitution considering a propor-

tion of invariable sites for the substitution rates; the num-

ber of substitution rates was six.

Results

Amplification and sequencing of the D1–D2 regions of the 28S

rRNA gene was successful for all the specimens selected for

molecular study. The size of DNA fragments was 630 bp, and

the sequences of the Italian specimens of Clitopilus giovanellae

were identical. Likewise, the two specimens of Omphalina farino-

lens were identical, and they were also identical to the

C. giovanellae sequences. The closest matches of these sequences

in GenBank were with sequences of several Clitopilus species.

To get a more detailed understanding on the phylogenetic

relationships of C. giovanellae with other Clitopilus and Ompha-

lina species, a number of sequences of members of these two

genera were retrieved from GenBank. These sequences were

aligned and this alignment was subjected to Bayesian analy-

sis to build the phylogenetic tree shown in Fig 5. The four

sequences of C. giovanellae and O. farinolens were grouped in

a monophyletic clade supported by a high PP support

(100 %). This clade was grouped together with all the other

Clitopilus species studied in a large branch also strongly sta-

tistically supported (100 %). All the Omphalina species were

Molecular phylogenetic studies show Omphalina giovanellae 1401

Fig 1 – Omphalia giovanellae (S, no F-14368 – lectotypus). (A) Basidiomata. (B) Basidia. (C) Details of the hymenophore.

(D) Structure of the pileipellis. (E) Caulocystidia.

grouped in a sister monophyletic clade with 94 % of statistical

support, except for two sequences of O. pyxidiata and O. rivu-

licola, which were grouped together in a clade rooted at the

base of the tree.

Amplification of the ITS1–5.8S–ITS2 fragment was positive

for the four specimens using primers ITS5/ITS4b. However, all

the sequences obtained from these amplifications showed the

presence of more than one amplification product, probably

due to contamination of the basidiomata during storage of

the material in collections. To select for the specific amplifica-

tion of the ITS region of C. giovanellae, we designed a specific

primer from the 28S rRNA gene sequence of these organisms

(OMP-1). The size of the ITS region amplified using this primer

was 670 bp. Only one specimen (S F-14368) provided a clean

sequence for the whole fragment. The remaining specimens

yielded a clean sequence for the ITS1, but the alignments of

the sequences of both the 5.8S rRNA gene and the ITS2 were

still ambiguous. In any case, the ITS1 sequences were identi-

cal for all the four isolates. FastA analysis of the whole ITS

sequence against GenBank showed that the closest matches

were two sequences of Clitopilus prunulus, AY228348 and

DQ202272, with 89 and 87 % similarity, respectively, in an

alignment of 581 bp. The ITS sequence of C. giovanellae S, no

F-14368 was deposited in GenBank with the accession code

EF413030.

Taxonomy

Clitopilus sect. Omphaloidei G. Moreno, Contu, A. Ortega,

Platas & Pelaez, sect. nov.

MycoBank no.: MB 510681.

Etym.: After the Omphalina-like form of the basidiomata.

Species sporis laevibus habitusque omphaloideus praeditis.

Typus: Clitopilus giovanellae (Bres.) Singer 1942.

Clitopilus giovanellae (Bres.) Singer, Mycologia 34: 66 (1942).

Basionym: Omphalia giovanellae Bres., Fungi Trid. 1: 9 (1881)

Synonyms: Omphalina farinolens G. Moreno & Esteve-Rav.,

Micologia 2000 (Trento): 393 (2000).

Gerronema farinolens (G. Moreno & Esteve-Rav.) Bon, Docu-

ments Mycologiques 31(122): 14 (2001)

1402 G. Moreno et al.

Fig 2 – (A–I) Omphalia giovanellae (NY 776561). (A) Basidioma fragments of Bresadola’s material. (B) Label of Bresadola’s

material. (C–D) Basidia. (E) Spores. (F–H) Spore tetrads. (I) Detail of gill trama. (J–O) Clitopilus giovanellae (AH 19780). (J) Detail

of hymenophore. (K) Basidia. (L) Detail of gill trama. (M) Basidia. (N–O) Spores. Bars [ (A) 5 mm; (C–O) 10 mm.

Molecular phylogenetic studies show Omphalina giovanellae 1403

Typus: Hispania, Segovia, Revenga Palacio de Riofrio, in

pascuis arenosis inter quercetum, 5 Nov 1998, A. Sanchez,

J. Diez, V. Escaso, M. M. Dios, F. Esteve-Raventos & G. Moreno,

(AH 19718 – holotypus).

Pileus 5–15 mm diam, almost without flesh, convex then

plane when expanded and with the centre clearly de-

pressed-cyathiform, never umbonate, when dried not striate,

pale grey-ash to grey-ochraceous, surface very slightly tomen-

tose and weakly sericeous. Lamellae rather thin, distant, some-

what crowded, decurrent, white or with some greyish hues,

not changing with age. Stipe 10–20� 1–2 mm, cylindrical,

with the base sometimes subbulbous, when dried opaque,

smooth, white or slightly paler than the cap, white mycelium

forming some mycelial strands. Flesh fragile, white, smelling

strongly of flour and with a similar taste, not bitter. Spore print

not obtained, but most likely white (Material GDA). Basidio-

spores 5–6.4–7.5(�8)� 3–3.8–4 mm, hyaline, not cyanophilic,

broadly ellipsoid, ovoid or amygdaliform, sometimes subna-

vicular (l:b¼ 1.6–1.8–2), with pointed top, smooth, thin-walled,

with one or more lipidic guttules, apiculus evident, isolated or

grouped in tetrads. Basidia 14–22� 6.5–9.5 mm, tetrasporic,

Fig 3 – Clitopilus giovanellae (AH 19780). Hymenium and

spores details under the scanning electronic microscope.

Bar [ 5 mm.

Fig 4 – Clitopilus giovanellae (AH 19780) basidiomata.

Bar [ 10 mm.

clavate or subpyriform (l:b¼ 1.9–2.3), without clamps at the

base; subhymenium with irregular texture, composed of

elongated elements. Hymenophoral trama parallel or irregular,

composed of rather thin hyphae, oleipherous hyphae sparse.

Cystidia and marginal cells absent. Pileipellis consisting of a xero-

cutis of cylindrical and very thin hyphae, interwoven and often

randomly straight, 2–3(–4.5) mm wide, with intraparietal

pigment and sometimes small crystals on the hyphae wall

surface. Stipitipellis with cylindrical hairs, sometimes bifurcate

at the apex and with intraparietal pigment. Clamp connections

absent (Figs 1–4).

Type material: Basidiospores 6.5–6.8–7.5� 3.5–3.7–4 mm

(l:b¼ 1.3–1.7–1.9). Basidia: 13–22� 6.5–9 mm (l:b 1.9–2.7).

Habitat: Gregarious in sandy soils with discontinuous vege-

tation cover. Autumn. Known from Italy and Spain.

Material studied: Italy: Prov. Trento, Trentino Alto Adige, Gioccia-doro, 25 Jun. 1888, G. Bresadola, (S, no F-14368 – lectotype of Ompha-lia giovanellae); loc. cit., locis herbidis, Jul. 1889, G. Bresadola (NY776561).Sardinia, Prov. Sassari, Aglientu, ‘Riu Li Saldi’, close tothe beach, between mosses and low grass, in sandy soil, 16 Oct.2005, M. Contu (AH 19780, GDA 52531). – Spain: Caceres, Madroneraat Erguijuela, in acidic sandy pastures with Bovista plumbea,17 May 1996, G. Moreno, L. Montoya, M. Lizarraga & V. Bandala (AH19593); Segovia, Revenga Palacio de Riofrıo, in open, sandy pas-tures with Silene colorata and Solantha guttata s. lat. in a Quercusilex subsp. ballota forest, 5 Jun. 1998, A. Sanchez, J. Diez, V. Escaso,M. M. Dios, F. Esteve-Raventos & G. Moreno (AH 19718 – holotype ofOmphalina farinolens, Herb. M. Bon – isotype).

Discussion

There is no doubt that Clitopilus giovanellae closely resembles

C. bigelowii, being different mainly from the micromorpholog-

ical standpoint, i.e. possessing completely smooth spores

lacking any longitudinal ridges. Macroscopically, it may be

difficult to distinguish the two species, but in our opinion,

whereas in C. bigelowii the gills are greyish with pinkish tones

when mature, in C. giovanellae they are white or at least totally

lacking any pink tones (and the spores are actually white or

pale). The morphology of the pileus surface is not a good dif-

ferential character, although there are some descriptions in

the literature reporting differences in this character. Clitopilus

bigelowii has been described as tomentose or tomentose-felted

(Josserand 1955; Contu 1992; Cetto 1993). Singer (1946), fol-

lowed by Baroni (1995), described the pileus cutis of C. giovanel-

lae in Singer’s sense (i.e. C. bigelowii) as ‘sometimes somewhat

shining, sericeous, smooth, but often subrugulose’; this char-

acter was absent in the Sardinian collection of C. giovanellae,

which instead had a pileus surface that was more sericeous

or only very slightly tomentose. This is a very subjective char-

acter and may depend on the prevailing weather conditions

when material was collected. However, spore morphology is

a much more stable and reliable character.

Our phylogenetic analysis, based on partial sequences of

the 28S rRNA gene, clearly supports the adscription of Bresa-

dola’s fungus to Clitopilus. The sequences of the material stud-

ied fell within a monophyletic clade containing all the

Clitopilus species we studied, including the type species

C. prunulus (Fig 5). These sequences were very close to the

1404 G. Moreno et al.

0.1

Agaricus campestris AY207134Omphalina rivulicola ORU66451Omphalina pyxidata OPU66450

Clitopilus scyphoides AF261288Clitopilus giovanellae S F-14368 (EF413027)

Clitopilus giovanellae AH 19780 (EF413026)

Omphalina farinolens AH 19718 (EF413028)

Omphalina farinolens AH 19593 (EF413029)

Clitopilopsis hirneola AF223164Clitopilopsis hirneola AF223163

Clitopilus apalus AF261287Clitopilus prunulus AY700181Clitopilus prunulus AF042645Clitopilus prunulus AY228348

Omphalina grossula OGU66444Omphalina foliacea AY542864

Omphalina rosella ORU66452Omphalina brevibasidiata OBU66441

Omphalina obscurata OOU66448Omphalina sphagnicola OSU66453

Omphalina philonotis OPU66449Omphalina ericetorum OEU66445

Omphalina velutina OVU66454Omphalina grisella OGU66443

Omphalina luteovitellina OLU66447Omphalina hudsoniana OHU66446

100

83

100

59

100

94

100

9992

94

66

100

65

100100

100100

97

10096

Fig 5 – Phylogenetic tree based on the D1–D2 domains of the 28S rRNA gene of Clitopilus and Omphalina species. The GenBank

accession numbers of the sequences obtained for this work are indicated in parenthesis. The alignment contained 630

characters, 403 of which were constant. The phylogenetic tree consisted of 545 steps. PP values are given above or at the

nodes. A sequence of Agaricus campestris was used to root the tree.

sequences of two specimens of Omphalina farinolens, being

grouped together in a monophyletic clade with high statistical

support. The rest of the Omphalina species included in the tree

were clustered in a branch separate from the clade containing

the Clitopilus species.

The topology of this phylogenetic tree was totally consis-

tent with the data reported by Moncalvo et al. (2002) based

on essentially the same sequences, included in a large phylog-

eny of agarics. Thus, their tree also included Clitopilopsis hir-

neola within the monophyletic group that contained all the

Clitopilus species studied. Likewise, the species of Omphalina

appeared divided into two well-differentiated clades, one

including only O. rivulicola and O. pyxidata (the type species)

and another large group containing the remaining species,

which would be more related to Arrhenia (Moncalvo et al. 2002).

O. farinolens was described by Moreno & Esteve-Raventos

(2000), collected in xerophilic pastures in Spain and character-

ized by very small basidiomata, greyish in colour, decurrent

lamellae, and flesh with a strong smell of flour. This species

was found growing on thin strata of mosses in forests of Quer-

cus ilex subsp. ballota in the spring. The rDNA sequences of the

type material of this taxon (Fig 5) showed it to be a later

synonym of Clitopilus giovanellae, and therefore, the known

distribution of that species should be extended to Spain.

The conclusions obtained from the study of the 28S rRNA

were also supported by partial sequences of the ITS region.

Although only the ITS1 could be amplified and sequenced

from all the specimens analysed, this spacer showed identical

sequence among the four isolates of C. giovanellae and O. fari-

nolens, convincingly supporting their co-specificity. Also, the

closest sequence of the ITS region of C. giovanellae in GenBank

was that of C. prunulus, consistently with the conclusions

inferred from the 28S rRNA sequences.

A related species could be O. albominutella, described from

Germany based on collections on moss mats (Ludwig 2001)

and previously described as resembling O. schyphoides ‘sensu

Bres.’ (Ludwig 1997). This fungus is characterized by its very

small size,whitish colour,decurrent gills, smallellipsoid spores,

and unclamped hyphae. At present, we are not in a position to

express a definitive opinion on the taxonomic position of this

species, not having studied DNA from authentic material.

In view of the phylogenetic distinctness of C. giovanellae,

and also its smooth basidiospores, we feel it is best accommo-

dated in a new section of Clitopilus for all the species with

smooth spores, sect. Omphaloidei.

Acknowledgements

We thank Luis Monje for his invaluable help in the digital

treatment of the photographs. We express our gratitude to

Molecular phylogenetic studies show Omphalina giovanellae 1405

Derek W. Mitchell for the revision of the manuscript, and wish

to thank Juan de Dios Bueno and Alicia Gonzalez for their in-

valuable help with the FESEM, and the curators of AH (Javier

Rejos) and GDA (Marıa Teresa Vizoso). We express our grati-

tude to David L. Hawksworth and Derek W. Mitchell for the

revision of the manuscript.

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