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Phylogenetic origins of two cleistothecial fungi, Orbicula parietina and Lasiobolidium orbiculoides, within the operculate discomycetes


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Parsimony, maximum-likelihood and Bayesian analyses of SSU rDNA sequences of representative taxa of Pezizomycetes, Eurotiomycetes, Dothideomycetes, Leotiomycetes and Sordariomycetes, all strongly support the cleistothecial fungi Orbicula parietina and Lasiobolidium orbiculoides to be of pezizalean origin. Previous hypotheses of close affinities with cleistothecial or highly reduced fungi now placed in the Thelebolales, Eurotiales or Onygenales are rejected. Orbicula parietina and L. orbiculoides are deeply nested within Pyronemataceae (which subsumes the families Ascodesmidaceae, Glaziellaceae and Otideaceae). LSU rDNA sequences suggest that Orbicula is nested within the apothecia-forming genus Pseudombrophila (including Nannfeldtiella and Fimaria) and that L. orbiculoides is closely related. Ascodesmis and Lasiobolus, which have been suggested as closely related to Orbicula and Lasiobolidium, are identified as a sister lineage to the Pseudombrophila lineage. Cleistothecial forms that have lost the ascus operculum and ability to discharge spores actively have evolved at least once in the Pseudombrophila lineage. Some species of Pseudombrophila produce subglobular ascomata initials that are closed early in development and open only in the mid-mesohymenial phase. We hypothesize that, in the Pseudombrophila lineage, ascomata forms that never open are derived from ascomata that open late in development. The placement of O. parietina and L. orbiculoides within Pseudombrophila is supported by morphological characters, ecology and temperature optima for fruiting.
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Phylogenetic origins of two cleistothecial fungi, Orbicula parietina and
Lasiobolidium orbiculoides, within the operculate discomycetes
K. Hansen
B.A. Perry
D.H. Pfister
Harvard University Herbaria, Cambridge,
Massachusetts 02138
Abstract: Parsimony, maximum-likelihood and Bayesian
analyses of SSU rDNA sequenc es of representative
taxa of Pezizomycetes, Eur otiomycetes, Dothideomy-
cetes, Leoti omycetes and Sordariomycetes, all strong-
ly support the cleistothecial fungi
Orbicula parietina
Lasiobolidium orbiculoides
to be of pezizalean
origin. Previous hypotheses of close affinities with
cleistothecial or highly reduced fungi now placed in
the Thelebolales, Eurotiales or Onygenales are
Orbicula parietina
L. orbiculoides
deeply nested within Pyronemataceae (which sub-
sumes the families Ascodesmidaceae, Glaziellaceae
and Otideaceae). LSU rDNA sequences suggest that
is nested within the apothecia-forming
L. orbiculoides
is closely
, which have been
suggested as closely related to
, are identified as a sister lineage to the
lineage. Cleistothecial forms that
have lost the ascus operculum and ability to
discharge spores actively have evolved at least once
in the
lineage. Some species of
produce subglobular ascomata in-
itials that are closed early in development and open
only in the mid-mesohymenial phase. We hypothe-
size that, in the
lineage, ascomata
forms that never open are derived from ascomata
that open late in developmen t. The placement of
L. orbiculoides
supported by morphological characters, ecology and
temperature optima for fruiting.
Key words: ascoma evolution, molecular phyloge-
netics, Pezizales,
, Pyronemataceae
Morphological and molecular evidence show that
cleistothecial fungi have evolved independently sev-
eral times within apothecial and perithecial lineages
of ascomycetes. Molecular phylogenetic analyses in-
dicate one major group (composed of Ascosphaer-
ales, Onygenales and Eurotiales) containing most
cleistothecial ascom ycetes but that other cleistothecial
fungi fall within other ascomycete groups (Berbee
and Taylor 1994, LoBuglio et al 1996). We investigate
the placement of two cleistothecial fungi,
Lasiobolidium orbiculoides
, that have
been suggested to have pezizalean affinities.
The genus
Cooke (1871) produces epige-
ous, globose ascomata, up to 2 mm in diam, without
an opening (F
IGS. 1–3). At first sight it resembles, and
is often mistaken for, a slime mold (a miniature
) (Hughes 1951). Asci lack an apical opening
mechanism and disintegrate at maturity. The ascomata
become filled with a dry mass of ascospores that are
liberated when the peridium is disturbed and broken
by an external force (F
IG. 2).
Orbicula parietina
, the
only accepted species, has been repeatedly described
as new and assigned to nine genera, four in the
myxomycetes (Hughes 1951). Moreover, the family
Orbiculaceae and order Orbiculales (Ascomycota)
were erected for
(Locquin 1974), but
invalidly published (ICBN Art. 36.1). Malloch and
Cain (1971) thought that
was closely related
to members of the Thelebolaceae and placed it, along
with other cleistothecial fungi—
the cleistothecial family Eoterfeziaceae, which they
considered Pezizales. Jeng and Krug (1976) trans-
ferred the genera of Eoterfeziaceae including
to the tribe Theleboleae sensu Korf (1972) of the
Pyronemataceae, following the notion that closely
related genera with exposed hymenia and cleistothe-
cial genera are better accommodated in one rather
than separate families. Benny and Kimbrough (1980)
in Eoterfeziaceae, while Dennis
(1981) placed it in Eurotiaceae (Plectascales). Arx
(1981) treated
in the Pezizales and Campbell
et al (1991) likewise suspected affinities with the
operculate discomycetes. Malloch (in Dissing and
Schumacher 1994) suggested that both
, with clearly pezizalean characteristics,
might better be included in Pyronemataceae or in the
Pezizales without assignment rather than in Eoterfe-
ziaceae. Currently
is placed in the Pyronema-
taceae, but its disposition is indicated tentatively with
a question mark (Eriksson 2005).
Accepted for publication 30 Aug 2005.
Corresponding author. E-mail:
Mycologia, 97(5), 2005, pp. 1023–1033.
2005 by The Mycological Society of America, Lawrence, KS 66044-8897
Lasiobolidium orbiculoides
was the second species
described in
(Malloch and Benny
1973). The specific epithet
refers to its
similarity to
O. parietina
L. orbiculoides
both have broadly oblate, uniseriate ascosp ores
(Malloch and Benny 1973).
Lasiobolidium orbiculoides
differs from the type species of
, by being fast-growing, producing ascomata
with wavy to flexuous, septate appendages, an
excipulum of one tissue type, cylindrical uniseriate
asci and oblate ascospores (Moustafa and Ezz-Eldin
was consider ed a cleistothecial
counterpart of
(Thelebolaceae, Pezizales),
but the genus was at first placed in the Eoterfeziaceae
(Malloch and Cain 1971). Jeng and Krug (1976)
to the Theleboleae sensu
Korf (1972) of the Pyronemataceae. Developmental
studies of
L. orbiculoides
however, have shown little
evidence of affinities with either
Thelebolaceae, rather they support a relationship
with other operculate discomycetes, especially with
(Janex-Favre and Locquin-Linard 1979).
The type genus of Thelebolaceae,
, was
found to be non-pezizalean based on ascoma de-
velopment and ascus structure (e.g., Samuelson and
Kimbrough 1978, Kimbrough 1981). Based on mo-
lecular phylogenetic analyses (Momol et al 1996,
Landvik et al 1997) the Thelebolaceae was found to
be closely related to Erysiphales and Leotiales, and
was moved to the Leotiomycetes (Eriksson and Winka
maintained in the Pezizales, in Otideaceae (Eriksson
1999) (5Pyronemataceae [Eriksson et al 2001,
Eriksson 2005]). Further studies by Landvik et al
(1998) confirmed the relationship of Thelebolaceae
with members of the Leotiomycetes, and grouped
in the Pezizales.
In the present study we performed phylogenetic
analyses of the SSU rDNA to address the phylogenetic
position of
O. parietina
L. orbiculoides
within the
ascomycetes; and analyses of the LSU rDNA to
address specific relationships of
O. parietina
within the Pyronemataceae.
Specimens.—Specimens sequenced and morphologically
examined are listed (T
I). To test hypotheses
regarding relationships of Orbicula and L. orbiculoides,
the SSU rDNA sequences were analyzed with 61
sequences retrieved from GenBank from represen tative
taxa of Pezizomycetes, Eurotiomycetes (Eurotiales and
FIGS. 1–6.
Orbicula parietina
. 1. Cleistothecia on dung of dove (?) 32.5. 2. Close-up of cleistothecia, globose with a flattened
base, glabrous over the upper surface and with hyphoid hairs originating from the base. In one ascomata the thin outer
excipulum is broken open to show the contents of the yellowish spores 35. 3. Median, longitudinal section through a mature
ascomata to show basal cushion on which asci and paraphyses are borne 315. (F
IGS. 1–3: JHP-01.013/PH01-001, C). 4. Light
microscopic slide (LM) of part of lower cleistothecial wall in surface view, with long, hyphoid hairs coming from the base of the
cleistothecia 3100. 5. LM of hyphoid, flexuous, pale brownish, thick-walled and remotely septate ascomatal hairs 3200. 6. LM
of outer excipulum cells seen in surface view; cells thick-walled, small and irregularly lobed (puzzle-like) with intercellular,
amorphous, brownish pigment. Hairs originating from outer excipulum cells, with forked bases 3400. (F
IGS. 4–6: C F-24441).
Photos: 1–3 J.H. Petersen. 4–6 K. Hansen.
Onygenales), Dothideomycetes (Pleosporales), Leotio-
mycetes (Helotiales, Erysiphales and Thelebolales) and
Sordariomycetes (Sordariales and Hypocreales): Ascobo-
lus carbonarius P. Karst., AY544720; Ascodesmis sphaeros-
pora W. Obrist., U53372; Ascozonus woolhopensis Renny,
AF010590; Barssia oregonensis Gilkey, U42657; Byssonec-
tria terrestris (Alb. & Schwein. : Fr.) Pfister, Z30241;
Caccobius minusculus Kimbr.; Chaetomium elatum Kunze,
M83257; Chalazion helveticum Dissing, AF061716; Cheily-
menia stercorea (Pers. : Fr.) Boud., U53375; Chorioactis
TABLE I. Source of material examined and sequenced by the authors. Numbers in parentheses indicate multiple collections
of a single taxon
Collection number (Herbarium),
date and collector LSU SSU
Ascodesmis nigricans
Tiegh. CBS 389.68, Netherlands, Wageningen,
29.I.1986, G. Tichelaar
Eleutherascus lectardii
(Nicot) Arx CBS 626.71, France, Moselle, I.1968, P. Lectard DQ168334
Geopyxis carbonaria
(Alb. & Schwein.)
C F-49793 (C), Denmark, Jutland, 13.XI.1982,
T. Læssøe
sp. KH.04.48 (FH, DBG), USA, Colorado,
13.IX.2004, K. Hansen, V. Evenson
Glaziella aurantiaca
(Berk. & Curt.)
PR-5954 (FH), Puerto Rico, Loquillo Mts.,
11.VI.1998, N.C. Clum, D.J. Lodge.
Greletia reticulosperma
Riousset & G. Riousset
Part of isotype (herb. Roy Kristiansen), France,
1984, G. Riousset
Lasiobolidim orbiculoides
Malloch & Benny CBS 344.73, USA, California, dung of deer,
10.VI.1953, G.L. Benny
DQ062995 DQ063000
Lasiobolus cuniculi
Velen. Rana 76.053 (C), Norway, Nordland, 9.IX,1976,
H. Dissing
Lasiobolus ciliatus
(Berk.) Sacc. KS-94-005 (C), Denmark, Møn, 5.IV.1994, K.
Hansen, S.K. Sandal
Orbicula parietina
(Schrad.) S. Hughes C F-24441 (C), Denmark, Zealand, on rush mat,
1988, H.F. Gøtzsche
DQ062988 DQ062998
Otidea onotica
(Pers.) Fuckel KH-98-107 (C), Denmark, Zealand, 21.VII.1998,
K. Hansen
Otidea umbrina
(Pers.) Bres. KH.01.09 (C), Denmark, Bornholm,
30.IX.2001, C. Lange
Paurocotylis pila
Berk. Trappe 12583 (OSC), New Zealand, South
Island, 24.IX.1993, M. Amaranthus
Pseudombrophila guldeniae
Svrcek (1) Kongsv. 85.10B (C), Norway, Oppdal,
23.VIII.1985. H. Dissing, S. Sivertsen
DQ062993 DQ063001
Pseudombrophila guldeniae
(2) s.n. (FH, part in C and TRH), Norway, Oppdal,
13.VI.1985, S. Sivertsen, I. Dissing, H. Dissing
Pseudombrophila merdaria
Brumm. (1)
s.n. (FH), USA, ME, on manured soil,
30.VII.1994, D.H. Pfister
Pseudombrophila merdaria
(2) s.n. (FH), USA, VE, on composted silage at
edge of hay field, VI.1979, M. Shemluck
Pseudombrophila merdaria
(3) s.n. (FH), USA, IA, no date, T.J. Farrell DQ062992
Pseudombrophila theioleuca
Rolland C F-70057 (C), Denmark, on deer dung,
25.IX.1982, H. Knudsen
DQ062989 DQ062999
Pulvinula constellatio
(Berk. & Broome.)
KH.03.64 (FH), Norway, Nordland,
22.VIII.2003, K. Hansen, C. Lange
Pulvinula convexella
(P. Karst.) Pfister KH.01.020 (C), Denmark, Fyn, 5.X.2001, K.
Smardaea amethystina
(W. Phillips)
KH-97-132 (C), Denmark, Zealand, 1997, C.
Lange, K. Hansen
Tarzetta catinus
(Holmsk.) Korf & J.K.
KS.94.10A (C), Denmark, Møn, 11.V.1994, K.
Hansen, S.K. Sandal
Tarzetta pusilla
Harmaja KH.03.66 (FH), Norway, Nordland,
22.VIII.2003, K. Hansen, C. Lange
Sequences from manus in prep.
geaster (Peck) Kupfer, AF104340; Cookeina speciosa
(Fr.:Fr.) Dennis (5C. sulcipes (Berk.) Kuntz e),
U53376; Desmazierella acicola Lib., AF104341; Discina
macrospora Buba´k, U42651; Elaphomyces maculatus Vittad.,
U45440; Erysiphe orontii Castagne, AB033483; Eurotium
herbariorum (F.H. Wigg.) Link, AB008402; Geopyxis
carbonaria (Alb. & Schwein. : Fr.) Sacc., AF104665;
Gymnoascus reessii Baran., AB015774; Gyromitra montana
Harmaja, U42652; Hymenoscyphus ericae (D.J. Read) Korf
& Kernan, AY524847; Iodophanus carneus (Pers. : Fr.)
Korf, U 53380; Lamprospora kristiansenii Benkert,
AF121075; Lasiobolus papillatus (Pers.:Fr.) Sacc.,
AF010588; Monascus ruber Tiegh., AB024048; Morchella
elata Fr. : Fr., U42641; Nectri a cinnabarina (Tode : Fr.) Fr.,
AB003949; Neottiella rutilans (Fr.) Dennis, AF061720;
Neurospora crassa Shear & B.O. Dodge, X04971; Helvella
lacunosa Afzel. : Fr., U53378; Leucangium carthusianum
(Que´l.) Paol., U42647 ; O nygena equina (Willd. : Fr.)
Pers., U45442; Paurocotylis pila Berk., U53382; Peziza
succosa Berk., U53383; Phyllactinia guttata (Wallr. : Fr.)
Le´v., AF021796; Plectania rhytidia (Berk.) Nannf. & Korf,
AF104344; Pleospora herbarum (Pers. : Fr.) Rabenh.,
U05201; Pulvinula archeri (Berk.) Rifai, U62012; Pyro-
nema domesticum (Sowerby : Fr.) Sacc., U53385; Reddello-
myces donkii (Malenc¸on) Trappe et al, U42660; Rhizina
undulata Fr., U42664; Sarcosphaera coronaria (Jacq.)
Boud., AY544712; Saccobolus sp., U53393; Sarcoscypha
austriaca (Berk.) Boud., AF006318; Sarcosoma globosum
(Schmidel : Fr.) Casp., U53386; Sclerotinia sclerotiorum
(Lib.) de Bary, L37541;
Scutellinia scutellata (L. : Fr.)
Lambotte, U53387; Sordaria fimicola (Roberge ex Desm.)
Ces. & De Not., AY545724; Sporormia lignicola W. Phillips
& Plowr., U42478; Talaromyces flavus (Klo¨cker) Stolk &
Samson, M83262; Tarzetta catinus (Holmsk. : Fr.) Korf &
J.K. Rogers, U53389; Thecotheus holmskjoldii (E.C. Han-
sen) Chenant., AF010589; Thelebolus crustaceus (Fuckel)
Kimbr., U53394; Thelebolus stercoreus Tode : Fr., U49936;
Trichophaea hybrida (Sowerby.) T. Schumach., U53390;
Tuber gibbosum Harkn., U42663; Wilcoxina mikolae (Chin.
S. Yang & Wilcox) Chin. S. Yang & Korf, U62014;
Xeromyces bisporus L.R. Fraser, AB024049; Zopfia rosatii
(Segretain & Destombes) D. Hawksw. & C. Booth,
Speg. and
Fr. were used to root the
SSU trees (
Neolecta vitellina
[Bres.] Korf & J.K. Rogers,
Neolecta irregularis
[Peck] Korf & J.K. Rogers,
Taphrina pruni
Tul., AJ495828). Representatives of
Saccharomycetes were also included (
Kluyveromyces lactis
(Boidin, Abadie, J.L. Jacob & Pignal) Van der Walt,
Saccharomyces cerevisiae
E.C. Hansen, V01335;
sp., AY227899).
Ingroup taxa in the LSU data set, in addition to the
lineage, were selected based on prelimi-
nary results of a large-scale molecular study of the
Pyronemataceae (in prep). In that study, based on analyses
of LSU rDNA sequences, the type species of
L. spirale
, is placed in a different lineage of Pyronemataceae
distant from
L. orbiculoides
(data not shown). Therefore,
is not included in the present study. The genus
sensu Brummelen (1995) includes two
species of both sections,
P. merdaria
P. guldeniae
, were
were used to root the LSU
trees, based on the results of our large-scale Pyronemata-
ceae study (in prep.), which resolved these taxa in a clade
basal to the ingroup of this investigation. The effect of
additional, alternative outgroups was explored (
Molecular methods and Analyse s.—Laboratory techniques
generally followed procedures outlined in Hansen et al
(2002). SSU rDNA was amplified using the PCR with
primer pair SL1 (Landvik et al 1997) and NS8 (White et
al 1990). In addition to the primers used for PCR,
internal primers SL122 and SL344 (Landvik et al 1997),
and NS2, NS4 and NS6 (White et al 1990) were used for
sequencing. The 5 9 end of the LSU-rDNA was amplified
using the primer pairs LROR and LR5 (Moncalvo et al
2002). These primers and two internal primers, LR3
and LR3R (Moncalvo et al 2002), were used for
sequencing. Electrophoresis and data collecting were
done on an ABI PRISMH 3100 or 3730 DNA sequencer
(Perkin-Elmer/ABI). To verify the LSU rDNA sequence
of Orbicula, DNA was extracted and sequenced twice,
on two different occasions, from coll. no. F-24441 (C).
Sequences were edited using Seq uencher 3.0 (Gene
Codes, Ann Arbor, Michigan). Sequences are deposited
in GenBank (T
I). Nucleotide sequences were
aligned by hand using the software program Se-Al v.
2.0a11 (Rambaut 2002). Alignments are available from
TreeBase ( as ac-
cessions M2365 (SSU) and M2366 (LSU). Analyses were
performed using PAUP* 4.0b 10 for Unix (Swofford
2002) and MrBayes 3.0b4 (Huelsenbeck and Ronquist
2001) on a G5 Macintosh computer. Maximum parsi-
mony (MP), Bayesian and Maximum likelihood (ML)
analyses were performed as in Hansen et al (2005),
except MP analyses of the LSU data used branch-and-
bound searches, ML model parameter valu es were
estimated on the LSU data set, and Bayesian MCMC
were run for 2 000 000 generations. To select the model
of nucleotide substitution with the least number of
parameters best fitting the SSU data set, hierarchical
likelihood ratio tests were performed as implemented in
the program MrModeltest 2.2 (Nylander 2004). In
Bayesian analyses of the SSU and LSU data, the first
2000 trees and 1000 trees were deleted respectively as
the ‘‘burn-in’’ period of the chain. In addition to
parsimony bootstrap proportions (BP) and Bayesian
posterior probabilities (PP), ML bootstrap proportions
(ML-BP) were generated using 100 bootstrap replicates
of ‘‘fast’’ stepwise sequence addition for the SSU data,
and using 500 bootstrap replicates of heuristic searches,
with 10 random addition sequences and TBR branch
swapping for the LSU data. The model parameters
estimated for the ML analyses were entered manually
into PAUP for the ML-BP searches. Topological ly
constrained MP and ML analyses of the LSU data set
were used to evaluate if the cleistothecial form
originated once or twice from apothecia-forming taxa
with loss of active spore discharge. MacClade 4.05
(Maddison and Maddison 2002) was used to construc t
a constraint tree with O. parietina and L. orbiculoides
forced to be monophyle tic. Parsimony and ML analyses
were performed under the constraint, using the same
settings as specified above. For the ML analyses the
estimated model parameters were used. The Kishino-
Hasegawa test (Kishino and Hasegawa 1989) was used to
compare the trees under the constrained and un -
constrained topologies in PAUP.
Morphological features.—The material of O. parietina
used for DNA extraction (C F-24441) agrees with
the description by Hughes (1951) and Udagawa
and Furuya (1972). This collection is fully mature;
asci have completely disintegrated and the dry
ascoma is filled with loose, powdery, yellow spores.
We want to highlight the following characters in
O. parietina. Dried mature ascomata show no signs
of breaking open (F
. 1), but a light touch
ruptures the thin and brittle outer excipular wall
. 2). Ascomata are glabrous and loosely
covered at the base with pale brown hyphal hairs,
that are up to 120 mm long or possibly longer, up
to 5 mm wide, flexuous, thick-walled (1.2 mm) and
remotely septate (F
originate from the base of the ascoma, are
rounded at the tips, abundant and often form
a loose ‘‘mat’’ at the base of the ascoma, almost
like a subiculum. The cells of the outer excipulum
are, in surface view, brown, thick-walled, small and
irregularly lobed in outline (puzzle-like) (F
. 6).
Phylogenetic position of Orbicula and L. orbiculoides
among the Ascomycetes; the SSU phylogeny.—-The SSU
rDNA data set included 1721 characters with 398
being parsimony informative. Parsimony analysis
resulted in 16 MPTs (1667 steps, CI 5 0.496, RI 5
0.702). ML analysis resulted in one optimal tree
(2lnL 5 11 857.5723, F
. 7) under the GTR + I +
G model of sequence evolution selected by
MrModeltest with base frequencies A 5 0.2593,
C 5 0.2042, G 5 0.2645, T 5 0.2720, and the
substitution model: [A–C] 5 1.2438, [A–G] 5
2.3987, [A–T] 5 1.5819, [C–G] 5 0.8876, [C–T] 5
4.9308, [G–T] 5 1.0000, with a proportion of
invariable sites I 5 0.4510, and a gamma distrib-
uted shape parameter 5 0.5648.
The Pezizomycetes form a moderately supported
monophyletic group in the MP and Bayesian analyses
(BP 77%, PP 100%), as a sister group to all other
Euascomycetes sampled (including Thelebolales) (PP
IG. 7). Although relationships between classes
are resolved with only weak support (BP , 50%), the
Dothideomycetes, Eurotiomycetes and Sordariomy-
cetes are highly supported (all 100%). Leotiomycetes
is resolved as monophyletic in all analyses but with low
support. The orders, Thelebolales and Erysiphales are
strongly supported as monophyletic (BP 92–100%,
ML-BP 86–100%, PP 100%) within the Leoti omycetes.
Orbicula parietina
L. orbiculoides
form a highl y
supported group with two species of the apothecial
in all analyses (BP 96%, ML-
BP 92%, PP 100%,F
IG. 7). The
lineage is deeply nested within a highl y supported
lineage of members of Pyronemataceae s.l., Sarcoscy-
phaceae and Sarcosomataceae (BP 92%, ML-BP 78%,
PP 100%) (lineage I) within the Pezizales. A few
potential sister lineages are supported e.g., the
lineage (BP 84%, ML-BP 75%,
PP 100% )andthe
lineage (PP
100%). The Pyronemataceae as sampled here, in-
cluding Ascodesmidaceae and Glaziellaceae, are
monophyletic and supported by Bayesian analyses
(PP 97 %,F
IG. 7). The families Discinaceae, Helvella-
ceae, Morchellaceae, Rhizinaceae and Tuberaceae are
highly supported (BP 90–100%, PP 98–100%) and
form a weakly supported sister group (II) to the
lineage I (F
IG. 7). Lineage s I and II form a moderately
supported group by MP and Bayesian analyses (BP
73%, PP 100%). Ascobolaceae and Pezizaceae are
strongly supported (each BP and PP 100%, ML-BP
98–100%) and form a weakly supported lineage (III,
BP 65%) that is sister to the lineages I and II.
Relationships of the Pseudombrophila lineage within
Pyronemataceae; the LSU phylogeny.—Branch and
Bound searches resulted in 2 MPTs (501 steps,
CI 5 0.653, RI 5 0.749) produced from 915 total
characters, of which 182 are parsimony informa-
tive. ML analysis resulted in one optimal tree
(2lnL 5 3797.8522, F
. 8) under the GTR + I + G
model with base frequencies A 5 0.2519, C 5
0.2051, G 5 0.3044, T 5 0.2386, and the
substitution model: [A–C] 5 0.6839, [A–G] 5
3.0327, [A–T] 5 1.9889, [C–G] 5 0.6644, [C–T] 5
6.2107, [G–T] 5 1.0000, with a proportion of
invariable sites I 5 0.5745, and a gamma distrib-
uted shape parameter 5 0.7125. The trees re-
covered by the different analyses of the LSU data
did not posses any conflict. Trees obtained in
analyses with Pyronema and Byssonectria as alterna-
tive outgroups (not shown) were identical to trees
obtained with Smardaea and Greletia as outgroup.
Confirming the SSU data,
O. parietina
form a highly supported group with
Pseudombrophila theioleuca
P. merdaria
P. gulde-
, in all analyses (BP 90%, ML-BP 77%, PP 100%,
groups with
P. theioleuca
high support (BP 96%, ML-BP 93%, PP 100%),
otherwise relationships within the
lineage are not supported. A clade of three lineage s,
lineages, is resolved as the sister group to the
lineage in all analyses, and highly
supported by MP bootstrap (BP 86%). The
are all highly supported (BP 92–100%, ML-BP 97–
100%,PP100% ), but relationships between the
lineages are unresolved in the strict consensus tree
of the MPTs and by Bayesian analyses, and with only
low ML-BP support (,50% ,FIG. 8).
a separate lineage, sister to the highly supported clade
of the
lineages (99–100%).
The most parsimonious interpretation of the
molecular LSU phylogeny suggests that the cleistothe-
cial form originated twice in the
lineage (FIG. 8). Nevertheless, constraint MP and ML
analyses forcing
O. parietina
L. orbiculoides
a monophyletic group could not be rejected using the
Kishino-Hasegawa test (
, 0.05). Forced monophyly
of the cleistothecial taxa did not yield trees that were
significantly less likely or longer than the uncon-
strained trees (MLT: 2lnL 5 3806.9008, difference in
2LnL 5 9.0486; MPT: 505 steps, 4 steps longer than
the unconstrained MPTs).
Evolutionary relationships.—Molecular phylogenetic
analyses confirm the evolutionary origins of O.
parietina and L. orbiculoides within Pyronemataceae
(Pezizales) as suggested by Malloch (in Dissing
and Schumacher 1994). Other hypotheses, such as
close affinities with cleistothecial or highly re-
duced fungi now placed in the Thelebolales,
IG. 7. Phylogenetic placement of
Lasiobolidium orbiculoides
within the ascomycetes inferred from SSU rDNA
sequences. The tree with the highest likelihood (2lnL 5 11 857.5723) obtained from maximum likelihood analyses. Branch
length corresponds to genetic distance (expected nucleotide substitutions per site). Numbers above branches are posterior
probabilities (PP $ 95%), obtained from the 50% majority rule consensus tree of the 18 000 trees sampled from a Bayesian
MCMC analysis. Numbers below branches that are before the backslash are ML ‘‘fast’’ bootstrap proportions (ML-BP . 70%)
and numbers after the backslash are MP bootstrap proportions (BP . 70%). Selected classifications are indicated on the tree
for discussion. The lineages I, II and III represents the pezizalean families: Pyronemataceae (including Ascodesmidaceae and
Glaziellaceae), Sarcoscyphaceae and Sarcosomataceae (I); Discinaceae, Helvellaceae, Morchellaceae, Rhizinaceae and
Tuberaceae (II); and Ascobolaceae and Pezizaceae (III).
FIG. 8. Phylogenetic relationships of
Lasiobolidium orbiculoides
among members of the Pyronemataceae (taxa
selection are based on a large-scale molecular study of the Pyronemataceae (in prep.) inferred from LSU rDNA sequences.
The tree with the highest likelihood (2lnL 5 3797.8522) obtained from maximum likelihood analyses. Branch length
corresponds to genetic distance (expected nucleotide substitutions per site). Numbers above branches are posterior
probabilities (PP $ 95%), obtained from the 50% majority rule consensus tree of the 19 000 trees sampled from a Bayesian
MCMC analysis. Numbers below branches that are before the backslash are ML bootstrap proportions (ML-BP . 70%) and
numbers after the backslash are MP bootstrap proportions (BP . 70%).
Eurotiales or Onygenales are rejected (F
. 7).
The position within the Pezizales is supported by
morphology; the genus Orbicula and L. orbiculoides
possess some clearly pezizalean characteristics
such as uniseriate, narrowly clavate to cylindrical
asci and unicellular, large, hyaline, thin-walled
ascospores (Hughes 1951, Udagawa and Furuya
1972, Malloch and Benny 1973). For the first time,
a close relationship between species of Pseudom-
brophila and O. parietina is suggested. Furthermore,
a close relationship between O. parietina and L.
orbiculoides, as indicated by Malloch and Benny
(1973), is shown.
Evolution of cleistothecial forms.—The cleistothecial
form, with loss of active spore discharge, has
evolved at least once in the Pseudombrophila lineage
. 8). Species of Pseudombrophila produce disc-
to cup-shaped apothecia with an exposed hyme-
nium at maturity (F
discharging, operculate asci. Epigeous, open
apothecia of various shapes with forcible discharge
are the most common ascoma form in the
Pezizales and presumably the ancestral state. Cain
(1956a, b, 1961) and Malloch (1979, 1981) were
among the first to suggest that certain cleistothe-
cial ascomycetes were derived from apothecial and
perithecial forms, a concept now widely accepted.
Cain (1956a) believed that the cleistothecial fungi
represented a large number of unrelated and
highly evolved lineages, arguing that these fungi
were adapted to passive spore dispersal in very
specific ecological niches. Malloch (1979, 1981)
placed several cleistothecial taxa in the Pezizales,
of which only Cleistothelebolus, Warcupia and Orbi-
cula are still retained in the order. Recent
molecular phylogenetic analyses, especially within
perithecial lineages, have strengthened the evi-
dence that the cleistothecial ascoma with concom-
itant loss of active spore discharge has arisen
independently from ostiolate forms multiple times
(e.g., Berbee and Taylor 1992 , Rehner and
Samuels 1995, Suh and Blackwell 1999).
An analogous situation within the Pezizales is the
repeated evolution of hypogeous or semi-hypogeous,
closed ascomata, with loss of active spore discharge.
Truffle and truffle-like forms evolved independently
at least 10 times in six different families (e.g. Trappe
1979, O’Donnell et al 1997, Landvik et al 1997,
Percudani et al 1999, Hansen et al 2001 and
unpublished). Molecular data supports the view
(e.g. Trappe 1979) that the truffles are all derived
from epigeous apothecial ancestors, through selec-
tion forces related to animal mycophagy, reduction in
water loss (Thiers 1984, Bruns et al 1989), or selection
in mycorrhizal fungi for deposition into the soil
spore-bank (Miller et al 1994).
The grouping of
O. parietina
L. orbiculoides
taxa that produce completely closed ascomata, with
, is not as surprising as it first might
seem. All spec ies of
produce sub-
globular ascomatal primordia and in
P. hepatica
P. cervaria
P. theioleuca
these are closed
at early stages. The excipular roof over the hymenium
opens in the mid-mesohymenial phase when the asci
are ripening (Brummelen 1995). In most species of
the evi-
dence of the tearing of the excipular roof is seen in
mature ascomata, by a ragged rim of the excipular
margin (F
IGS. 9, 10). We hypothesize that in the
FIGS. 9–10. Apothecia of
Pseudombrophila ripensis
(E.C. Hansen) Brumm. developing from large sclerotia, on old dung of
cow (JHP-95.104, C). 9. Apothecia at first sub-globular and closed, here deeply cup-shaped to urnulate; the excipular roof over
the hymenium has opened and left the margins raised and ragged 31. 10. Apothecia fully expanded, flattened to curved-
lobate, showing dentate to irregular laciniate prominent margin from the disruption of the excipular roof, and receptacles
covered with woolly hairs 31. Photos: J.H. Petersen.
lineage, ascomata forms that never
open (F
IG. 1) are derived from ascomata that open
late in development (F
IGS. 9, 10). Once the opening
of the ascomata is lost, relaxation of selection for
forcible spore discharge may permit loss of some or
all of the accompanying morphological traits, such as
the loss of the operculu m and/or loss of a distinct
hymenial layer (Malloch 1981). In a transition from
open apothecia to cl eistothecial forms,
be considered a morphological intermediate and
a more derived form. In
O. parietina
arise in a hymeni um at the base of the ascoma
accompanied by parap hyses, whereas in
L. orbiculoides
asci are not arranged in a parallel layer but rather
arise in small clusters at several points within the
ascoma and paraphyses are lacking (Malloch and
Benny 1973).
Morphology and ecology.—The excipular hairs found
in Orbicula (F
. 4, 5) are of the same type as
found in some species of Pseudombrophila, most
notably in species previously placed in Fimaria, e.g.
P. theioleuca, P. hepatica and P. dentata (Brummelen
1962, Jeng and Krug 1977 with P. dentata as Fimaria
trochospora Jeng & Krug, Pfister 1984). All hairs in
Pseudombrophila are pale brown, hyphoid and
originate from the outermost layer of the excipu-
lum from base to margin (Brummelen 1995).
Ascomatal hairs of L. orbiculoides also originate
from the outermost excipular cells, are flexuous to
wavy, remotely septate, thick-walled, very long (2
to 3 mm; Malloch and Benny 1973), and scattered
all over the ascoma.
species the outer excipulum is
differentiated from the underlying medullary excipu-
lum (Brummelen 1995) and is comparable to the
excipulum of
O. parietina
L. orbiculoides
composed of thick-walled cells, subglobose to irregu-
larly lobe d in outline in surface view (consisting of
closely compacted oblong or isodiametric cells)
IG. 6).
Orbicula parietina
has a more differentiated
pezizalean excipulum than
L. orbiculoides
, which has
a simple one-celled excipulum (Malloch and Benny
1973). In
O. parietina
the outermost cells of the
excipulum are thick-walled, small and brown (F
IG. 6),
compared to the inner cells, which are thin-walled,
large and hyaline (Hughes 1951). In all species of
and in
O. parietina
the pigment is
intercellular and amorphous (Brummelen 1995, and
IG. 6).
The similarities in ecological requirements and
substrate also support the close relationships between
species of
O. parietina
. All are likely saprobes, fruiting on dung
or well-rotted material, and species of
O. parietina
fruit in cold conditions. The
optimal temperature for developing fruit-bodies of
is between 10 and 15 C, and thus
they are found more frequently in spring and in mild
winters (Brummelen 1995). Arx (1981) listed
as psychrophilous and Campbe ll et al (1991)
fruiting in early spring on
-inoculated oat kernels left on sand over
winter, and considered it to be psychrophilic. Species
are strictly coprophilous (FIGS.9
and 10); occur on soil or vegetable debris contami-
nated with dung, urine or urea; on decaying stems
and leaves of plants; or on rotting materials (Brum-
melen 1995).
Orbicula parietina
is most commonly
reported on a variety of rotting substrates, including
damp paper, cardboard, straw, willow baskets and
leaves, or directly on dung (Hughes 1951, Udagawa
and Furuya 1972, F
IGS. 1–3). Hughes (1951) found
the best medium for fruiting of
to be tap-
water agar with ground weathered rabbit pellets.
Lasiobolidium orbiculoides
occurs on dung but also has
been reported from soil (in Yaguchi et al 1996).
Conclusions and taxonomic directions.—Based on
LSU rDNA sequence data, morphology and
ecology we consider Orbicula and Pseudombrophila
sensu Brummelen (1995) to be congeneric. The
genus Pseudombrophila sensu Brummelen (1995)
includes the formerly recognized genera Fimaria
and Nannfeldtiella. Further sampling of species of
Pseudombrophila for molecular phylogenetic study
may reveal that these genera represent separate
lineages that could deserve recognition. Orbicula
groups wi th P. theioleuca, a specie s placed in
Fimaria by Brummelen (1962). The type species
of Fimaria, P. hepatica, was not sampled. Ascomata
that open late in development and similarities in
excipular hair morphology supports the place-
ment of O. parietina in Fimaria.InthePseudom-
brophila lineage Orbicula (Cooke 1871) is the
earliest generic name and thus has priority. If this
lineage is recognized as a single genus, we will
propose to conserve Pseudombrophila against Orbic-
ula,becausePseud ombrophila includes a larger
number of species (28 total, Brummelen 1995).
Lasiobolidium orbiculoides also may be considered
congeneric with Pseudombrophila. However, based
on LSU rDNA sequences L. orbiculoides is not
unambiguously nested within Ps eudombrophila
. 8) and therefore no combination will be
made. To further understand phylogenetic rela-
tionships in the Pseudombrophila lineage, and to
determine the number of times the cleistothecial
form has arisen, a larger sampling of taxa within
the lineage is needed. Detailed developmental
studies of Pseudombrophila species and L. orbicu-
loides have been done (Brummelen 1995, Janex-
Favre and Locquin-Linard 1979), but comparable
studies with O. parietina are lacking. Studies of
ascomatal development in O. parietina would
provide data that could further clarify the evolu-
tion of closed forms in the apothecia-forming
operculate lineage.
We wish to thank the curator at C for arranging loan of
material, Jens H. Petersen for use of his photographs (F
1–3, 9 and 10), Gustavo Romero for assembling the
photographic plates, and Thomas Læssøe and Katherine
F. LoBuglio for comments on the manuscript. This research
was financially supported by an NSF grant to KH and DHP
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Rosa roxburghii Tratt., a deciduous shrub of the family Rosaceae, is usually used as food and medicinal materials and also cultivated as an ornamental. Plant endophytic fungi are a large class of microbial resources not fully researched, with great potential applications. Two strains of Ascodesmis and Talaromyces were isolated during a survey of biodiversity on endophytic fungi of R. roxburghii in China. Multigene phylogenetic analyses showed that each of the two fungi formed a distinct lineage and separated from known congeneric species and they are proposed as two novel taxa. Ascodesmis rosicola sp. nov. usually has one or two conspicuous simple or branched ridges extending to the majority of the ascospore surface and remarkably small asci, distinguishing it from the previously-described species in the genus Ascodesmis . Talaromyces rosarhiza sp. nov., of the section Talaromyces, is closely related to T. francoae . It differs from the latter by having both monoverticillate and biverticillate conidiophores, while those of T. francoae are biverticillate. Both novel endophytes are illustrated and described.
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Recent collections of unknown Trichophaea-like discomycetes made in Europe enabled the authors to explore the taxonomy of Trichophaea and allies, based on morphological, ecological and molecular data. Our 3-gene phylogeny confirms the paraphyly of the genus Trichophaea and designs a new systematics for this group of cup-fungi. Three new genera are published to accommodate several species previously assigned to Trichophaea or morphologically close genera: Perilachnea gen. nov. with Lachnea hemisphaerioides as type-species, Aurantiolachnea gen. nov. with Lachnea solsequia as type-species, and Parawilcoxina gen. nov. with P. inexpectata sp. nov. as type-species. some species of the genus Paratrichophaea belong to the cleistothecial genus Lasiobolidium, and furthermore two new species, L. trachysporum and L. coprophilum, are described. Paratrichophaea macrocystis is also combined in Lasiobolidium. Finally, three new species of Chaetothiersia, C. laricina, C. cupressicola and C. eguttulata are described. a new species of Perilachnea, P. ochraceoflava, is outlined from Italy, and a new species of Trichophaeopsis, T. asturiensis, is described from Spain. A total of 15 species are described and illustrated herein. Keys are provided.
The class Pezizomycetes is monophyletic within the subdivision Pezizomycotina. The main distinguishing character of this class is operculate asci, although in some taxa this character has been lost. The circumscription of the families and generic level delimitation in Pezizomycetes is still controversial, although several molecular phylogenetic studies have been published on this group. This paper reviews 21 families of Pezizomycetes including five new families, which are introduced here, viz. Kallistoskyphaceae, Pseudombrophilaceae, Pulvinulaceae, Strobiloscyphaceae and Tarzettaceae. Moreover, this study provides a modified backbone tree based on phylogenetic analysis of five combined loci. Descriptions and illustrations of representative taxa for the families are provided from collections made in China, Thailand and the UK, herbarium material from international herbaria (FH, FLAS, H, HKAS and MA) and the literature. Pezizales separates into six major clades. Clade 1 of Pezizales includes the families Ascobolaceae and Pezizaceae. Clade 2 is the new family Kallistoskyphaceae. Clade 3 comprises the families Caloscyphaceae, Karstenellaceae and Rhizinaceae. Clade 4 represents the families Discinaceae, Helvellaceae, Morchellaceae, Tuberaceae and Underwoodia columnaris lineage. Clade 5 includes Chorioactidaceae, Sarcoscyphaceae and Sarcosomataceae and Clade 6 comprises Ascodesmidaceae, Glaziellaceae, Otideaceae, Pseudombrophilaceae, Pulvinulaceae, Pyronemataceae, Strobiloscyphaceae and Tarzettaceae. New sequence data belonging to ITS, LSU, SSU, TEF, RPB2 gene regions from 40 pezizalian species are provided here. The paper provides a working document for apothecial Pezizomycetes which can be modified as new data comes to light. It is hoped that by illustrating taxa we provide stimulation and interest in the operculate discomycetes, so that further research is carried out on this remarkable, but poorly studied group of fungi.
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In a mycological research performed in the Sjeverni Velebit National Park, Croatia, a new species of Coprotus was discovered, described here as C. epithecioides. Along with the microscopic examination, phylogenetic analysis of the type material, based on ITS and LSU sequences, was performed in order to evaluate its relationship with the type species, C. sexdecimsporus. The type species was sequenced in this study for the first time, providing ITS and LSU sequences from two separate collections which displayed differences in macroscopic characters and content of paraphyses. An extended description of C. sexdecimsporus based on Croatian material is also provided. A worldwide identification key to the species assigned to the genus Coprotus is presented, along with a species overview, containing a data matrix. The phylogenetic position of Coprotus in the Boubovia-Coprotus clade within Pyronemataceae s.l. is discussed. Coprotus sexdecimsporus is also reported here as new to the Croatian mycobiota.
The Pezizomycetes (order Pezizales) is an early diverging lineage within the Pezizomycotina. A shared derived character, the operculate ascus, supports the Pezizales as monophyletic, although functional opercula have been lost in certain taxa. Phylogenetic relationships within Pezizales were studied using parsimony and Bayesian analyses of partial SSU and LSU rDNA sequences from 100 taxa representing 82 genera and 13 of the 15 families currently recognized. Three primary lineages are identified that more or less correspond to the A, B and C lineages resolved in previous analyses using SSU rDNA: (A) Ascobolaceae and Pezizaceae; (B) Discinaceae-Morchellaceae and Helvellaceae-Tuberaceae; (C) Ascodesmidaceae, Glaziellaceae, Pyronemataceae, Sarcoscyphaceae and Sarcosomataceae. In contrast the monotypic Rhizinaceae and Caloscyphaceae are resolved as two independent lineages. Bayesian analyses support a relationship among Rhizina and two species of Psilopezia (Pyronemataceae). Only lineage C is highly supported. The B and C lineages form a strongly supported monophyletic group. None of these lineages corresponds to earlier proposed suborders. The A and B lineages are supported by certain morphological features (e.g. ascus bluing reaction in iodine, cytology of spores and paraphyses, septal pore structures and excipulum structure); these characters have been subject to homoplasy. Lineage C is the largest and most heterogeneous, and no unifying morphological features support its recognition. The Pyronemataceae, in which almost half of the species in the order are found, is not monophyletic because the Ascodesmidaceae and Glaziellaceae are nested within it. The relationships among all families in the C lineage remain uncertain. The origin of various forms of ascomata, including hypogeous forms (truffles and truffle-like), epigeous cleistothecia, simple reduced apothecia and highly elaborate, stipitate forms (helvelloid and morchelloid), are discussed.
The classes Orbiliomycetes and Pezizomycetes are early diverging lineages in the Pezizomycotina. The Orbiliomycetes, with a single order and single family, produce small ascomata, small unitunicate asci that open without an operculum, and minute ascospores. They are saprobic or predaceous on invertebrates. The order exhibits a range of conidial states, all of which develop holoblastically. Some of the anamorphs are found routinely in aquatic habitats. The Pezizomycetes are organized under a single order with 13 families. Families are distinguished based on characters of the asci, ascospores, and general morphology of the ascomata. Many members are mycorrhizal. Saprobes occur on plant material and dung. There are a few plant parasitic species. Of note in the class is the multiple origin of hypogeous taxa, nearly all of which occur within clades that are known to be mycorrhizal. Mitosporic states developing holoblastically are known in most families.
Ample evidence from both morphological and molecular characters has accumulated to establish that the cleistothecial ascoma has been derived independently on different occasions from perithecial and apothecial ascomycetes. In order to clarify the phylogenetic position of additional species of cleistothecial ascomycetes, particularly those placed in the Cephalothecaceae and Pseudeurotiaceae of the Eurotiales, partial sequences of the small and large subunit ribosomal DNAs of fourteen taxa were compared with those of other ascomycetes. Phylogenetic trees from both sequence sets showed that some species in Pseudeurotiaceae are closely related to the taxa of four different orders of the perithecial ascomycetes (Hypocreales, Sordariales, Ophiostomatales, and Xylariales). Others (species of Pleuroascus, Connersia, Leuconeurospora, and Pseudeurotium) were not closely related to perithecial ascomycetes or Eurotiales, but to discomycetes and loculoascomycetes. Cephalotheca sulfurea, the type of the Cephalothecaceae, formed a monophyletic group with pseudeurotiaceous species of Cryptendoxyla and Albertiniella. The phylogeny provided in this study suggests strongly that all of these fungi are excluded from the Eurotiales where they have been placed and that they do not form a natural group.
Locations of epigeous basidiocarps of five common ectomycorrhizal fungi with substantial spore deposits beneath them and of two hypogeous species were marked in the fall. Subsamples of the litter and mineral soil at 0- to 3- and 3- to 6-cm depths were subsequently taken with a soil corer from locations marked for epigeous basidiocarps, and extracted in the fall and again following snowmelt with a procedure developed to enumerate propagules of ectomycorrhizal fungi from the soil. Spores of most epigeous species were plentiful in the litter layer in the fall but were much less abundant in the spring. Spores of Suillus brevipes, Suillus tomentosus and Lactarius scrobiculatus were still present at the 0- to 3-cm depth in the spring. No spores of any of the epigeous fungi were found at the 3- to 6-cm depth in the spring. Subsamples of mineral soil were also taken in the spring at locations marked for the hypogeous fungi. Spores of both Rhizopogon subcaerulescens and Rhizopogon rubescens were present in high numbers in the lowermost soil fractions after snowmelt. In a second study sclerotia of two species of fungi were extracted from soil in a burned area and a nearby unburned forest for 2 years following fire. Sclerotia of Cenococcum geophilum and a species of Morchella were more numerous in the burn than in the unburned forest in both years. Both basidiospores and sclerotia persisted in the soil for at least 2 years. Basidiospores of hypogeous fungi appear to persist in the soil for longer periods than those of epigeous fungi due to in situ dispersion deeper into the soil profile. Although maximum longevity of spores in the soil has not been determined, observed differential persistence of spores from epigeous and hypogeous ectomycorrhizal fungi could play a role in soil mycorrhiza-forming potential and population dynamics of ectomycorrhizal fungi.
Several minute dung-inhabiting discomycetes have been classified in the family Thelebolaceae, which has traditionally been included in the order Pezizales. The non-operculate type-genus Thelebolus has recently been excluded from the Pezizales. The phylogenetic distribution of other genera associated with Thelebolaceae is still obscure. We have analysed ca. 580 bp from a variable part of the nuclear SSU rRNA gene from Ascozonus, Ceccobius, Lasiobolus and Thecotheus, and compared these with ca. 1700 bp sequences from Thelebolus, Pleospora, Pezizales, Leotiales and Leotiales-related taxa. In the resulting trees, Ascozonus and Caccobius group with Thelebolus and the inoperculate discomycetes; Lesiobolus groups with Ascodesmis, and Thecotheus with Ascobolus within Pezizales. SEM pictures of fruit-bodies and ascus apices of Ascozonus, and ascospores from Thecotheus are presented to illustrate characteristic features of these taxa.