Phylogenetics of the Pezizaceae,
with an Emphasis on Peziza
The Harvard community has made this
article openly available. Please share how
this access benefits you. Your story matters
Citation
Hansen, Karen, Thomas Laessoe, and Donald H. Pfister. 2001.
Phylogenetics of the Pezizaceae, with an emphasis on Peziza.
Mycologia 93, no. 5: 958-990.
Published Version
http://dx.doi.org/10.2307/3761760
Citable link
http://nrs.harvard.edu/urn-3:HUL.InstRepos:3153300
Terms of Use
This article was downloaded from Harvard University’s DASH
repository, and is made available under the terms and conditions
applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-ofuse#LAA
Mycological Society of America
Phylogenetics of the Pezizaceae, with an Emphasis on Peziza
Author(s): Karen Hansen, Thomas L[ae]ssoe, Donald H. Pfister
Source: Mycologia, Vol. 93, No. 5 (Sep. - Oct., 2001), pp. 958-990
Published by: Mycological Society of America
Stable URL: http://www.jstor.org/stable/3761760
Accessed: 05/06/2009 20:11
Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at
http://www.jstor.org/page/info/about/policies/terms.jsp. JSTOR's Terms and Conditions of Use provides, in part, that unless
you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you
may use content in the JSTOR archive only for your personal, non-commercial use.
Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at
http://www.jstor.org/action/showPublisher?publisherCode=mysa.
Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed
page of such transmission.
JSTOR is a not-for-profit organization founded in 1995 to build trusted digital archives for scholarship. We work with the
scholarly community to preserve their work and the materials they rely upon, and to build a common research platform that
promotes the discovery and use of these resources. For more information about JSTOR, please contact support@jstor.org.
Mycological Society of America is collaborating with JSTOR to digitize, preserve and extend access to
Mycologia.
http://www.jstor.org
Mycologia, 93(5), 2001, pp. 958-990.
? 2001 by The Mycological Society of America, Lawrence, KS 66044-8897
Phylogenetics of the Pezizaceae, with an emphasis on Peziza
Karen Hansen'
Thomas Laess0e
Departmentof Mycology,Universityof Copenhagen,
0sterFarimagsgade2 D, DK-1353 Copenhagen
K,
Denmark
tions were found to support different rDNA lineages,
e.g., a distinct amyloid ring zone at the apex is a synapomorphy for group IV, an intense and unrestricted
amyloid reaction of the apex is mostly found in
group VI, and asci that are weakly or diffusely amyloid in the entire length are present in group II. Other morphological features, such as spore surface relief, guttulation, excipulum structure and pigments,
while not free from homoplasy, do support the
groupings. Anamorphs likewise provide clues to higher-order relationships within the Pezizaceae. Several
macro- and micromorphological features, however,
appear to have evolved several times independently,
including ascomatal form and habit (epigeous, semihypogeous or hypogeous), spore discharge mechanisms, and spore shape. Parsimony-based optimization of character states on our phylogenetic trees suggested that transitions to truffle and truffle-like forms
evolved at least three times within the Pezizaceae (in
group III, V and VI). The 9 hypogeous species included are nested in lineages with epigeous pezizaceous taxa. Species with apothecia of various shapes
and with forcible spore discharge are spread among
all groups and the apothecium is suggested to be
symplesiomorphic in the Pezizaceae. The results indicate that the apothecia forming Pezizaceae have
given rise to at least 3 different forms of hypogeous
ascomata without forcible spore discharge: ptychothecia, stereothecia and exothecia.
Key Words: ascus amyloid reactions, epigeous-hypogeous evolution, molecular phylogeny, nLSU
rDNA, Pezizales, systematics
Donald H. Pfister
Harvard UniversityHerbaria,Cambridge,
Massachusetts,02138 USA
Abstract: Phylogenetic relationships among members of the Pezizaceae were studied using 90 partial
LSU rDNA sequences from 51 species of Peziza and
20 species from 8 additional epigeous genera of the
Pezizaceae, viz. Boudiera, Iodophanus, Iodowynnea,
Kimbropezia,Pachyella, Plicaria, Sarcosphaeraand Scabropezia,and 5 hypogeous genera, viz. Amylascus, Cazia, Hydnotryopsis, Ruhlandiella and Tirmania. To
test the monophyly of the Pezizaceae and the relationships to the genera Marcelleina and Pfistera (Pyronemataceae), 6 species from the families Ascobolaceae, Morchellaceae and Pyronemataceae were included. Maximum parsimony and maximum likelihood analyses of these sequences suggest that the
Pezizaceae is paraphyletic, because the non-amyloid
Marcelleina is nested within it. If Marcelleina were
transferred to the Pezizaceae, then the family would
be monophyletic. Although the Pezizaceae is traditionally characterized by amyloid asci, our results indicate that the amyloid reaction is a symplesiomorphy, which has been lost in some lineages, e.g., in
those including Marcelleina and Cazia. Nodes deep
in the tree could not be resolved, but 7 groups of
species (I-VII) are generally well supported or present in all trees. Peziza species, which constitute the
core of the family, are present in all groups except
group III, confirming the non-monophyly of the genus. The analyses suggest that the other included
genera of the Pezizaceae are all nested within Peziza,
the placement of lodophanus being unresolved. The
morphologically distinct Peziza gerardii, which forms
a clade with Marcelleina, appears to be the sister
group to the rest of the Pezizaceae. Morphological
features were studied and evaluated in the context of
the phylogeny. Distinct types of ascus amyloid reac-
INTRODUCTION
The Pezizaceae (Pezizales) displays great variation in
ascomatal forms. It includes taxa that produce epigeous, sessile or stipitate, cupulate, discoid, turbinate,
pulvinate or sparassoid ascomata, or semi-hypogeous
to hypogeous, closed, folded to solid ascomata (FIGS.
1-21). The ascomata range in size from a few mm to
more than 10 cm in diam, and are often fleshy, soft
and brittle. Anatomical and biochemical diversity is
found in characters such as spore discharge mechanism; amyloid reaction of the ascus; spore shape, color, ornamentation, and guttulation; pigmentation of
the paraphyses; and excipulum structure (TABLE I).
Accepted for publication April 3, 2001.
1 Corresponding author, Email: karenh@bot.ku.dk
958
959
OF PEZIZACEAE
HANSEN ET AL: PHYLOGENY
I
I
EwI
V,
,,.w)
4i
IiNfw.'e'^1
,
N. ^tc&I
^I, <-an
.. c
r
,,.*?,, *
?.I'O1
o
,.
w
FIGS.1-12. Ascoma forms and color variation in the Pezizaceae (group I-IV). 1. Pulvinate apothecia with protruding asci.
Iodophanus carneus (JHP-00.027, C) X 15. 2-3. Group I. Discoid apothecia on broad obconical bases. 2. Peziza gerardii (KH98-86, C) X3. 3. Marcelleina persoonii (TL-5346, C) X3. 4. Group II. Peziza subisabellina (RK83.115, herb. Roy Kristiansen)
X1.3. 5-8. Group III. 5. Pachyellapunctispora (KH-98-77, C) X 1.5. 6. Turbinate apothecia with a convex hymenium. Boudiera
dennisii (JHP-93.095, C) X6. 7. Highly pustulate outer surface. Scabropeziaflavovirens (Fuckel) Dissing & Pfister (KH-97-68,
C) X2. 8. Stereothecium. Amylascus. (5728, OSC) Xl.5. 9-10. Group IVa. 9. Cup-shaped, shortly stipitate apothecia. Peziza
arvernensis (KH-98-08, C) x0.4. 10. Peziza ammophila (KH-98-88, C) x0.8. 11-12. Group IVb. 11. Peziza exogelatinosa (KH97-75, C) x0.8. 12. Peziza subcitrina (KH-97-133, C) X 1.3.
Photos: 1, 2, 6, 7, 11 J.H. Petersen. 5, 9, 12 K. Hansen. 8 M. Castellano. 10 S.A. Elborne. 3 T. Laess0e. 4 R. Kristiansen.
Ecologically, the family covers a broad range of niches, fruiting on all types of soil, sand, clay, limestone,
burnt ground, dung and wood. Many species are
known to prefer soil with a high pH and in some
cases a low content of organic matter (Petersen 1967,
1985). The family is primarily restricted to temperate
zones and arctic-alpine areas, although a few strictly
tropical taxa are known. The majority of species are
considered to be saprotrophs, although most hypogeous and a few epigeous species are claimed to be
(Maia et al 1996 and references
ectomycorrhizal
therein), but in general their trophic statuses are not
960
MYCOLOGIA
TABLEI.
Characters used in the delimitation
of genera in the Pezizaceae
(based on published
and original observations)
Ascoma:
positionb;
+/-
forcible
spore discharge
Ascoma: type;
shape when
epigeous
Ascus: +/- amyloid;
type of
amyloid reaction
Amylascus
hy, se;
-
Ptychothecium to
stereothecium
+; weakly over the
entire length
Globose; 1 or several
Hyaline to pale
brown; + spines,
rods or cones
Boudiera
ep; +
Apothecium,
+; weakly over the
entire length
Globose; 1 or several
hy; -
Pale brown; + echinulate to reticulate
Cazi&a
bose, turbinate to
pulvinate
Stereothecium
Ellipsoid; 1
Hyaline; + minutely
Hapsidomycese
ep; +
Apothecium,
Genus
subglo-
turbi-
nate
Spore: shape;
guttulation
+; weakly over the
entire length
Globose; 0
Hydnobolites
hy; -
Stereothecium
+/-; weakly (2%
KOH pretreatment)
Globose; 0
Hydnotryopsis
hy; -
Stereothecium
+; diffusely over the
entire length
Ellipsoid; ?
Iodophanus
ep; +
Apothecium, pulvinate
+; weakly over the
entire length
Ellipsoid to broadly
ellipsoid; 0
lodowynneca
ep; +
Kimbropezite
ep; +
Apothecium, convoluted to highly
folded, arising
from a hypogeous
stipe
Apothecium, discoid
Mycoclelandia
hy, se;
Ptychothecium
Pachyella
ep; +
Pachyphloeus
hy; -
Apothecium, discoid,
flat to pulvinate,
broadly attached
to the substrate
Stereothecium
Peziza
ep,
rarely
se,
hy; +
rarely
Apothecium, discoid
to cupulate, rarely
sparassoid, or
rarely ptychothecium
Spore: color; +/ornamentation
warty-reticulate
Hyaline to pale yellow; + reticulate
and spiny
Hyaline to pale
brown; + reticulate
Hyaline to brownish
yellow; + warty to
pitted to ridged
Hyaline; + warty or
episporium
wrin-
+; intensely over the
entire length
Ellipsoid; 1 or 2,
rarely several
kled
Hyaline; + longitudinally ridged and
warty
+; intensely over the
apex
Ellipsoid; 0
Hyaline; -
+; diffusely, more
intense near the
apex
+; diffusely over the
entire length
Broadly ellipsoid to
subglobose; 1 or 2
Hyaline;
Ellipsoid; 2
Hyaline;
ridged
+; weakly over the
entire length
Globose; 0 ?
+; ring zone at the
apex, intensely unrestricted over the
apex or evenly
over the entire
length (diffusely,
weakly or intense-
Ellipsoid or fusiform; 0, 2-or
multi-guttulate,
rarely 1
Hyaline to pale
brown; + spinose
to verrucose, +/embedded in mucilage
Hyaline or pale
brown; -/ +
warty, ridged to
reticulate, spiny,
apiculate
ly)
+ warty,
961
HANSEN ET AL: PHYLOGENY OF PEZIZACEAE
TABLEI. Extended
Paraphyses: pigments;
gelatinous
matrix; epithecium
Excipulum: outer
Excipulum: medullary
Trophic status;
substratec
Anamorphs
Outermost textura globulosa
to angularis, giving rise to
surface tomentum. Inner
layer of broad hyphae,
mixed with inflated cells
Textura globulosa to angularis
Textura intricata, +/large cells
ec ?; te
Unknown
Dense textura intricata
sa ?, ec ?; te
Unknown
Paraphyses present:
pigments ?
Yellowish contents
Textura intricata, at septa,
the cells often inflated
Textura globulosa to angularis
Textura intricata
ec ?; te
Unknown
Textura intricata
sa; co
Unknown
Absent
Textura globulosa (+ isodiametric cells) with brownish, amorphous material
in the intercellular spaces
Textura globulosa (? isodiametric cells), sometimes
with interwove hyphae
Textura globulosa to angularis, sometimes with interwoven hyphae
Textura globulosa intermixed with hyphae, outermost cells arranged in
short chains
Textura globulosa (?
isodiametric cells),
or textura intricata
ec ?; te
Unknown
Textura intricata
ec ?; te
Unknown
Textura intricata
sa ?; te, co,
Oedocephalum
Absent
Hyaline or brownish
walls
Paraphyses present:
pigments ?
Usually containing carotenoid pigments
Hyaline
Hyaline
Textura intricata, with very
short hyphae, extending
towards outside
Hyaline
Textura intricata or textura
globulosa
Dark granules in the
apical cells
Textura globulosa to angularis, outer cells terminating
in flexuous hairs embedded in a gelatinous matrix
Subangular to globose isodiametric cells, on outside
scabrous to verrucose
Absent
+/-
pigmented gran-
ules; +/-
embed-
ded in granular matrix
Textura globulosa or textura
angularis, of large, thinwalled cells +/- interwoven hyphae, or smaller
globose cells elongating
to a hyphoid layer towards the outside
PY
Textura globulosa intermixed with hyphae
ec ?; te
Unknown
A layer of textura intricata, between two
layers of textura angularis to globulosa
Textura globulosa, of
large, thin-walled
cells
Textura intricata +/gelatinous matrix
sa ?; te
Unknown
ec ?; te
Unknown
sa ?; li
Oedocephalumlike
Isodiametric cells
ec ?; te
Glischroderma?
One, two or more lay-
sa, ec; te, co,
Oedocephalum,
Chromelosporium, Chromelosporium-like,
or hyaline to
colored, 0-3
septate resting
ers, of textura globu-
losa or textura angularis, of large, thin
walled cells +/- interwoven hyphae, or
of parallel hyphae
py, li
spores
MYCOLOGIA
962
TABLEI.
Continued
Ascoma:
positionb;
+/-
Genus
Plicaria
forcible
spore discharge
ep; +
Ascoma: type;
shape when
epigeous
Apothecium,
discoid
to cupulate
Ascus: +/- amyloid;
type of
amyloid reaction
+; weakly over the
entire length
Spore: shape;
guttulation
Globose; few to mul-
ti-guttulate
Rhodopezizaa
ep; +
Apothecium, cupulate
+; over the entire
length
Broadly ellipsoid; 1
small evanescent
guttule
Ruhlandiella
se; -
Exothecium
Globose; 1
Sarcosphaer&a
ep, se;
Apothecium, hollow,
+; weakly over the
entire length
+; weakly over the
+
initially hypoge-
entire length (a
ous, becoming exposed and split
into acute rays
Apothecium, cupulate
little stronger at
the apex)
Scabropezia
ep; +
Sphaerozone~
Exothecium
Terfezia
hy, se;
hy; -
Tirmania
hy; -
Stereothecium
Stereothecium
Ellipsoid; 2
Spore: color; +/ornamentation
Pale brown; -/+
warty, spiny, reticulate
Pale yellowish; + tuberculate
Pale brown; + reticulate-areolate
Hyaline; + low warty
+; over the entire
length
Globose; 1
Pale brown; + truncate or delicate
warts
+; weakly, over the
entire length
-
Globose; 0
Globose; 0
+; diffusely, +/weakly
Globose to broadly
ellipsoid; 1
Reddish brown; +
irregular warty
Hyaline to pale
brown; + spiny,
papillate, reticulate, -/+ mucilage-embedded
Hyaline; -/+ minutely roughened
a Monotypic genus.
b Position of
ascoma; ep = epigeous, hy = hypogeous, se = semihypogeous.
c Trophic status: sa = saprobic, ec = ectomycorrhizal. Substrate: te = terrestrial, co =
coprophilous, li = lignicolous, py
=pyrophilous.
well studied. A study by Warcup (1990), with single
spore isolation and incubation experiments did, however, show several species, both epigeous and hypogeous, to be ectomycorrhizal.
Eriksson (2000) lists 19 genera in the Pezizaceae,
with a circumscription based mainly on Rifai (1968),
Kimbrough (1970) and Trappe (1979). Recent molecular phylogenetic studies have led to the inclusion
of Pachyphloeus and Terfezia in the family (Norman
and Egger 1999, Percudani et al 1999) (TABLE I).
The Pezizaceae is chiefly characterized by amyloid
asci, a feature shared only with the Ascobolaceae
within the Pezizales. The spores are uninucleate, usually thin-walled, globose, ellipsoid or fusiform, hyaline or pale brown, smooth or with cyanophilic or-
naments. The excipulum consists, at least partly, of
large isodiametric cells. In addition, ultrastructural
and refined cytochemical studies have led to further
delimiting characters such as the presence of electron-opaque convex and biconvex bands in septal
pores of the ascal bases (so far recorded in Peziza,
Plicaria, lodophanus, Sarcosphaera, Pachyella and Hydnobolites) (Curry and Kimbrough 1983, Kimbrough
and Curry 1985, Kimbrough et al 1991, Kimbrough
1994).
Molecular phylogenetic studies of the Pezizales to
date have included relatively few taxa from the
groups with amyloid asci. With regard to the Pezizaceae, the work of Norman and Egger (1996, 1999)
deserves special note, because it provides broader
963
HANSEN ET AL: PHYLOGENY OF PEZIZACEAE
TABLE I.
Extended
Continued
Paraphyses:pigments;
gelatinous
matrix; epithecium
Embedded in dark
granular matrix
Orange granules (carotenoid granules)
Excipulum: outer
Textura globulosa to angularis, and intermixed
broad hyphae
Textura globulosa to angularis, of subglobose to polygonal cells
Excipulum: medullary
Textura globulosa and
intermixed broad
hyphae
Small textura globulosa angularis, occasionally intermixed
with hyphae
Textura globulosa to
angularis
Textura globulosa
Trophic status;
substratec
Anamorphs
sa ?, ec; py
Chromelosporium
sa ?; te
Unknown
ec; te
Chromelosporium-like
Unknown
Gelatinous sheath; exceeding the asci
Brownish contents
Textura globulosa to angularis, thick-walled cells
Textura epidermoidea, to
textura prismatica towards
the outside
Small brownish guttules; embedded in a
coloured, granular
matrix
Exceeding the asci
Textura globulosa, outermost cells thick-walled,
forming conical pustules
Textura intricata
sa ?, ec ?; te,
li
Glischroderma?
Textura globulosa to angularis, cells thick-walled
Textura intricata, of broad
hyphae with inflated cells
Textura globulosa to
angularis
Textura intricata, of
broad hyphae with
inflated cells
ec ?; te
Unknown
ec ?; te
Unknown
Textura intricata, of broad,
thin-walled hyphae and
many inflated cells
Textura intricata, of
broad, thin-walled
hyphae and many
inflated cells
ec ?; te
Unknown
Absent
Absent
sampling of the family than any previous study. Nevertheless, further sampling of Peziza species and inclusion of additional genera, as described here, are
necessary to critically assess generic limits within the
family and to work toward a phylogenetic classification. The main objectives of this research were: i) to
resolve the major lineages within the Pezizaceae, especially within Peziza, using LSU rDNA sequences,
and to compare these with previous morpho-taxonomic classifications, ii) to explore the circumscription of Peziza itself, and iii) to evaluate and refine
morphological characters previously used in the delimitation of genera within the Pezizaceae. The results should give direction to further morphological
and molecular studies.
BACKGROUND INFORMATION
The family: delimitation and important morphological
characters.-For nomenclatural purposes the Peziza-
ec ?; te
ceae can be traced to Dumortier (1829) (Hawksworth and David 1989), as a family of the order Fungarieae (fungi with a distinct external hymenium), to
include genera with the spores born on the upper
surface of a cup-shaped receptacle. Dumortier's circumscription was essentially the same as used by Fries
(1822, as "Pezizeae"), but Fries did not use a consistent method of ranking in his system, and therefore
Fries is not accepted as author of the Pezizaceae contrary to the use by, e.g., Eckblad (1968), Kimbrough
(1970) and Korf (1972). Dumortier (1829) listed Peziza and Ascobolus, along with other genera of the
discomycetes, Stictis, Bulgaria, Tympanis, Cenangium,
Patellaria and Helotium, and two genera of basidiomycetes, Solenia and Ditiola. This system was based
solely on macroscopic features and has long been regarded as artificial.
Boudier (1885) was the first to delimit a group,
"the Aleuries," which more or less circumscribes the
964
MYCOLOGIA
core of the Pezizaceae as we recognize it today. The
group was placed in the "division Opercules," distinguished by asci that open with an operculum. Besides
the presence or absence of an operculum, Boudier
introduced and used further important microscopic
characters, such as the amyloid reaction of asci, spore
shape and number of oil guttules in the spores in his
classification of the "fleshy discomycetes." The
"Aleuries" was distinguished by amyloid asci and in
general having cup-shaped, sessile ascomata without
hairs on the receptacle. The genera Lepidotia Boud.,
Aleuria (Fr.) Gill. and Galactinia (Cooke) Boud. (=
Peziza), Sarcosphaera, Sphaerosoma Klotzsch, and Pli-
caria were included. Later Boudier (1907) treated
the group at tribus level ("Aleuriees") and added his
new genus Pachyella, but Sphaerosoma was excluded.
Pachyella was segregated from Peziza and Psilopezia
Berk. on the basis of the pulvinate to convex apothecia and lack of or only weakly amyloid asci. The absence of an amyloid reaction for taxa in the tribe was
contrary to the definition of "Aleuriees," but Boudier also knowingly included species in Plicaria that
had non-amyloid asci.
Boudier's (1907) circumscription of the Aleuriees
was generally followed by Le Gal (1947, as tribus
Aleurieae), Rifai (1968), Eckblad (1968) and Korf
(1972, 1973b) (as Pezizaceae), and Dennis (1981, as
tribus Pezizeae). Additional characters, such as spore
ornamentation of cyanophilic material (Le Gal
1947), uninucleate spores (Berthet 1964a), absence
of carotenoid pigments (Arpin 1969) and the anatomy of the ascomata, consisting of mostly large isodiametric cells, were discovered and used to refine
the limits of the family. In culture, many species of
the Pezizaceae were found to produce Oedocephalum
or Chromelosporiumconidial states (Rifai 1968).
Trappe (1979) emended the family to include hypogeous taxa with chambered or marbled glebae and
cylindric to saccate asci which lacked functional opercula. He recognized the artificial nature of the Tuberales, abandoned the order and assigned most taxa
to families within the Pezizales. Hypogeous species of
Peziza, and the genera Mycoclelandia, Hydnotryopsis,
Amylascus and Tirmania were accepted in the Pezizaceae. Trappe considered the hypogeous taxa to be
derived from various epigeous taxa within the family.
This hypothesis was based primarily on similarities in
the anatomy of the excipulum and on the presence
of amyloid asci. Currently 10 genera, almost half of
the genera accepted in the Pezizaceae, have semi-hypogeous to hypogeous ascomata (Eriksson 2000, TABLEI), while six hypogeous species are placed in Peziza (e.g., FIGS. 17-18) (Trappe 1979).
The amyloid reaction. Amyloid asci were described
by Nylander (1868), Karsten (1869) and Boudier
(1879) in a number of species of Peziza and Ascobolus
Pers. Korf (1972) attached some importance to ascus
amyloid reactions. He recognized the Pezizaceae for
taxa with ascus amyloid reactions: i) in an apical ring,
ii) over the apex, or iii) rarely in their whole length.
Taxa in the Ascobolaceae tribus lodophaneae were
described as having diffusely amyloid asci (tribus Ascoboleae showed non-amyloid or diffusely amyloid
asci). Nevertheless, Korf included Pachyella, characterized by diffusely amyloid asci, in the Pezizaceae.
Except for the genera Pachyella, Iodophanus and Boudiera, the location and extent of the amyloid reaction
have not been used as characters to delimit taxa within the Pezizaceae. Within the Ascobolaceae, differences in ascus reactions were noticed for some sections in the genus Ascobolus: intensely amyloid in
sect. Dasyobolus (Sacc.) Brumm. (in all but one species), faintly amyloid or none at all in several mainly
terrestrial and lignicolous species of sect. Ascobolus,
and non-amyloid in sect. PseudoascodesmisBrumm.
(van Brummelen 1967). All species in the remaining
four sections of Ascobolus showed a "general" amyloid reaction of the ascus wall. Van Brummelen
(1967) found the intensity of the reaction to be constant for each species although significant differences
existed between related species. A diffuse amyloid reaction was described by Pfister (1973) in Pachyella as
"not restricted to the apex of the ascus, nor is it in
the form of aJ+ ring at the apex of the ascus," and
that it "is present either as an external layer on the
ascus wall (which may separate from the ascus wall
proper), or occurs in the gel which surrounds the
asci and paraphyses and is not restricted to the wall."
Morphological and cytochemical studies of amyloid asci were performed on five species of the Pezizaceae and Ascobolaceae, using light microscopy and
transmission electron microscopy (TEM) (Samuelson
1978). The amyloid site in Peziza succosa, Saccobolus
depauperatus (Berk. and Broome) E.C. Hansen and
Thecotheuspelletieri (Crouan) Boud. appeared to be
an exogenous mucilaginous coat, whereas in Iodophanus granulipolaris Kimbrough it appeared to be
the ascal wall proper. However, Samuelson stated that
the amyloid reaction may be the result of more than
one site of activity. Peziza succosa was strongly but unrestrictedly amyloid over the apex ("10-14 Ipmdown
the sides of the ascal wall"). Peziza species with a distinct amyloid ring zone at the apex were not studied.
According to van Brummelen (1978) the site of the
amyloid reaction is strictly confined to an extra-ascal
layer, the periascus (a mucilagenous substance),
which varies in thickness. In Boudiera and Iodophanus
it is more or less uniform, but in Peziza ammophila
and P succosella it develops into a ring-shaped thickening at the apex. In P ammophila a thick ring with
HANSEN ET AL: PHYLOGENY OF PEZIZACEAE
an indented central portion forms, whereas in P. succosella the periascus is considerably thickened but
uniformly so over the ascus apex (van Brummelen
1978: figs. 7, 8). Such ultrastructural studies help to
explain the variation in the amyloid reaction of asci,
which is observed in the Pezizaceae.
The genera: delimitation and relationships.-Epigeous
genera. Peziza Fr. (1822) is the largest genus within
the Pezizales, with approximately 100 currently accepted species (Hohmeyer 1986, Korf 1972). It is a
complex and heterogeneous assemblage, that constitutes the core of the Pezizaceae. Although many keys
(Dissing 2000, Hohmeyer 1986, Haffner 1995, Le Gal
1941, Maas Geesteranus 1967, Moser 1963, Romagnesi 1978) and descriptions (e.g., Donadini 1978,
1979a, b, 1980a, Moravec 1985) of European species
exist, Peziza has never been monographed and the
infrageneric relationships are poorly understood. A
number of subgenera and sections have been proposed, but only few attempts to provide a coherent
system have been made (Donadini 1977, 1978). Furthermore, controversy has surrounded the generic
delimitation of Peziza. Recent molecular studies suggest that the genus is not monophyletic, but rather
is composed of at least two distinct clades (Norman
and Egger 1996, 1999).
The nomenclature of Peziza is complicated on several counts. It is a pre-starting point name originating
from Dillenius (1719). It has been applied to a number of cup-shaped fungi including both basidiomycetes and ascomycetes. Linnaeus (1753) adopted the
name Peziza in Species Plantarum, the starting point
work, for a group of fungi now referred to Cyathus
Willd. (Basidiomycota) (Dennis 1983). Post-linnaean
authors, particularly Bulliard (1791) and Persoon
(1801), included primarily discomycetes, both operculate and inoperculate, in Peziza, thus making important contributions to the modern concept of the
genus. Since Fries (1822) adopted the concept of
these authors (not Linnaeus's concept) and placed
what became the lectotype of Peziza L. (= Peziza lentiferaL. = Cyathus olla Pers.) in Nidularia Fr. (Dennis
1983), Fries can be considered as having described
Peziza as a new genus with a sanctioned status (Yao
et al 1995). Peziza in the sense of Fries has been lectotypified by P vesiculosa Bull.: Fr. (Wakefield 1939).
Boudier (1885, 1907) used two genera for taxa
now placed in Peziza. Aleuria emend. Boud. non
Fuckel for ellipsoid-spored species without oil guttules and Galactinia for ellipsoid-spored species with
two guttules. Le Gal (1941, 1947) followed Boudier,
but later (1953a) united the genera Aleuria and Galactinia in Galactinia with two sections: "Eguttulisporae" nom illeg. and "Guttulisporae" nom illeg.
965
(corresponding to Aleuria and Galactinia sensu
Boud. respectively). Le Gal abandoned Peziza as a
"nomen confusum." The most recent classifications
(Dennis 1981, Dissing and Pfister 1981, Dissing 2000,
Eckblad 1968, Rifai 1968) follow Le Gal's (1953a) circumscription of the genus, but under the name Peziza. As such, Peziza is defined as having epigeous,
discoid to cupulate apothecia, a few mm to ca 15 cm
in diam, sessile or with a short stipe (FIGS. 2, 4, 9-12,
15, 20-21), amyloid asci, ellipsoid or rarely fusiform
spores and an excipulum most often stratified in two
or more layers of globose thin-walled cells intermixed
with hyphae or a hyphal layer; lactiferous hyphae may
be present. We have adopted this circumscription as
the starting point for taxonomic comparisons in this
study. Nevertheless, a few species are placed in the
genus which produce more unusual ascomatal forms,
i.e., sparassoid (Boudier 1899, Korf 1973a) or hypogeous, closed, hollow, often folded ascomata (FIGS.
17-18) (Burdsall 1968, Trappe 1979). The hypogeous forms have persistent hymenia and amyloid asci,
but lack forcible spore discharge (TABLE I).
SarcosphaeraAuersw. (1869) was segregated from
Peziza on the basis of its macro-morphology and initial hypogeous development. It produces large, enclosed, hollow apothecia, initially underground,
which as they mature, usually become exposed and
split into irregular acute rays (FIG.13). The asci mature before the apothecia open, but are in organized
hymenia, and the spores are forcibly discharged.
Whether to recognize Sarcosphaeraas distinct from
Peziza has been questionable. Eckblad (1968) did not
recognize Sarcosphaeraas a distinct genus, and treated the species in Peziza. However, most contemporary taxonomists accept Sarcosphaera (Pouzar 1972,
Korf 1973b, Pfister 1979a, Dissing 2000).
Plicaria Fuckel (1870) was introduced to accommodate species mostly treated by modern authors as
Peziza and Plicaria, with both ellipsoid and globose
spores, united by a few characters, e.g., thick-walled,
brownish, ornamented spores. Peziza vesiculosa and
others were placed in Pustularia Fuckel, while Peziza
in Fuckel's classification included small stipitate inoperculate discomycetes. Fuckel erected Aleuria
Fuckel for the non-amyloid, P aurantia Pers. (the
modern accepted concept of Aleuria). Boudier
(1885) restricted Plicaria to the globose-spored species and was followed by most contemporary and later workers. Rehm (1894) used Plicaria, for only ellipsoid-spored species with amyloid asci, and erected Plicariella (Sacc.) Rehm for the globose-spored species;
he rejected the name Peziza. Some authors have
maintained Plicaria emend. Boud. as a separate genus (Eckblad 1968, Rifai 1968, Dennis 1981, Dissing
and Pfister 1981, Hirsch 1985, Norman and Egger
966
MYCOLOGIA
/6
0
I
Aem
.
-VI
. ,
. -Oip.rt--q.. .
.-""%
11.
.
L?,,. -, /,
.I
.
r
-
.
.,-.
i
-
"-
.
#1
K^esfffRf7
C
?. tt
',
.
::,',.::.;'
171
~ ! r ^; ^ y
W
:.-,i~~"-
4
18
;". 711
, J ,.,;
". .;r;.
1Iw-;-..
.e-I %-,
'Mlil.,-.
- mg
19
'
-
r'
,
W.M,
coronFIGS.13-21. Ascomaforms and color variationin the Pezizaceae(groupV-VII). 13-14. GroupV. 13. Sarcosphaera
setchellii(Trappe19134, OSC) X1.2. 15-19. GroupVI. 15. Pezizasaniosa(JHParia X0.4. 14. Stereothecium.Hydnotryopsis
93.193, C) XI. 16. On burnt ground. Plicariatrachycarpa
(KH-97-89,C) X1.3. 17. Ptychothecium.Pezizaellipsospora
(Trappe
12996, OSC) X1.3. 18. Ptychothecium.Pezizawhitei(M1445,OSC) Xl. 19. Exothecium.Ruhlandiellaberolinensis(12355,
OSC) XI. 20. GroupVIIa.Pezizapolaripapulata(KS-95-10,C) X0.8. 21. Pezizaretrocurvata(KS-94-182,C holotype, K, FH
isotypes) X0.6.
Photos: 15, 16J.H. Petersen.21 K. Hansen. 14, 17, 18 M. Castellano.20 T. Lass0e. 13 D.S. Hibbett. 19 D. Luoma.
1996, Dissing 2000), while others have regarded it as
a part of Peziza in the sense adopted here (e.g., following Korf 1960, 1972, Le Gal 1953b, 1962). Whether the shape of the spores is a reliable character at
the generic level has been a persistent controversy.
Other characters have been proposed to support the
recognition of Plicaria. These are pale brownish
spores, paraphyses embedded in a granular matrix,
dark-colored apothecial pigments (brown pigment
on the walls of the asci), Chromelosporiumanamorphs
and a carbonicolous habitat (FIG. 16). Egger (1987)
found that Plicaria trachycarpa,P. endocarpoidesand
some species of Peziza (P. atrovinosa, P. ostracoderma,
Peziza sp. #2), exhibited a positive tyrosinase reaction,
while other species of Peziza had a negative reaction.
Egger noted that if correlating characters were
found, the concept of Plicaria might have to be modified to include ellipsoid-spored species as in the orig-
inal concept. Moravec and Spooner (1988) reached
the same conclusion based on a morphological study
of brown-spored species of Peziza (P. atrovinosa, P.
vacinii, P. retiderma Cooke, P. rifaii J. Moravec &
Spooner and P ostracoderma)and stated that if Plicaria is to be recognized "the inclusion in it of species with non-globose spores appears unavoidable."
Phylogenetic analyses by Norman and Egger (1996)
including most of these taxa, indicated that the globose-spored Plicaria is monophyletic, but nested
within Peziza. They accepted the genus ad interim
even though it implied a paraphyletic Peziza. In this
study we are using the name Plicaria as emended by
Boudier.
Boudiera Cooke (1877) has been placed in the Ascobolaceae (Boudier 1907, Korf 1972, 1973b) due to
the turbinate to pulvinate ascomata (FIG.6), protruding asci and pale brownish spores, but its relationship
OF PEZIZACEAE
HANSENETAL:PHYLOGENY
has been much disputed. Eckblad (1968) stated that
Boudiera did not seem to be closely related to any
other genus with protruding asci, but he, nevertheless, referred it to the Ascobolaceae. Boudiera was revised (Dissing and Schumacher 1979) and placed in
the Pezizaceae by Dissing and Korf (1980) and
Hirsch (1980) along with other genera with globose
spores and amyloid asci. Dissing and Korf emphasized characters of the anatomy and morphology of
the ascomata and suggested that Boudiera is in a line
with Plicaria on the one hand, and Ruhlandiella and
Sphaerozoneon the other. Hirsch (1980) considered
Boudiera closely related to Plicaria, as did Rifai
(1968). Hirsch also pointed out similarities in the excipulum structure, besides spore color, amyloid reactions of asci (which blue evenly over the entire
length in both Plicaria and Boudiera) (TABLE I), agglutination of paraphyses and often a complex spore
ornamentation. He did not find protruding asci, a
consistent generic character in Boudiera. Molecular
phylogenetic analyses (Landvik et al 1997, Norman
and Egger 1999) support the placement of Boudiera
in the Pezizaceae.
Pachyella Boud. (1907) (FIG. 5) was synonymised
with Peziza (Donadini 1980b, Eckblad 1968, Seaver
1928), but was accepted by Le Gal (1947, 1953a),
Korf (1972) and Pfister (1973) based on the presence of a well-developed cortical zone of globose
cells which on the outside terminate in hyaline, hyphoid hairs, embedded in a gel and asci usually diffusely amyloid. Donadini (1980b) gave Pachyella subgeneric rank under Peziza. Most taxonomists have followed Pfister (1973). Hirsch (1984) even regarded
Pachyella as so isolated within the Pezizaceae that he
proposed a tribus Pachyelleae for this taxon alone,
opposed to tribus Pezizeae including all other genera
of the family.
Iodophanus Korf (Kimbrough and Korf 1967) was
segregated from Ascophanus Boud. (tribus Pseudoascoboleae, Ascobolaceae) and was, along with Thecotheus Boud., assigned to the tribus "Pezizeae" (Pezizaceae) rather than the tribus Theleboleae (Pezizaceae), because they possess amyloid asci, spores with
cyanophilic spore markings and an Oedocephalumconidial state (Gamundi and Ranalli 1964). Korf (1972,
1973b) later treated Iodophanus in the Ascobolaceae
(tribe Iodophaneae), emphasizing the thick-walled
spores in early stages of development, the carotenoid
pigments, and asci that protrude prominently at maturity (FIG. 1). Furthermore, Korf stated that the
tribe Iodophaneae (Iodophanus, Boudiera, Thecotheus
and Sphaerosoma)seems surely to represent the link
between Pezizaceae and Ascobolaceae. On the contrary, Iodophanus has been regarded as a "primitive
type" within the Pyronemataceae by Eckblad (1968),
967
based on the paraphyses' containing carotenoid pigments, the excipular structure, and the diffusely amyloid type of ascus. A close relationship between Iodophanus, Peziza and Plicaria has been indicated by
septal ultrastructure of asci and ascogenous hyphae
(Kimbrough and Curry 1985). Two ascal septum
types were found; the pezizoid and the ascoboloid.
The genus Thecotheus shared the ascoboloid type
with Ascobolus and SaccobolusBoud. Molecular phylogenetic studies support the relationship of Iodophanus to the Pezizaceae and of Thecotheusto Ascobolaceae (Landvik et al 1997, 1998).
ScabropeziaDissing & Pfister (1981) was segregated
from Plicaria and Peziza, based on the anatomy of
the apothecia. In this genus the outer surface of the
ascomata is pustulate (FIG. 7), the pustules being
composed of aggregates of globose to angular cells
clearly separated from the medullary excipulum of
textura intricata. Only two species are known.
HapsidomycesJ.C. Krug & Jeng (1984) was erected
for H. venezuelensisKrug & Jeng and assigned to the
Pezizaceae based on the amyloid asci and the thinwalled, pale-colored spores. Krug and Jeng considered it to be closely related to Boudiera (see TABLE
I), but sufficiently distinct by its early cleistohymenial
development; a distinct apothecial margin; short,
narrow asci with a short stipe; a rather narrow operculum; filiform paraphyses with yellowish content;
small, eguttulate spores, and a coprophilous habitat.
As in Boudiera, the asci protrude at maturity.
KimbropeziaKorf & Zhuang (1991) was described
on the basis of a cyanophilic and Congo Red-staining
lens-shaped thickening ("opercular lens") within the
ascus operculum. The asci were further noted to be
unusually brittle and with rather abruptly narrowed
and often twisted bases. The genus was assigned to
the Pezizaceae, differing from Peziza only by the very
peculiar asci (see TABLEI). Van Brummelen (1998)
studied the ascus apical structure by TEM and found
it to be unique. He considered Kimbropezia best
placed as a rather aberrant genus in the Pezizaceae.
On the contrary, molecular analysis by Norman and
Egger (1999) placed Kimbropeziawithin Peziza.
RhodopezizaHohmeyer & Moravec (Moravec 1994)
was described for R. tuberculata (Gamundi) Moravec
& Hohmeyer (= Aleuria tuberculataGamundi). It differs from Aleuria in having amyloid asci and tuberculate spores which are not regularly biguttulate. According to Moravec (1994) it has a special position
in the Pezizales, with its carotenoid granules in the
paraphyses and asci being amyloid over the entire
length. It shares characters with Iodophanus in particular, especially the presence of carotenoids, ornamented spores and diffusely amyloid asci. It was
968
MYCOLOGIA
placed in the Pezizaceae by Eriksson and Hawksworth
(1995).
Iodowynnea Medel, Guzman & Chac6n (Medel et
al 1996) was segregated from Peziza based on its
large, convoluted to highly folded apothecia, arising
from a buried stipe, and longitudinally verrucose-striate spores. One of the authors, Korf, reserved judgement on whether the new taxon should be considered a subgenus of Peziza or merit generic rank. Le
Gal (1953a) and Pfister (1974) treated the species in
bolitesfrom other genera of the Terfeziaceae (Trappe
1979). However, amyloid asci occur in some species
of Pachyphloeus (e.g., in the type species), and based
on this and anatomical features, the genus has been
placed in the Pezizaceae by Dissing and Korf (1980)
and Dissing (2000). Molecular phylogenetic analyses
has recently confirmed this placement (Norman and
Egger 1999, Percudani et al 1999).
Terfezia(Tul. & C. Tul.) Tul. & C. Tul. (1851) was
Galactinia and Peziza respectively.
Tul. to generic rank. It was distinguished by having
pockets of fertile tissue separated by sterile, but otherwise undifferentiated veins, highly ornamented
spores and non-amyloid asci (Trappe 1971, 1979). It
is the type genus of Terfeziaceae, and the most heterogeneous genus of the family. Recent molecular
phylogenetic analyses suggest that Terfezia is not
monophyletic, and that it is nested within Pezizaceae
(Norman and Egger 1999, Percudani et al 1999).
SphaerozoneZobel (Corda 1854) was described for
Marcelleina Brumm., Korf & Rifai (van Brummelen
1967) is included in this study to test the hypothesis
of a close relationship to Peziza gerardii.
Hypogeousgenera. The truffle or truffle-like genera
presented below, except for Mycoclelandiaand Cazia,
were originally described in the order Tuberales
(Tuberaceae or Terfeziaceae) due to their hypogeous
habit and lack of forcible spore discharge.
Hydnobolites Tul. & C. Tul. (1843) was described
for H. cerebriformisTul. & C. Tul. It was referred to
Tuberaceae by Gilkey (1954b) and Korf (1973b), but
placed in the Terfeziaceae by Hawker (1954) and
Trappe (1971, 1979) because the spores are globose
and there is no organized hymenium with paraphyses
at any stage of development. This position has been
accepted by most subsequent authors. The genus was
distinguished from others in the Terfeziaceae by having an interior layer of solid undifferentiated tissue
in which the asci are randomly distributed. Meandering veins deeply penetrate the interior of the ascomata from the outer surface. These are lined with outer
excipulum tissue. The asci are non-amyloid without
pretreatment in KOH, therefore Hydnoboliteswas not
placed in the Pezizaceae. Trappe (1979) regarded
Hydnobolites to be close to both Pachyphloeus and Ter-
fezia. The recent discovery of several cytological and
ultrastructural features by Kimbrough et al (1991)
made them suggest that Hydnobolitesbelongs in Pezizaceae. These features are the electron-dense, biconvex bands in septal pores of the asci, dextrinoid
and weakly amyloid asci (only after pretreatment with
2% KOH) and a type of spore wall deposition similar
to members of Pezizaceae.
PachyphloeusTul & C. Tul. (1844) was erected for
P melanoxanthus (Tul.) Tul & C. Tul. It was, like Hydnobolites, referred to Tuberaceae by Gilkey (1954b)
and Korf (1973b), and placed in the Terfeziaceae by
Trappe (1979). Hawker (1954) placed the genus in
Eutuberaceae E. Fisch. It lacks a hymenium and asci
irregularly line veins or are randomly produced in
the medullary excipulum. An apical depression or
pore leading into a few open veins is characteristic
of the ascomata. The verrucose outer excipulum of
isodiametric cells separates Pachyphloeusand Hydno-
elevated from Choiromyces section
Terfezia Tul. & C.
S. tulasnei Zobel (= Sphaerosoma ostiolatum Tul. & C.
Tul.), which has reddish brown spores. Dissing and
Korf (1980) studied the type species and discovered
that the asci are amyloid, contrary to previous reports
by Korf (1972, 1973b). Under the impression that
asci were non-amyloid Korf treated the genus in Pyronemataceae, rather than a genus in Pezizaceae as
was subsequently done by Dissing and Korf (1980).
It differs from Ruhlandiella by having paraphyses
without gelatinous sheaths and persistent asci.
Tirmania Chatin (1891) was described for T. africana Chatin (= Tuberniveum Desf.). It was placed in
the Terfeziaceae by Trappe (1971) and Korf (1973b).
It resembles Terfezia,but differs by having smooth or
at most minutely roughened spores and amyloid asci.
Based on the amyloid reaction and its anatomy, Trappe (1979) transferred the genus to the Pezizaceae.
Ruhlandiella Henn. (1903) was redescribed and
emended by Dissing and Korf (1980), who selected
a neotype for the type species R. berolinensis.They
found the asci to be amyloid and the paraphyses to
have characteristic gelatinous sheaths (features not
mentioned in Hennings' original description). The
ascomata are relatively small, hypogeous or semi-hypogeous, solid, pulvinate to globose ascomata and
with the hymenium covering the outer surface and
permanently exposed (FIG. 19) (Dissing and Korf
1980, Galan and Moreno 1998, Warcup and Talbot
1989 as MuciturboTalbot). Asci are evanescent at maturity. Dissing and Korf (1980) referred Ruhlandiella
to the Pezizaceae, with other globose-spored, amyloid
genera. A close relationship to Sphaerozone,in a lineage with Boudiera and Plicaria, was suggested.
Hydnotryopsis Gilkey (1916) was erected for H.
setchellii, but later synonymized with the globose-
HANSEN ET AL: PHYLOGENY OF PEZIZACEAE
(Gilkey
spored Choiromyces Vittad. (Tuberaceae)
1954b). Trappe (1975a), however, showed that it has
amyloid asci and, based on this and its ellipsoid
spores, the genus was reinstated. Fischer (1938) and
Trappe (1979) placed the genus in the Pezizaceae.
The ascomata are solid, but the asci and paraphyses
are arranged in hymenia.
Amylascus Trappe (1971) (FIG. 8) was segregated
from Hydnobolites on the basis of its thick-walled, amyloid asci, echinulate spores and the verrucose outer
excipulum of large inflated cells which gives rise to
a tomentose outer surface (Trappe 1971, 1975b, Beaton and Weste 1982). It was described in the Terfeziaceae for a single species, H. herbertianus Cribb.
Later, Trappe (1975b) considered the genus to be
closely related to the Geneaceae in both form and
anatomy, and the similarities between Hydnobolites
and Amylascus to be the result of parallel evolution.
When restricting Terfeziaceae to include only genera
with non-amyloid asci, however, Trappe (1979) transferred Amylascus to Pezizaceae.
Mycoclelandia Trappe & Beaton (1984) was proposed to replaced Clelandia Trappe (1979), a homonym of Clelandia J.M. Black (Violaceae). It was
placed in the Pezizaceae, and noted to resemble Peziza s.l. in several anatomical features, but differing
in its "cleistothecial" ascomata and indehiscent asci
(Trappe 1979). The asci are diffusely amyloid with
the reaction strongest near the apex.
Cazia Trappe (1989) was assigned initially to the
Helvellaceae due to the non-amyloid asci and large
isodiametric cells in the excipulum. It differed from
other similar hypogeous genera by its hyaline, minutely ornamented spores, crooked asci and a yellow
KOH reaction of the fresh medullary excipulum. The
genus was identified as a member of the Pezizaceae,
using sequences from the nLSU and nSSU rDNA
(O'Donnell et al 1997).
969
celleina and Pfistera Korf & W.Y. Zhuang (currently placed
in the Pyronemataceae), species belonging to the Ascobolaceae (Ascobolus), Morchellaceae (Verpa Fr.) and Pyronemataceae (Aleurina Massee, Otidea (Pers.) Bonord., Smardaea Svrcek) were included. Generic type species were used
as far as possible (TABLEII). Neolecta vitellina was used to
root the tree because phylogenetic analyses at more inclusive levels places NeolectaSpeg. basal to the rest of the fruitbody producing ascomycetes and the budding yeasts (Landvik et al 1993, Landvik 1996). To test the influence of the
outgroup on the analyses, Neolectawas replaced with Orbilia
Fr., which has been suggested as the sister group to the
Pezizales (Harrington et al 1999, Platt and Spatafora 2000).
Several collections of most included Peziza species have
been studied morphologically, and existing types or other
original material have been examined. The amyloid reactions of asci have been examined in additional specimens
of other included genera. A list of the specimens is available
from KH. The published voucher specimen for Pachyella
clypeata (ALTA 9029, GenBank SSU: AF133144, LSU:
U40619) (Norman and Egger 1999) was examined by KH
and DHP and was redetermined as Peziza subisabellina.The
voucher specimens for the other sequences obtained from
GenBank have not been studied morphologically by us.
AND METHODS
MATERIALS
Morphological methods.-The voucher material was studied
using light microscopy, and was identified or identifications
were verified by KH. All species of Pachyella were further
confirmed by DHP. Special notice was paid to the intensity
and location of the amyloid reaction in asci by using Melzer's reagent (1.5 g iodine, 5.0 g potassium iodide, 100 g
chloral hydrate, 100 mL distilled water), applied with or
without KOH pretreatment (2% or 10% KOH). Other
chemicals used were Cotton Blue in lactic acid (1% in 875
mL lactic acid, 63 mL glycerol, 62 mL water) and Congo
Red in ammonia (0.3% in commercial ammonia cleaner).
For critical species the spore surface was viewed by an AMRay 1000 scanning electron microscope (SEM). The anatomical structures of the excipulum were studied using
hand sections (for fresh material) or sections made on a
freezing microtome. Microanatomical terminology follows
Korf (1973b). Photographs of the ascus amyloid reactions
were made with an Olympus BH 2 microscope and an
Olympus C35AD-4 camera, and processed in Photoshop
(Adobe Photoshop 4.0).
Material studied.-Forty eight collections of Peziza representing 39 species were selected to cover the range of macro- and micromorphological characters, and habitats and
substrates recognized within the genus. To critically assess
the monophyly of Peziza and define its sistergroup relationships, representatives of the genera Amylascus, Boudiera,
Hydnotryopsis, Iodowynnea, Pachyella, Ruhlandiella and Tirmania were included (Tables I, II). For a few selected species more than one collection was sequenced to verify the
LSU rDNA sequences and estimate the intraspecific variation. LSU rDNA sequences from 12 additional species of
Peziza and representatives of the genera Cazia, lodophanus,
Kimbropezia,Plicaria, Sarcosphaeraand Scabropeziawere obtained from GenBank (TABLEII). To assess the monophyly
of the Pezizaceae and the relationship to the genera Mar-
Molecular methods.-DNA was isolated from dried or fresh
ascomata and was extracted as outlined in Hansen et al
(1999). Serial dilutions of DNA (1:10, 1:100, 1:1000) were
used as template for the polymerase chain reaction (PCR).
The 5' end of the nuclear large subunit rDNA (nLSUrDNA), spanning domains Dl and D2, was amplified using
the primers LROR and LR5 (for a few taxa LR5 was replaced with LR3 or LR7) (Moncalvo et al 2000). PCR products were purified either directly or following agarose gel
electrophoresis and band excision using QIAquick spin columns (Qiagen 1997). In addition to the primers used for
PCR, internal primers PNL2 (O'Donnell et al 1997) and
LSU0354-5' (5'- GCA AGA GAC CGA TAG CGC ACA AGT
AG-3') designed for Peziza, were used for BigDye terminator cycle sequencing (Applied Biosystems, Foster City,
970
MYCOLOGIA
California) following the manufacturer's protocol, except
that reaction volumes were 10 pJL.Sequencing reactions
were purified using ethanol/sodium precipitation (ABI protocol). Sequencing reactions were electrophoresed and
data collected on an Applied Biosystems 377 automated
DNA sequencer (Perkin-Elmer/ABI).
Analytical methods.-Sequences were edited and contigs assembled using Sequencher 3.0 (GeneCodes, Ann Arbor,
Michigan). Sequences are deposited in GenBank (TABLE
II). Nucleotide sequences were aligned manually in the data
editor of PAUP*4.0b3a (Swofford 1998) with alignment
gaps inserted to optimize aligned sites. The data matrix is
available from TreeBASE (http://phylogeny.harvard.edu/
treebase) as accessions S612 and M947. Nucleotide diversity
(e.g., Nei 1987, equation 10.6), as the average number of
nucleotide differences per site between two LSU rDNA sequences, were calculacted within and between selected spe-
epigeous and a hypogeous habit, with equally-weighted
transformations. Topologically constrained parsimony analyses were used to evaluate the hypothesis that all hypogeous
Pezizaceae have been derived (repeatedly) from epigeous
taxa, with loss of forcible spore discharge. MacClade was
used to construct constraint trees. Parsimony analyses were
performed under the constraints, using the same settings
as specified above, but with MAXTREESset to only 400 in
the second step of the search. The Kishino-Hasegawa test
(Kishino and Hasegawa 1989) was used to compare 400
trees under the constrained and unconstrained topologies
in PAUP 4.0b3a. Maximum likelihood analyses were also
performed under the constraints (with a transition:transversion ratio of 1.9:1) and compared to the optimal ML
tree.
RESULTS
cies.
Phylogenetic analyses were performed in PAUP*4.0b3a
(Swofford 1998) on an iMac 350 MHz (unless otherwise
stated). Maximum parsimony (MP) analyses with heuristic
searches were performed, with 1000 random stepwise addition replicates and TBR branch swapping. All characters
were equally weighted and unordered. Gapped positions
were included, treated as missing data (gap = missing), or
excluded in separate analyses. A two-step search was performed based on strategies designed by Maddison et al
(1992) and Olmstead et al (1993): first, only up to two trees
per replicate were kept, then exhaustive swapping was performed on all of the most parsimonious trees discovered,
with MAXTREES set to 15 000. Relative robustness of individual branches was estimated by bootstrap, using 500 replicates, with heuristic searches, 10 random addition sequences, TBR branch swapping and MAXTREESset at 100.
Maximum likelihood (ML) analyses were performed using two different starting trees for TBR branch swapping:
one of the most parsimonious trees and a tree generated
with one random taxon addition sequence. Empirical nucleotide frequencies were used, with the Felsenstein (1984)
two-parameter model for unequal base frequencies. The
proportion of invariable sites was set to zero and an equal
rate of substitution was assumed for all sites. The starting
parameter values were obtained using the Rogers-Swofford
method (Swofford 1998). To find the optimal tree, searches
were repeated by varying the transition:transversion ratio
until the best ML score was reached. Due to the large number of taxa, ML bootstrap values were generated using a
"fast" stepwise sequence addition, with 100 replicates and
a transition:transversion ratio of 1.9:1 (the optimal value
from the previous analyses). This method does not optimize
trees by swapping branches (Swofford 1998) and is therefore fast but not thorough. Equal bootstrap values (+ 5%)
were obtained with ML analysis using fastDNAml V. 1.0.8
(Olsen et al 1994), with 100 replicates. The latter analysis
however, required a 14-d search on a Pentium 600 MHz
computer, whereas the "fast" bootstrapping was conducted
on an iMac 350 MHz in 6 d.
MacClade 3.04 (Maddison and Maddison 1992) was used
to infer historical patterns of transformation between an
dataset consisted of partial LSU
Alignment.-The
rDNA sequences from 90 specimens. Sixty-eight new
sequences were determined in this study. For most
specimens, a 900 bp region at the 5' end of the LSU
rDNA was sequenced on both strands. For eight species of Peziza (P cerea, P. depressa, P granulosa, P
proteana, P. saniosa, P subviolacea, P succosella and
Peziza sp. 1) only a c 620-680 bp. region at the 5'
end was determined.
obtained from
Sequences
GenBank (27 specimens) were likewise c 620 bp.
Missing data were spaced out with gaps in the alignment, and these positions were therefore either treated as missing data or excluded from analyses. The
aligned length of all sequences including inserted
gaps was 966 bp. The missing data could have introduced error in the phylogenetic estimates (Maddison
1993, Wiens 1998), however analyses of only the 620
bp region for the full dataset resulted in the same
basic topologies
and bootstrap values, with only
slightly fewer resolved nodes.
No size variability due to introns was observed.
LSU rDNA sequences from the same morphological
species showed either no (e.g., P depressa, P echinispora) or several substitutions or deletions (e.g., P
gerardii, P subisabellina, Pachyella babingtonii). Intraspecific genetic variation was in some cases larger
than interspecific variation, e.g., the nucleotide diversity within P. gerardii was 3.1% and within Pachyella babingtonii 1.7%, whereas the average nucleotide
diversity between the Peziza species within group IVa
was only 1.3%. Identical sequences of the same morphological species were left out of the analyses.
Phylogenetic analyses.-Under
gap = missing coding
there were 319 parsimony informative characters.
With gapped positions omitted this number decreased to 249. Maximum parsimony analysis produced over 15 000 equally most parsimonious trees
HANSEN ET AL: PHYLOGENY
OF PEZIZACEAE
971
TABLEII. List of species used in the molecular phylogenetic study, with voucher specimen information and GenBank
accession numbers. Information on new sequences are listed first, followed by information on sequences obtained from
GenBank. Numbers in parentheses are used to indicate multiple collections of a single taxon.
Species
Voucher and geographic origin
Year and collector
GenBank
T. Laess0e 6236 (C). Malaysia
CUP-CH 2333 (CUP). China
1999, T. Laess0e
1981, R-y. Zheng, R.P. Korf
AF335111
AF335112
Trappe 18084 (C, dubl. OSC).
Australia
HK 90.074 (C). Russia
1996, J. Trappe
AF335113
1990, H. Knudsen
AF335114
Trappe 19134 (C, dubl. OSC).
USA
Trappe 17231 (C, dubl. OSC).
USA
CUP-ME566 (CUP). Mexico
1996, J. Trappe
AF335115
1995, J. Trappe
AF335116
1986, Ram6n-Farias
AF335117
18510 PAN (FH). India
HD S85-96 (C). Svalbard
1981, R. Kaushal
1985, H. Dissing
AF335118
AF335119
All-94-8 (C, type). Denmark
1994, K. Hansen, S.K. Sandal
AF335120
KH-98-107 (C). Denmark
KS-94-45 (C). Denmark
1998, K. Hansen
1994, K. Hansen, S.K. Sandal
AF335121
AF335122
KH-99-09 (C). USA
KH-98-77 (C). Austria
s.n. (FH). Switzerland
1999, K. Hansen, D.H. Pfister
1998, K. Hansen
1979, A. Nyffenegger
AF335123
AF335124
AF335125
SAE-1245 (C). Denmark
KH 00.011 (C). Denmark
JHC 92-386 (C). Denmark
Winterhoff 86239 (herb. Winterhoff). Germany
KH-98-08 (C). Denmark
1984, S.A. Elborne
1994, C. Lange
1992, J. Heilmann-Clausen
1986, W. Winterhoff
AF335126
AF335127
AF335128
AF335129
1998, A. Storgaard
AF335130
1998, B. Klug-Andersen
1992, S.-A. Hanson
1979, V. Demoulin, L. Smeets
AF335131
AF335132
AF335133
1997, J.H. Petersen
1998, H. Knudsen
1998, K. Hansen, T. Laess0c
1989, P.M. Petersen
1994,J. Vauras
AF335134
AF335135
AF335136
AF335137
AF335138
1993, J. Trappe
AF335139
1994, G. Piepenbroek-Groters
AF335140
Peziza exogelatinosaK. Hansen &
Sandal
Peziza gerardii Cooke (1)
Peziza gerardii (2)
KH-98-12 (C). Denmark
KH-98-113 (C). Sweden
V. Demoulin 5529 (FH). New
Guinea
KH-97-54 (C). Denmark
KH-98-28 (C). Denmark
KH-98-18 (C). Denmark
C no. 52152 (C). Denmark
Jukka Vauras 911 OF (TURA).
Finland
Trappe 13017 (C, dubl. OSC).
USA
Brummelen 1921 (L) The
Netherlands
KS-94-149 (C, dubl. FH) Denmark
KH-98-86 (C). Denmark
KH-97-90 (C). Denmark
1994, K. Hansen, S. K. Sandal
AF335141
AF335142
AF335143
Peziza gerardii (3)
Peziza granulosa Schumach.
Peziza howsei Boud.
KH-98-42 (C). Denmark
KH 00.012 (C). Denmark
KH-97-98 (C). Denmark
1998, K. Hansen
1997, M. Christensen, K. Hansen
1998, T. Laess0e, K. Hansen
1994, K. Hansen, S.K. Sandal
1997, J.H. Petersen, C. Lange
Nov. gen. ?
Aleurina imaii (Korf) W.Y.
Zhuang & Korf
Amylascus tasmanicus (Rodway)
Trappe
Boudiera dennisii Dissing & Sivertsen
Hydnotryopsis setchelliiGilkeya
Hydnotryopsissp.
Iodowynnea auriformus (Le Gal)
Medel, Guzman & Chac6na (1)
Iodowynnea auriformis (2)
Marcelleina persoonii (P. Crouan &
H. Crouan) Brumm.a
Marcelleina tuberculisporaK. Hansen & Sandal
Otidea onotica (Pers.: Fr.) Fuckela
Pachyella babingtonii (Berk. &
Broome) Boud. (1)
Pachyella babingtonii (2)
Pachyella punctispora Pfister
Pachyella violaceonigra (Rehm)
Pfistera
Peziza ammophila Durieu & Mont.
Peziza ampelina Quel.
Peziza ampliata Pers.: Fr.
Peziza apiculata Cooke
Peziza arvernensis Roze & Boud.
(1)
Peziza arvernensis (2)
Peziza badiofusca (Boud.) Dennis
Peziza bananicola (Rehm) Sacc.
Peziza cereaBull.: Fr.
Peziza depressaPers. (1)
Peziza depressa (2)
Peziza domiciliana Cooke
Peziza echinisporaP. Karst (1)
Peziza ellipsospora(Gilkey) Trappe
Peziza emileia Cooke
AF335144
AF335145
AF335146
972
MYCOLOGIA
TABLEII. Continued
Species
Peziza limnaea Maas Geest.
Peziza luteoloflavida Svcek
Peziza michelii (Boud.) Dennis
Peziza micropusPers.: Fr. (1)
Peziza micropus (2)
Peziza natrophila A.Z.M. Khan (1)
Peziza natrophila (2)
Peziza nivalis (Hein & Remy)
M.M. Moser
Peziza phyllogena Cooke (1)
Peziza phyllogena (2)
Peziza polaripapulata (J. Moravec)
K. Hansen
Peziza proteana (Broud). Seaver f.
proteana
Peziza retrocurvataK. Hansen &
Sandal
Peziza saniosa Schrad.: Fr.
Peziza subcutrina (Bres.) Koif (1)
Peziza subcitrina (2)
Peziza subisabellina (Le Gal)
Blank, Haffner & Hohmeyer
Voucher and geographic origin
HFG 94.2 (C). Denmark
T. Schumacher & K. 0stmoe D
170/83 (0). Norway
KH-98-13 (C). Denmark
KH-97-107 (C). Denmark
KH-99-04 (C). USA
Kew 59522 (K, Isotype). Bangladesh
JHP 93.021 (C). Denmark
KH-97-44 (C). USA
Year and collector
1994, H.F. Gotzsche
1983, T. Schumacher, K. Ostmoe
1998,
1997,
1999,
1970,
T. Laess0e
K. Hansen
Z. Yang
G.B. Hants et al.
GenBank
AF335147
AF335148
AF335149
AF335150
AF335151
AF335152
1993, J. Vesterholt et al.
1997, K. Hansen
AF335153
AF335154
KH-99-03 (C). USA
s.n. (TUR, type of Peziza hallioi). Finland
KS-95-10A (C). Denmark
1999, K. Hansen, D.H. Pfister
1965, K. Makinen
AF335155
AF335156
1995, H. Knudsen, M. Sasa
AF335157
Ginns 4156 (FH). Canada
1997, J. Ginns
AF335158
KS-94-182 (C). Denmark
1994, K. Hansen, S.K. Sandal
AF335159
KH-97-137 (C). Denmark
KS-94-133 (C). Denmark
KH-97-133 (C). Denmark
RK 96.54 (herb. Roy Kristiansen). Norway
1997,
1994,
1997,
1996,
C. Lange
K. Hansen, S.K. Sandal
C. Lange, K. Hansen
R. Kristiansen
AF335160
AF335161
AF335162
AF335163
Winterhoff 8844 (herb. Winterhoff.). Germany
KH-98-29 (C). Denmark
KH-98-07 (C). Denmark
KH-97-139 (C). Denmark
1988, W. Winterhoff
AF335164
1998, B. Klug-Andersen
1998, A. Storgaard
1997, C. Lange
AF335165
AF335166
AF335167
1995,J. Trappe
AF335168
1998, K. Hansen
1982, S.A. Elborne, H. Knudsen
1997, P. Marstad
AF335169
AF335170
AF335171
1985, J.H. Petersen, K. Hansen
1985, H. Dissing
1976, R.P. Korf et al.
AF335172
AF335173
AF335174
1976, R.P. Korf et al.
AF335175
1997, C. Lange, K. Hansen
AF335176
1983, C.H. de Vries
AF335177
1982, H. Bakhary
AF335178
Unknown
USA
USA
Norway
AF133159
U42694
AF133160
AF133161
(1)
Peziza subisabellina (2)
Peziza subviolacea Svrcek (1)
Peziza succosa Berk.
Peziza succosella (Le Gal & Romagn.) Aviz.-Hersh. & Nemlich
Peziza whitei (Gilkey) Trappe
Peziza sp. 1
Peziza sp. 2
Peziza sp. 3
Peziza sp. 4
Peziza sp. 5
Pfistera pyrophila Korf & W.Y
Zhuanga
Ruhlandiella berolinensisHenn.
Smardaea amethystina (W. Phil-
Trappe 17049 (C, dubl. OSC).
Australia
KH-98-30 (C). Denmark
C. no. 52153 (C). Denmark
PM-120-97 (Herb. Roy Kristiansen). Norway
KH-97-85 (C). Denmark
HD S.85.41 (C). Svalbard
CUP-MM 854 (CUP, type). Canary Islands
Mycoflora of Macaronesia 1230
(C, isoneotype). Canary Islands
KH-97-132 (C). Denmark
lips) Svrceka
Tirmania nivea (Desf.: Fr) Trappea
Tirmania pinoyi (Maire) Malencon
Published sequences:
Ascobolus lineolatus Brumm.
Cazia flexiascus Trappea
Glischrodermasp.
Iodophanus carneus (Pers.) Korfa
Trappe 23190 (C, dubl. OSC).
Israel
Trappe 13587 (C, dubl. OSC).
Saudi Arabia
NRRL A23604
JMT 12993
CUP62651
ARON 2102
HANSEN ET AL: PHYLOGENY
OF PEZIZACEAE
973
TABLEII. Continued
Species
Year and collector
Voucher and geographic origin
GenBank
CUP MM 2761
Canary Islands
AF133163
NSW 6359
USA
U42695
ALTA 8477
DAOM 199606
KNE 2205
UC 1475104
KNE 2143
DAOM 199749
ALTA 9066
Canada
USA
Canada
Sweden
Canada
Canada
Canada
U40615
U40613
U40614
U42692
U40617
AF133165
AF133170
Peziza griseoroseaGerard
Peziza lobulata (Velen.) Svcek (as
P violacea Pers. ss. Dennis
1978)
Peziza aff. merdaeDonadini
Peziza ostracodermaKorf
Peziza petersii Berk.
Peziza quelepidotiaKorf &
O'Donnell
CUP 62472
DAOM 199673
USA
Canada
U40616
AF133171
CUP RPK-206
DAOM 199608
DAOM 195796
NRRL 22205
USA
Canada
Canada
USA
AF133166
U40612
AF133167
U42693
Peziza subisabellina (3)b
ALTA9029
DAOM 195816
Canada
Canada
U40619
U40618
NSW 6752
C 530 (C)
USA
Denmark
U40611
U40607
20.X.1986 (C)
Denmark
U40608
DAOM 199089
DAOM 195830
ALTA 9605
Canada
Canada
Canada
U40610
U40609
AF133172
Pfister 13.8.83 (FH)
USA
AF133173
NRRL 20858 = CBS 551.72
Germany
U42672
KimbropeziacampestrisKorf & W.Y.
Zhuanga
Neolecta vitellina (Bres.) Korf &
J.K. Rogers
Peziza alaskana Cash
Peziza atrovinosa Cooke
Peziza badia Pers. (1)
Peziza badia (2)
Peziza aff. brunneoatraDesm.
Peziza echinispora (2)
Peziza fimeti (Fuckel) E.C. Hansenc
Peziza subviolacea (2) (as P pratervisa Bres. ss Dennis 1978)
Peziza vacinii (Velen.) Svrcek
Plicaria acanthodictya Dissing &
Hauerbach
Plicaria carbonaria (Fuckel)
Fuckel
Plicaria endocarpoides(Berk.) Rifai
Plicaria trachycarpa(Curr.) Boud.a
Sarcosphaeracoronaria (Jacq.)
Boud.a
Scabropeziascabrosa (Cooke) Dissing & Pfistera
Verpabohemica(Krombh.) Schr6t.
Generic type species.
Published as Pachyella clypeata (Schw.) Le Gal (Norman & Egger 1996, 1999).
c Published as Peziza vesiculosa Bull. (Norman & Egger 1999).
a
b
(MPT) of 1832 steps under gap = missing coding
(consistency index [CI] = 0.362, retention index
[RI] = 0.650), and 1366 steps with gapped positions
omitted ([CI] = 0.354, [RI] = 0.658). Although the
degree of resolution varied in parts of the trees, no
conflict between the strict consensus trees of the two
sets of analyses was observed (FIG. 23, trees with
gapped positions excluded are not shown). Trees obtained in analyses with Orbilia as the outgroup (data
not shown) were identical to trees obtained with Neolecta as the outgroup. The large number of equally
parsimonious trees reflects the inability of the data
to resolve the higher order relationships, and relationships between closely related species (FIG. 23).
However, 7 clades were identified (groups I-VII) that
were generally well supported (as measured by bootstrapping) or present in all trees (FIGS.22-23). The
Pezizaceae was suggested to be paraphyletic (bootstrap MPT 98%, ML 94%), because Marcelleina was
nested within it (FIGS.22-24). Ascobolaceae (represented by Ascobolus under this sampling) was confirmed to be its sister group. The included, currently
accepted genera in the Pezizaceae (TABLEI) were all
nested within Peziza, with Iodophanus (FIG. 1) in a
basal position, either as the sister group to the rest
of the Pezizaceae or nested within the Pezizaceae. Peziza gerardii, which forms a clade with Marcelleina
(FIGS.2-3) (bootstrap 96%), is the sister group to
974
MYCOLOGIA
95
Pezizaceae
Epigeous,
withforcible
spore dischalrge
Hypogeous,
withoutforcib
Ale
spore discharge
...-----
Equivocalstaate
20 steps
I
t
98
Neolectavitellina
82
Aleurinaimaii
I'Smardaea
I
amethystina
100
r- Otideaonotica
Nov.gen. ?
Verpabohemica
Ascoboluslineolatus
lodophanuscarneus
100
r- Peziza natrophila(1,2)
L Pezizaquelepidotia
65 peZiZa
-!
Pezizapolaripapulata
-1
Pezizasp.4
a
4
L_
Peziza
luteoloflavida
_j
VIIl
* 100
auriformis
lodowynnea
(2)
=! b
L
(1) J
odowynneaauriformis
berolinensis
56. Ruhlandietla
Peziza ostracoderma
Pezizawhitei
Pezizaellipsospora
__[-- Caziaflexiascus
-- -- Peziza badiofusca
r Peziza sp. 1
I Peziza badia (2)
- Peziza iimnaea
Peziza badia (1)
r
Pezizaalaskana
53 53
- Pezizadepressa (1,2)
VI
Peziza saniosa
- Pezizagriseorosea
Peziza atrovinosa
99
pinoyi
- Tirmania
Tirmania
nivea
- Pezizavacinii
*
.gg 1 Peziza phyllogena(2)
Peziza phyllogena(1)
Plicariaendocarpoides
- Plicariacarbonaria
I
4LJ Plicaria
trachycarpa
I
651 Plicariaacanthodictya
setchellii
Hydnotryopsis
100 r4...........
70 - Hydnotryopsis
sp.
L_
Sarcosphaeracoronaria
.....
Peziza sp. 3
100 r Pezizasuccosella
Pezizasuccosa
69
ot
Peziza aff. brunneoatra
i*jb
Peziza michelii ,
Peziza
howsei, emileia - 571
92
Pezizaexogelatinosa
J-i
(
Peziza proteana,petersii
*
subviolacea (1)
- Peziza
b
Peziza lobulata
*-~ r--'_ -i-- Peziza subviolaea (2)
_
'Peziza ampelina
Sr Peziza subcitrina(2)
Peziza subcitrina(1) -I
9- Peziza echinispora(1,2)
97 ,rPeziza micropus(2)
- Peziza
micropus(1)
IV
97[ Pezizacerea
Pezizaaff. merdae
80
*- Pfisterapyrophila
Peziza arvernensis(1,2)
. Pezizasp. 2
a
i' Pezizafimeti
- Peziza sp. 5
Peziza granulosa
5 - Peziza ampliata
91 [- Pezizanivalis
91
- Pezizadomiciliana
j
Peziza admophlila
90
Kimbropezia
campestris
- Peziza retrocurvata
50
Amylascustasmanicus
100
Glischroderma
sp.
_9161
I
Scabropeziascabrosa
!
Boudieradennisii
-- Pachyellababingtonii
94
100
(1)
L Pachyellababingtonii
I
(2)
*
100
!
/
Pachyellaviolaceonigra
Pachyellapunctispora Pezza apculata
*
Peziza
apiculata
.
Peziza
bananicola
ate
9
9
1
9Z7Pezizasubisabellina(1)
9L9 -L Peziza subisabellina(3)
I
L Peziza subisabellina(2)
100
Peziza
r
gerardii(1,2)
96
Peziza gerardii(3) 1
98
-98
I
Marcelleina
tuberculispora
Marcelleina
persoonii -
975
HANSEN ET AL: PHYLOGENY OF PEZIZACEAE
the rest of the Pezizaceae in the MLT (FIG. 24). In
the strict consensus of the MPTs, the basal node of
the Pezizaceae is unresolved, with Iodophanus, the
clade of P gerardiiand Marcelleina, and the rest forming a trichotomy (FIG.23). Species of Peziza occurred
in all groups except group III, often along with other
genera of the Pezizaceae. Several taxa constitute separate lineages of uncertain placement: P. apiculata,
P. retrocurvata (FIG. 21), and the clade of P natrophila and P quelepidotia (FIGS. 22, 23).
The optimal MLT's found under the two sets of
analyses using different starting trees do not have significantly different likelihood scores (Kishino-Hasegawa test, P < 0.05). The optimal MLT was found
with a log likelihood score of -11308.898, under a
transition:transversion ratio of 1.9:1 (FIG. 24), with
the starting tree generated with one random taxon
addition sequence. In analyses with one of the most
parsimonious trees as starting tree for branch swapping, the optimal MLT was found with a log likelihood score of -11312.599, under a transition:transversion ratio of 2.0:1. The ML analyses recovered the
groupings found by the MP analyses, with some minor changes. Deeper nodes in the MLT were resolved, but not strongly supported by bootstrapping
(FIG.24), and correspond to those branches that collapse in the strict consensus tree of the MPT's (FIG.
23).
Amyloid reaction types.-Distinct types of ascus amyloid reactions were found to correspond to different
rDNA lineages (FIG. 24). The location and intensity
of the amyloid reaction are constant in a given species, but varies between species. Four types of reactions were identified: (i) faintly amyloid over the entire length (sometimes more strongly in upper half),
but intensely and unrestrictedly amyloid over the
apex (most taxa in group VI, P succosa and P michelii); (ii) faintly amyloid over the entire length, except intensely amyloid in a distinct ring zone at the
apex, restricted to the area of dehiscence of the operculum (a synapomorphy for group IV); (iii) evenly
amyloid over the entire length, either: (iiia) weakly,
perhaps slightly stronger at the apex, or diffusely amyloid [periascus and hymenial gel amyloid (Pfister
1973)], or rarely non-amyloid (lodophanus, P gerar-
dii, group II, Pachyella, Boudiera, Amylascus, Hydnotryopsis, Plicaria, Tirmania, Ruhlandiella, P. natrophila, P. polaripapulata and Peziza sp. 4); (iiib) strongly
amyloid (Iodowynnea, P ellipsospora,P. luteoloflavida,
P. retrocurvata, P whitei and Sarcosphaera);(iv) Nonamyloid (Marcelleina and Cazia). The amyloid reaction is a fairly stable character, but rarely may be absent due to change of humidity (e.g., long periods of
rain) or an overripe state of the hymenium, e.g., presumedly in Pfistera (van Brummelen 1998) and other
Peziza species. The periascus (an extra-ascal mucilagenous coat, as defined by van Brummelen 1978) can
also disappear after heating (Donadini 1985) or be
removed in microscopic preparation by applying
pressure on the cover slip and sliding it back and
forth (Samuelsen 1978).
DISCUSSION
Overview.-The Pezizaceae appear to be paraphyletic
as currently circumscribed (Eriksson 2000) because
Marcelleina is nested within it. If Marcelleina is transferred to the Pezizaceae, then the family would be a
strongly supported monophyletic group (FIGS.2224). Our data fail to resolve the higher level relationships within the Pezizaceae (FIG.23) or result in resolutions of relationships that are not well-supported
by bootstrap analyses (FIG.24). The weakness in our
estimate of the phylogeny may be due to having too
few informative characters, or to missing key taxa in
our sampling (especially species of Peziza with apiculate spores). Addition of more characters from other molecules (such as the protein-coding nuclear
genes Beta-tubulin or RPB2), and expansion of the
set of taxa to include more aberrant species of Peziza,
may improve resolution deep in the tree and "break
up" long branches (Graybeal 1998). Nevertheless,
seven groups of species are identified that may eventually be formally recognized. Confirming recent molecular studies, Peziza is not supported as monophyletic (Norman and Egger 1996, 1999). Peziza species
are spread among all groups, except group III, and
it is evident from this that Peziza will have to be subdivided into several genera, or a very wide concept
FIG. 22. Phylogeny of the Pezizaceae inferred from nLSU-rDNA sequences. One of more than 15 000 equally parsimonious
trees (1832 steps), generated under gaps = missing coding. Terminal taxa represent individual specimens (TABLEII). Branches with asterisks collapse in the strict consensus of all most parsimonious trees. Numbers by branches are bootstrap frequencies
(values greater than 50%). Branch lengths are proportional to the number of steps (character changes) along the branch.
Branch colors and dotted branches represent character state optimizations (using MacClade). Species of Peziza are in boldface. Taxa in grey are hypogeous, without forcible spore discharge. Bracketed groups I-VII are discussed in the text. Diamond
indicates suggested position of P vesiculosa from subsequent analysis (see note added after acceptance of the manuscript).
976
MYCOLOGIA
82
1100oo
75
'
100
100
61
94
1 91
100
100
56
[
|
53
95
53
99
j
99
|
65 r
'
50
65
1
j00
100
.98
100
Pezizaceae
1
"99
99
97
97
91
90o
96
i
97
'
99
Neolectavitellina
Aleurinaimaii
Smardaeaamethystina
Otideaonotica
Nov.gen. ?
Verpabohemica
Ascoboluslineolatus
lodophanuscarneus
Peziza natrophila(1,2)
Peziza quelepidotia
Amylascustasmanicus
Glischroderma
sp.
Scabropeziascabrosa
Boudieradennisii
Pachyellababingtonii(1)
Pachyellababingtonii(2)
Pachyellaviolaceonigra
Pachyellapunctispora
Ruhlandiellaberolinensis
Peziza ostracoderma
Peziza whitei
Peziza ellipsospora
Caziaflexiascus
Peziza badiofusca
Peziza sp. 1
Peziza badia (2)
Peziza limnaea
Peziza badia (1)
Peziza alaskana
Peziza saniosa
Peziza depressa (1,2)
Peziza atrovinosa
Peziza griseorosea
Tirmaniapinoyi
Tirmanianivea
Peziza vacinii
Peziza phyllogena (2)
Peziza phyllogena (1)
Plicariaendocarpoides
Plicariacarbonaria
Plicariaacanthodictya
Plicariatrachycarpa
Peziza retrocurvata
Peziza apiculata
Peziza polaripapulata -- a
Pezizasp.4
Peziza luteoloflavida VII
auriformis
I
b
(2)
lodowynnea
lodowynneaauriformis(1) 1
Peziza bananicolaPeziza subisabellina
(1)
"Pezia subisabellina (3)
Peziza subisabellina (2)
setcheilti
^100 {Hydnotryopsis
70
sp.
Hydnotryopsis
Sarcosphaeracoronaria
Peziza sp. 3
Peziza succosella
100
Peziza s!!ccosa
Peziza aff. brunneoatra
Peziza michelii -.Peziza howsei, emileia 57 (
92
Peziza exogelatinosa
Peziza proteana, petersii
Peziza subviolacea (1)
Peziza lobulata ?
Peziza subviolacea (2)
Peziza ampelina
,
Peziza subcitrina (2)
Peziza subcitrina (1)
Peziza echinispora (1,2)
64
Peziza micropus (2)
97 1
Peziza micropus (1)
66
Peziza cerea
{
Peziza aff. merdae
80
Pfisterapyrophila
[
Peziza arvernensis (1,2)
Peziza sp. 2
Peziza sp. 5
Peziza ranulosa
Peziza fimeti
Peziza ampliata
Peziza nivalis
*
Peziza domiciliana
Peziza
anmlophila
I
Kimbropezia
campestris
100 _
Peziza gerardii (1,2)
Peziza gerardii (3)
98 j
Marcelleinatuberculispora /
Marcelleinapersoonii
V
-
b
IV
a
-
FIG. 23. Strict consensus of 15 000 equally parsimonious trees generated under gaps = missing coding. Numbers by
branches are bootstrap frequencies (values greater than 50%). Species of Peziza are in boldface. Taxa in grey are hypogeous,
without forcible spore discharge. Bracketed groups I-VII correspond to groups I-VII in FIG.22. Diamond indicates suggested
position of E vesiculosa from subsequent analysis (see note added after acceptance of the manuscript).
HANSENETAL:PHYLOGENY
OF PEZIZACEAE
would have to be accepted, to better reflect phylogeny.
Ascomata forms and habit.-Four different forms of
ascomata exist within the Pezizaceae. Epigeous apothecia of various shapes (TABLEI) with forcible spore
discharge, are the most common and occur in groups
I-VII. This form is likely to be symplesiomorphic, implying that the apothecia-forming members of the
Pezizaceae have given rise to at least three different
forms of hypogeous ascomata without forcible spore
discharge (sensu Weber et al 1997): (i) ptychothecia:
enclosed, hollow, subglobose to globose, often convolute ascomata, which may have one or a few open
chambers, and a persistent, recognizable hymenium
of asci and paraphyses typically lining the hollow
chambers (FIGS. 17, 18) (in group VI); (ii) stereothecia: more or less solid, fleshy ascomata, with the asci
either solitary, scattered relatively evenly throughout
the medullary excipulum or grouped in dispersed
pockets or meandering veins (FIGs.8, 14) (in group
III, V, VI); (iii) exothecia: solid, pulvinate to globose
ascomata, in which the hymenium covers the outer
surface (leaving no discernible margin) and permanently exposed, with the asci distinctly shorter than
the paraphyses (FIG. 19) (in group VI).
At least three independent origins of hypogeous
forms within the Pezizaceae were supported by the
LSU rDNA gene trees (in group III, V and VI) (FIGS.
22, 24). The most parsimonious interpretations of
the molecular phylogenies suggest that forcible spore
discharge has been lost once in group III; lost twice,
or lost once and gained once in group V; and either
lost twice and gained once, or lost once and gained
twice in group VI (FIGS.22, 24). However, separate
constraint analyses forcing the hypogeous taxa within
either group V or VI into monophyletic groups,
could not be rejected using the Kishino-Hasegawa
test (TABLEIII). Forced monophyly of the hypogeous
taxa within group VI, did not yield trees that were
significantly worse (P < 0.05) than the unconstrained
trees (TABLEIII). Likewise, forced monophyly of the
two species of Hydnotryopsisin group V could not be
rejected (P < 0.05). Trees with constrained monophyly of the hypogeous taxa within groups V and VI,
respectively, but analyzed together, were not significantly longer than the unconstrained MPTs (P <
0.05). However, under this constraint the MLT was
significantly worse than the unconstrained optimal
MLT (P < 0.05) (TABLE III). The most conservative
conclusion is thus, that forcible spore discharge has
been lost only once within each of the groups V and
VI.
Phylogeny within the Pezizaceae.-Group I. This is a
highly supported clade (bootstrap 96%) of the mor-
977
phologically distinct Peziza gerardii and two species of
Marcelleina, M. persoonii and M. tuberculispora.It is a
likely sister group to the rest of the Pezizaceae (FIG.
24). Contrary to the current classification, which accepts Marcelleina in the Pyronemataceae (Eriksson
2000) or Otideaceae (Hawksworth et al 1995), our
analyses place the non-amyloid Marcelleina in the Pezizaceae. The genus is restricted to species with small
purplish apothecia, 2-10 mm in diam (FIG. 3), with
light brown pigments in the walls of the hyphae in
the outer excipulum and hyaline spores (Moravec
1987). The purplish (-brownish) pigments are found
in the paraphyses and subhymenium. Smardaea and
Aleurina, presumed to be closely related to Marcelleina (Zhuang and Korf 1986, Moravec 1987, Hansen
et al 1998), are supported in the Pyronemataceae
(grouping with Otidea and an undescribed genus (TL
6236)) (FIGS.22, 23). Smardaea and Greletia Donadini (the latter not included in this study) are distinguished by purplish pigments in all parts of the apothecia, including the spore ornamentation, and by a
clearly differentiated medullary excipulum (Svrcek
1969, Donadini 1979c, Moravec 1987). Korf (1972)
placed Marcelleina (as Pulparia P. Karst.) in the subfamily Ascodesmidoideae (Pyronemataceae). To him,
the genus seemed to provide a link between Sphaerozoneand Aleurina (as Jafneadelphus Rifai). Moravec
(1987) revised and placed Marcelleina in a new subfamily, Aleurinoideae (Pyronemataceae), along with
Aleurina, Eoaleurina Korf & W.Y. Zhuang, Greletia,
Smardaea and Sphaerosoma.
Although the Pezizaceae has generally been characterized by amyloid asci, these results indicate that
the amyloid reaction might be lost (type iv) in some
lineages, such as in the branch leading to Marcelleina
(FIGS.22, 24). Boudier (1907) placed species of Marcelleina (as Plicaria), including the type species M.
persoonii, in the "Aleuri&es" (= Pezizaceae), presumably based on the globose spores and the excipulum
structure. He fully realized that the asci were nonamyloid (Boudier 1905-1910, plate 308). Eckblad
(1968) also regarded Marcelleina to be very close to
Plicaria, the non-amyloid asci being one of only a few
differentiating characters. He placed it in the Pezizaceae, despite the non-amyloid asci, and concluded
that it would be ". . . attaching too much importance
to a single, widely distributed character to keep Marcelleina out of its otherwise close affinity to Plicaria
and Peziza." Other contemporary taxonomists have
considered Marcelleina as logically placed among the
non-amyloid genera. However, morphological similarities between P gerardii and Marcelleina (FIGS.2,
3), Smardaea and Greletiawere noted by Schumacher
and Jenssen (1992).
Macroscopically, and in habitat requirements (en-
978
MYCOLOGIA
94
69
99
,
Neolectavitellina
Aleurinaimaii
Smardaeaamethystina
Verpabohemica
F- Otideaonotica
Nov.gen. ?
Ascoboluslineolatus
IAS
A-
SAE
;arleus
lupropanldlus
p- Pezizanatrophila(1,2)
100
Pezizaquelepidotia
1
100
19494 -
84
Amylascus tatsmanicus
Glischrode
ermasp.
l
Sc
scabrosa
____
-
100
I55 |
100
abropezia
Boudieradennisii
Pachyella babingtonii
(1)
Pachyella tbabingtonii
(2)
-
Pezizaapiculata
Pezizabananicola
Pezizasubisabellina(1)
_9199
Pachyellaviolaceonigra
Pa
Pachyellapunctispora
II
..
L._ Peziza subisabellina (3)
Pezizasubisabellina(2)
setchellii
Hydnotryopsis
Lqg{Hydnotryopsis
sp.
_
Sarcosphaeracoronaria
Pezizasp. 3
100 r Pezizasuccosella
69 L|
Pezizasuccosa
Pezizaafft.brunneoatra
Pezizamichelii
41X
99
64 Peziza polaripapulata
96
Peziza sp. 4
88
Pezizaluteoloflavida 100
(2)
lodowynneaauriformis
lodowynnea auriformis(1)
54i- Ruhlandiella
berolinensis
Pezizaostracoderma
Pezizawhitei
Pezizaellipsospora
Caziaflexiascus
- Peziza
- Pezizabadiofusca
sp. 1
1 Pezizabadia(2)
-
a
I
b I
b
VII
-' Peziza limnaea
-5
51
67
-
Pezizaceae
00
94
VI
Plicariacarbonaria
Peziza sp. 2
Hypogeous,
withoutforcible
sporedischarge
_-
Pezizafimeti
- Pezizasp. 5
Pezizagranulosa
Peziza ampliata
- Pezizanivalis
Pezizadomiciliana
?
--- Pezizaammophila
79 Kimbropezia
campestris
-55
Equivocalstate
I
Peziza alaskana
Pezizadepressa (1,2)
Pezizasaniosa
Pezizagriseorosea
Pezizaatrovinosa
93
Tirmania
pinoyi
- _i_
Tirmania
nivea
Pezizavacinii
Plicariaendocarpoides
~
Plicariaacanthodictya
~-Plicariatrachycarpa
_
9__ Peziza phyllogena (2)
Pezizaphyllogena(1) Pezizaretrocurvata
Pezizahowsei, emileia 511
exogelatinosa
93 i--Peziza
JI
Pezizaproteana,petersii
r Pezizasubviolacea (1)
Pezizalobulata
b
-Pezizasubviolacea (2)
Pezizaampelina
94 Pezizasubcitrina(2)
Pezizasubcitrina(1)
Pezizaechinispora(1,2)
I
886Er Pezizamicropus(2)
Pezizamicropus(1)
_. 84'r Peziza cerea
58 Pezizaafft.merdae
- j69Pfisterapyrophila
' Pezizaarvernensis(1,2)
Epigeous,
withforcible
sporedischarge
0.02
Pezizabadia(1)
--
r-
81
100
|
96
rI
Peziza gerardii (1,2)
Pezizagerardii(3)
Marcelleina
tuberculispora
Marcelleina
persoonii
a
IV
V%~
979
HANSEN ET AL: PHYLOGENY OF PEZIZACEAE
TABLEIII. Evaluation of different constrained tree topologies in MP and ML analyses, compared to the MPTs and the
optimal MLT, respectively, using the Kishino-Hasegawa test (p < 0.05)
ML
MP
Tree
Tree lengtha
Unconstrained optimal MLT
Unconstrained MPT
Hypogeous taxa within group VI monophyletic
Hypogeous taxa within group V monophyletic
Hypogeous taxa within group VI and V both
monophyletic
1832
1839 (+7)
1835 (+3)
1842 (+10)
a Difference
Significantly
worse?
Ln likelihood
Difference
in LnL
Standard
deviation
Significantly
worse?
-40.078
-17.243
-43.602
27.051
25.651
16.248
no
no
yes
-11308.898
no
no
no
-11348.976
-11326.140
-11352.500
in length between MPTs and constrained trees in parentheses.
riched, often calcareous soil), species of Marcelleina
(FIG.3) are very similar to P gerardii (FIG.2). Peziza
gerardii shares anatomical characters with Marcelleina, although the fusiform spores are very different.
Within the Pezizaceae, P. gerardii has a unique spore
shape and ornamentation (observed by SEM): large,
slightly inequilateral, narrowly fusiform spores, with
an ornamentation of low, longitudinal, cyanophobic
ribs (Berthet 1970), and 1 to 3 large and several small
guttules. Spores in Marcelleina are globose, smooth
or ornamented, with one large guttule. The purplish
(-brownish) pigments in P gerardii are located in the
paraphyses, subhymenium and excipulum. Peziza gerardii and species of Marcelleina have an excipulum
composed entirely of globose to angular cells, which,
toward the outer surface, become smaller (outer excipulum hardly discernible), and from which scattered short rows of cells project (appressed, hyphoid,
septate hairs). The weakly amyloid, general staining
of the asci (type iiia) in P. gerardii is also a distinct
and rare feature within the genus Peziza.
GroupII. This strongly supported (bootstrap 99%)
group includes two unusual species of Peziza, P subisabellina (FIG. 4) and P bananicola. Both species
have asci that are diffusely amyloid (type iiia) (FIG.
24). Presumably based on this feature, P subisabellina
has recently been combined in Pachyella (FIG. 5)
(Trimbach 1999), a placement in contradiction with
our molecular results (FIGS. 22, 24) as well as the
morphological characters. Diffusely amyloid asci are
typical for the genus Pachyella, but P. subisabellina
does not possess other pachyelloid characters (see
Background Information). Spore shape and guttulation in Pachyella is uniform, broadly ellipsoid containing two large guttules, and differs from P. subisabellina which has almost fusiform spores with many
small guttules. Our analyses support Pachyella in
group III.
Peziza sect. Purpureodiscus G. Hirsch (Hirsch 1992)
was proposed for P. subisabellina and P kreiselii G.
Hirsch, based on the diffusely amyloid asci, reddish
pigments in the apothecia (FIG. 4), smooth spores
without guttules and clavate paraphyses. A segregation of P. subisabellina from the "core group of Peziza
species" is also supported by our analyses. Sequences
from three different collections of P subisabellina are
included in this study, one occurring on wood (Winterhoff 8844) and two on soil (RK96.54, ALTA9029).
Several differences in the LSU have accumulated between these individuals, with the two specimens on
soil grouping together, although all three form a
well-supported monophyletic group (bootstrap 99%)
(FIG. 22).
Peziza bananicola is one of the few strictly tropical
Peziza species. It has two unique features: an extensive subiculum that covers and binds debris from ba-
FIG.24. The tree with the highest likelihood (lnL-11308), obtained from maximum-likelihood analyses. Branch lengths
correspond to genetic distance (expected nucleotide substitutions per site). Numbers by branches are "fast" bootstrap
frequencies (values greater than 50%). Branch colors represent character state optimizations (using MacClade). Taxa in red
are hypogeous, without forcible spore discharge. Species of Peziza are in boldface. Diamond indicates suggested position of
P vesiculosa from subsequent analysis (see note added after acceptance of the manuscript). Bracketed groups I-VII correspond to groups I-VII in FIG. 22. Types of ascus amyloid reactions supporting group II, IV and VI are shown. Microscopic
photos: diffusely amyloid (type iii, P. subisabellina, RK96.54, at the top) ca X400; intensely and unrestrictedly amyloid over
the apex (type i, P. badia, KH-97-84, in the middle) ca X600; intensely amyloid in a distinct ring zone at the apex (type ii,
P cerea, KH-97-01, at the bottom) ca X550. Photos: K. Hansen.
980
MYCOLOGIA
nana plants, and a peculiar spore ornamentation of
sparse, irregularly spaced, very thin, acutely pointed
spines (Pfister 1991). The spores are narrowly ellipsoid and eguttulate. Peziza subisabellina and P. bananicola share certain excipular features, being very
thick and dense. However, the medullary excipulum
is stratified in three layers in P bananicola, but is
composed of a single uniform layer in P subisabellina. Other less known tropical species, e.g., P luteorosella (Le Gal) Pfister and Galactinia tapesioidesLe
Gal (Le Gal 1960), are morphologically similar to P
bananicola (Pfister 1991) and may belong to group
II. Peziza luteorosellahas spore ornamentation much
like P. bananicola, but has an unstratified excipulum
like P. subisabellina. Hymenial colors in the three
tropical species and P subisabellina range from whitish, yellowish, rose, reddish to purplish brown.
Group III. Four genera and one form-genus are
represented in this clade (bootstrap MPT 56%, supported by all MPT, FIG. 23). Boudiera dennisii (FIG.
6) forms a monophyletic group with three species of
Pachyella, P. violaceonigra, P punctispora (FIG.5) and
P babingtonii (bootstrap MPT 91%, MLT 84%). This
is in contradiction with the ML analysis by Norman
and Egger (1999) based on nSSU rDNA sequences.
In their analysis Boudiera (B. acanthosporaDissing &
Schumacher) clusters with Iodophanus (FIG. 1), basally in the Pezizaceae (<50% bootstrap), which supports the placement of these genera in a tribe, lodophaneae (Korf 1972, 1973b). However, Pachyella is
not represented in the study by Norman and Egger
(1999), since the included Pachyella is Peziza subisabellina (see Materials and Methods). A close relationship between Boudiera and Plicaria, as has been suggested (see Background Information), is also not
supported by our analyses. Species of Boudiera lack
gelatinous excipular tissues and hyphoid hairs embedded in a gel, typical in the genus Pachyella. Nevertheless, the lineage is united by asci evenly amyloid
over the entire length (type iiia) and the overall anatomical structure of a medullary excipulum of textura intricata, an outer excipulum of textura angularis to globulosa and an indistinct subhymenium
(TABLEI). Species of Boudiera and Pachyella occur
on water-soaked, regularly inundated substrates, although Pachyella species most commonly occur on
wood, and Boudiera species on sand. The relationship
between the two genera is unresolved in the strict
consensus tree, but monophyly of Pachyella is weakly
supported in the MLT (55% bootstrap). The two subclades of Pachyella species are both highly supported
(100% bootstrap). Some of the longest branches in
the MPT (FIG. 22) occur in group III. The branch
leading to Boudiera is 63 steps. The branches uniting
the lineages of P. violaceonigra and P punctispora,
and the lineages of P babingtonii are each 42 and 26
steps. The branch uniting the lineages of Boudiera
and Pachyella is 39 steps. Long internode lengths
could most probably be obviated in this clade by including more species of Boudiera and Pachyella.
Glischroderma
Scabropezia and the mitosporic
Fuckel form a highly supported clade with the truffle
Amylascus tasmanicus (bootstrap 100%). Another
truffle, Pachyphloeus melanoxanthus (type of Pachyphloeus), is suggested as closely related to Scabropezia,
based on nSSU rDNA sequences (Norman and Egger
1999) and morphology (Dissing and Pfister 1981).
Both truffles produce ascomata with a tough-fleshy
medullary excipulum and asci randomly distributed
throughout the tissue (= stereothecia) (FIG(.8). The
asci are weakly amyloid over the entire length (type
iiia), but some other species of Pachyphloeusare described with non-amyloid asci (Dissing and Korf
1980, Dissing 2000). Dissing and Pfister (1981) noted
the similarities in the structure of the outer excipular
layers in Pachyphloeus and Scabropezia, which is highly
pustulate or verrucose (FIG.7), of thick-walled, large,
globose cells, a feature also shared with Amylascus
(Trappe 1971, 1975b, Beaton and Weste 1982). Scabropezia scabrosa, A. tasmanicus and P melanoxanthus
have globose spores, with an ornamentation of prominent conical or truncate warts, which in P melanoxanthus and A. tasmanicus are embedded in mucilage.
The spores in Scabropezia and P. melanoxanthus are
brownish at maturity. Scabropeziaoften occurs on calcareous soil, or rarely on decaying wood, and attempts to germinate spores of Scabropeziafailed (Dissing and Pfister 1981). Based on these observations,
and the fact that most hypogeous fungi are thought
to be ectomycorrhizal (Trappe and Maser 1977), we
believe this lineage could be mycorrhizal.
Group IV This group is weakly supported by bootstrap analyses (<50%), but is present in all MPTs
(FIG. 23) and in the MLT (FIG. 24). The presence of
a distinct amyloid ring zone at the apex of the asci
(type ii, FIG. 24) is a synapomorphy for group IV.
Such a reaction is present in all species, except in
Kimbropezia,in which asci are strongly amyloid apically (Korf and Zhuang 1991), and in P emileia, P
howsei and P ampelina, which show a less restricted
amyloid reaction, although P howsei (KH-97-98)
showed a distinctly amyloid ring zone in fresh material. Asci with a strongly amyloid ring were noted previously by Svrcek (1970) in several of the species in
clade IVa. Group IV is also united by the type of anamorph produced. All species with known anamorphs produce an Oedocephalumstate. An exception is Peziza aff. merdae which produces a Chromelos-
porium state (Norman and Egger 1999). Oedocephalum anamorphs are reported for species in both clade
HANSEN ET AL: PHYLOGENY
OF PEZIZACEAE
IVa (for P ammophila, P ampliata, P cerea, P echinispora, P micropus, P repanda, P sylvestris, P varia
and P vesiculosa) and in IVb [for P. subviolacea (as
P praetervisa), P. petersiiand P lobulata (as Galactinia
violacea (Pers.) Le Gal)] (e.g., Berthet 1964a, b, Paden 1972, 1973, Webster et al 1964). Additionally,
spores with a unilateral gelatinous coating have only
been found in group IV. The gelatinous spore coating was observed in P. ampliata, P granulosa, P. subcitrina and P vesiculosa. This character has rarely
been reported, probably because it is difficult to observe in dried material.
Group IV includes one strongly supported clade,
IVa, with bootstrap MPT 91%, MLT 55% (equivalent
to Boudier's genus Aleuria pro parte 1907). This
clade contains common Peziza taxa, characterized by
mostly large, yellowish brown disc- to cup-shaped
apothecia (FIG. 9), finely warted or smooth, eguttulate spores (P. domiciliana has two small guttules in
the spores), and simple, hyaline or pale yellowish paraphyses. They occur on rich soil mixed with litter or
woody debris or mortar, on strongly decayed trunks,
manured substrate or dung. A subset of these taxa
was also highly supported as monophyletic in ML
analysis by Norman and Egger (1999). Since the type
species of Peziza, P vesiculosa, is included, this clade
will have to serve as a core group for a future circumscription of the genus.
Morphological, ecological, and molecular divergence (nLSU rDNA) based on the branch lengths
(FIGS.22, 24) and nucleotide diversity are low within
group IVa, which may suggest a recent radiation. Additionally, species limits are controversial and the
number of species recognized varies. The group is
taxonomically problematic due to lack of diagnostic
morphological characters. Peziza species with ellipsoid, smooth, small (ca 15 izm) and eguttulate spores
were referred to as the "Peziza repanda-varia-micropus-cerea complex" by Rifai (1968). He stated that
this complex needs a prolonged field and laboratory
study to establish species limits. The most detailed
study of this complex is by Svrcek (1970), who also
included species with finely warted spores. A detailed
study of group IVa using morphology and more rapidly evolving sequences is in progress.
Peziza ammophila differs in its habitat and development. It fruits partly buried in sand (FIG. 10),
among Ammophila (Poaceae). The (pseudo-) stipitate
ascomata are deeply cup-shaped and the margins
split into irregular rays, becoming flattened on the
sand surface. Based on this characteristic development, P ammophila has been placed in Sarcosphaera
(FIG. 13) (e.g., Kotlaba and Pouzar 1963, Seaver
1928). Pouzar (1972) considered P. ammophila and
another species, P austrogaster(Rodway) Rifai, to be
981
a "unique" entity within Peziza. He proposed a new
subgenus of Peziza, AsterosphaeraPouz., with P ammophila as the type species. Most authors have considered the similarities in macromorphological development between Sarcosphaeraand P ammophila to
be a result of convergent or parallel evolution. These
hypotheses is congruent with the molecular results.
Furthermore, P ammophila shares all other morphological characters of clade IVa. Peziza echinisporadiffers from other members of this clade in its spore
ornamentation of isolated, regular spines and its occurrence on burnt ground. Other characters such as
eguttulate spores, an Oedocephalumanamorph, and
the distinctly amyloid ascus ring zone, justify its placement in this group. Apart from the guttulate spores,
P. domiciliana is also aberrant in this clade by having
a pinkish (violet) tinge in the hymenium otherwise
characteristic of clade IVb.
The monotypic genera Kimbropeziaand Pfistera,
described on the basis of peculiar ascus apices (Korf
and Zhuang 1991) are nested within clade IVa. Kimbropezia campestris was assigned to the Pezizaceae,
while Pfistera was excluded due to the supposedly
non-amyloid asci. Asci in Pfistera pyrophila were described as having a very thin, arched apical dome at
maturity, which may split vertically, horizontally or irregularly. Van Brummelen (1998) studied the ascus
apical structures in these species, and in species he
supposed might be related [P michelii, P. emileia and
Geopyxismajalis (Fr.) Sacc.] using TEM. Our results
support van Brummelen's conclusion, that Pfistera
campestrisbelongs in Peziza. It was found to display
two different types of ascus dehiscence, the second
type perhaps developing from the first type, resulting
from less than optimum conditions during development. The apical structure of the "typical" ascus was
of the Peziza type. In addition, Pfisterapyrophilashows
several characters that are typical for clade IVa and
microscopic examination (by DHP and KH) revealed
a few small patches of faintly amyloid material in the
hymenium, around some ascal tips (pretreated with
2% KOH). Van Brummelen observed "remains of a
moderately reactive periascus, too thin to be observed by light microscopy." Contrary to the phylogenetic analysis by Norman and Egger (1999) and to
our results, van Brummelen considered Kimbropezia
to be distinct from Peziza. He found that asci of Kimbropezia,in addition to having the unique "opercular
lens," was more strongly amyloid than those of Peziza, and the annular indentation or weakened zone
characteristic of Peziza was completely absent. Dehiscence was found to be by a circumscissile, rather irregularly delimited rupture in the thin part of the
ascus wall, around the opercular lens. We suggest that
this apical structure and the mechanism of dehis-
982
MYCOLOGIA
cence are derived within clade IVa from a typical Peziza-type and that the "opercular lens" is an autapomorphy. The excipulum structure in Kimbropezia
is, as also pointed out by Korf and Zhuang (1991),
typical for species in clade IVa; the spores are ellipsoid, smooth and eguttulate (TABLE I). Therefore,
Kimbropeziashould be included in Peziza. The operculate dehiscence has been lost completely several
times within Peziza (e.g., see Group VI) and the Pezizales (e.g., Trappe 1979, O'Donnell et al 1997, Percudani et al 1999). It should be noted that in ML
analysis by Norman and Egger (1999) the truffle Terfezia terfezioides(Matt.) Trappe is supported within,
or as a sister lineage to, group IV based on nSSU
rDNA.
An assemblage of nine Peziza species, clade IVb, is
a sister group to clade IVa. Clade IVb, like IVa has
mostly smooth or warted spores, but mostly have two,
small guttules. Common to this group are apothecia
with violaceous to vinaceous pigments in the hymenium (FIG. 11), either dark and intense or at least
with a pink, bluish or lilaceus tinge. Peziza subcitrina
deviates in its golden yellow ascomata (FIG. 12) and
eguttulate spores. Peziza lobulata has eguttulate
spores, while P proteana and P. petersii have spores
with low, short branched ridges and warts. Four of
the species are pyrophilous, P petersii, P proteana, P
subviolacea and P lobulata. Peziza exogelatinosaoccurs
on naked calcareous soil, with a high pH and a low
content of organic matter similar to the conditions
on burnt ground. Peziza ampelina, P subcitrina, P
emileia and P howsei are found on naked, clayey soil
in rich deciduous forests.
The two clades in group IV recall to some degree
Boudiers (1907) classification, based mainly on presence or absence of guttules. Peziza species in clade
IVa were placed in the genus Aleuria (P domiciliana
and P ammophila were not treated by Boudier),
whereas species in clade IVb were treated in both
Aleuria and Galactinia. Although these phylogenetic
results suggest that guttulation is a homoplasious
character that has been lost and gained several times,
a second interpretation is possible. The oil guttules
in clade IV are smaller and less prominent than those
in, e.g., Peziza species in group VI, and these two
types may not be homologous. Guttules may, however, always be present to a greater or lesser degree,
but the character may not be expressed in some species. If such is the case, lack of guttules is not phylogenetically informative. The "outlines" of two guttules have been observed (by KH in fresh material)
in the spores of several specimens of group IVa.
These "outlines" appear as aggregations of many
smaller oil drops, after pretreatment in 10% KOH,
staining with Sudan black in alcohol (a lipid stain)
and clearing with 50% alcohol.
Group V In these analyses, Sarcosphaeraand two
species of the hypogeous Hydnotryopsis, H. setchellii
and H. sp. form a highly supported clade within
group V (bootstrap 100%) (FIGS.22-24). The ascomata of Hydnotryopsishave hymenia located in labyrinthiform chambers or meandering veins, but these are
folded and compressed, making them solid (= stereothecia) (FIG. 14) (Trappe 1979). Hypogeous fruiting of Sarcosphaeramay occur (Trappe 1975a, 1979).
In such cases the apothecia occasionally become folded and form chambers, thus resembling ptychothecia, but the asci retain functional opercula. Sarcosphaera coronariafruits in spring to early summer and
its development is likely associated with selection for
reduction in water loss, as it is thought to be in truffles (e.g., Thiers 1984). The excipulum is thick and
tough, probably also correlated with microclimatic
aridity and initial development underground. It is
composed of a distinct outer layer (ca 750 [Lm) of
textura epidermoidea to textura prismatica on the
outermost region, a medullary layer (ca 450 iLm)of
large globose cells and a subhymenium of smaller
globose cells. A close relationship between Sarcosphaera and Hydnotryopsishas not previously been suggested. Trappe (1979) suggested that, "in its evolution to a solid gleba, Hydnotryopsisis related to Peziza
a step farther away than Mycoclelandia." Sarcosphaera
differs anatomically from Peziza in the outer excipular layer. Hydnotryopsis and Sarcosphaeraboth have
asci evenly amyloid over the entire length (type iii),
and ellipsoid, amorphous ornamented spores (in
SEM spores of Sarcosphaerahave an ornamentation
of low, rounded warts, covered by a thin amorphous
layer). Sarcosphaerais only found associated with mycorrhiza-forming trees, and is often locally abundant
where it occurs, year after year in the same spot, suggesting that it is ectomycorrhizal. Basal to these taxa
is an as yet unidentified species of Peziza sp. 3, collected on experimental plots treated with urea (supported by all MPT's and the MLT, but bootstrap
<50%). It shares features with species of Peziza in
group IV (a whitish, cup-shaped apothecium,
smooth, biguttulate spores, and hyaline or pale yellowish paraphyses, slightly enlarged and curved towards the apices), but has asci strongly amyloid over
the apex.
The Hydnotryopsis/Sarcosphaeraclade appears as a
sister group to common epigeous species of Peziza
sharing features with those in group VI. However, Peziza succosa, P. succosellaand P michelii possess lactiferous hyphae in the excipulum, which yield a yellow
juice when cut or bruised. Peziza species with apothecia that yield a colored juice are also present in
HANSEN ET AL: PHYLOGENY OF PEZIZACEAE
group VI (for example P. saniosa (FIG. 15) and P
badiofusca), but in these hyphae yield a bluish or watery juice. This character however, needs further
study, since lactiferous hyphae are likely to be present
in many species of Peziza. Thus, Le Gal (1937, 1953a)
described and illustrated lactiferous hyphae also in
species without or seemingly without staining flesh or
juice. Peziza succosa, P. succosella and P michelii have
spores with irregular, elongated warts and two large
guttules, asci strongly amyloid over the apex (type i),
and brownish colors in the hymenium (yellowish
brown, olivaceous yellow to greyish brown, or first
violet, then brownish). They occur on rich disturbed
soil in deciduous forests, always in association with
ectomycorrhizal fungi. Peziza aff. brunneoatra deviates by having dark brown apothecia, lacking a yellow
juice and in having finely warted spores packed with
numerous small guttules (sensu Dennis (1978) in
Norman and Egger (1999)).
Group VI. Although group VI has low bootstrap
support (< 50%), it is present in all equally parsimonious trees (FIG.23) and in the MLT (FIG.24). It
includes one weakly supported clade (bootstrap MPT
53%, MLT 51%) of 11 epigeous species of Peziza
(equivalent to Boudier's genus Galactinia pro parte
1907) and several truffles and truffle-like taxa. The
epigeous Peziza species are united morphologically
by mostly dark brownish, disc- to cupshaped apothecia, often with an olivaceous or purple tinge; highly
warted, ridged to reticulate spores, with two, rarely
one, large guttules; dark pigmented paraphyses; and
asci intensely and unrestrictedly amyloid over the
apex (type i). The hypogeous taxa have asci that are
evenly amyloid over the entire length, either weakly
or strongly (type iii), or are non-amyloid (type iv).
The relationship of several of the epigeous ellipsoid-spored species has been studied, and discussed
in relation to the globose-spored Plicaria (Pfister
1979b, Egger 1987, Moravec and Spooner 1988, Norman and Egger 1996, 1999) (see Background Information for previous hypotheses). Our analyses confirm a close relationship of Plicaria (FIG. 16) to the
more inclusive Peziza clade within group VI (FIG. 15).
Plicaria is supported as monophyletic in the MLT
(bootstrap 67%) (FIG.24), but the placement of the
smooth-spored P. endocarpoides (the only smoothspored species in group VI) is unresolved in the
MPTs (FIGS.22, 23). The three Plicaria species with
ornamented spores are supported as monophyletic
with a 65% bootstrap value. Globose, ellipsoid to fusiform-spored taxa group together in several lineages
within the Pezizaceae (in group I, III, VI) (TABLE I),
indicating that spore shape is not phylogenetically informative at these higher taxonomic levels. As shown
by Norman and Egger (1996, 1999) to maintain Pli-
983
caria as a monophyletic group will leave Peziza (or
the Peziza species in group VI) as a paraphyletic
group. Some important characters of Plicaria are
shared with some of the Peziza species, e.g., pale
brownish spores at maturity and paraphyses encrusted with amorphous matter. Such characters seem to
be synapomorphic to the Peziza species in group VI.
Pale brownish spores are found in P. atrovinosa, P.
vacinii and P alaskana. Paraphyses encrusted with
amorphous matter are present in P. alaskana, P badiofusca, Peziza sp. 1, P vacinii, P depressa and P
phyllogena. Other Peziza species reported with pale
brownish spores, e.g., P retidermaand P rifaii (Moravec and Spooner 1988), and paraphyses encrusted
with amorphous matter, e.g., P badiofuscoidesDonadini (Donadini 1979b), are likely members of group
VI, but have not been sequenced by us. In contrast
to the rest of the epigeous taxa in group VI, the asci
in Plicaria are weakly amyloid over their entire length
(type iiia).
The 11 epigeous Peziza species in the more inclusive clade of group VI constitute a homogeneous
group, both in macro- and microscopical features,
and levels of divergence in the nLSU rDNA sequences are low based on the branch lengths (FIGS.22, 24).
The clade of P badiofusca, Peziza sp. 1, P. badia, P
limnaea and P alaskana (FIGS. 22-24) represents a
complex of species centering around P. badia (see Le
Gal 1937, Donadini 1979b, Pfister 1979b), that possesses spores with ornamentations of warts elongated
into short ridges, which may anastomose to form an
incomplete to almost complete, irregular reticulum.
Less known unsampled species that may belong to
this clade are P. phlebospora (Le Gal) Donadini, P
badiofuscoides and P badioides Donadini. A closer
study of these species, mainly distinguished by small
differences in spore size and ornamentation and in
habitat, is needed. Peziza depressadiffers from members of this group by having spores ornamented with
distinct, regular, high warts, and a warm reddish
brown hymenium. Peziza saniosa, P badiofuscoides
and P badiofusca are distinct by possessing apothecia
that yield a bluish, blue opalescent or watery juice
when cut or bruised (FIG. 15).
Peziza phyllogena (syn. P. kallioi Harmaja, P badioconfusa Korf ss. auct.) is included in group VI, but
outside the core group of Peziza species (next to Plicaria) in the MLT (FIG. 24); its relationship to the
rest of the clade is not resolved in the strict consensus
tree (FIG.23). It shares many features with the group
VI Peziza species, and it has been confused with P
badia (Korf 1954, Elliott and Kaufert 1974). It is recognized by the bluish violet, pubescent to villose base
of the apothecium (seen easily on fresh material);
spores ornamented with delicate, irregular warts,
984
MYCOLOGIA
which become fused towards the poles, often forming apical caps; and its fruiting habit, on decaying
wood or woody debris in wet habitats (Wells and
Kempton 1967, Elliott and Kaufert 1974, Ginns 1980,
Harmaja 1986). Its spores do not possess two large
guttules as is typical for the group VI Peziza species,
but rather have many small guttules that aggregate
towards the poles.
The truffles and truffle-like taxa nested within
group VI show diverse ascomatal forms. Two hypogeous species of Peziza, P. whitei (FIG. 18) and P ellipsospora (FIG. 17), produce ptychothecia (TABLEI)
(Gilkey 1916, Trappe 1975a, 1979, Beaton and Weste
1982). Both species have hyaline, broadly ellipsoid
spores, which are minutely papillose in P ellipsospora
and reticulate in P whitei. Gilkey (1916) described P
ellipsosporain Hydnotrya Berk. & Broome, based on
the macro-morphology, but erected a new genus,
Hydnoplicata Gilkey for P. whitei (Gilkey 1954a). Peziza whitei has been placed in Hydnocystis Tul. and
Hydnotrya (see Trappe 1975a). Burdsall and Korf
(Burdsall 1968) and Trappe (1975a, 1979) transferred the two species to Peziza, based on the amyloid
reaction of the asci, spore ornamentation, ascocarp
structure, and supposedly operculate asci. Later
Trappe (pers comm) found that these species lack an
operculum and the spores are not forcibly discharged. Our molecular results support the placement of the two species among the core group of
epigeous Peziza species in group VI (FIGS. 22-24).
Peziza ellipsosporais a sister species to Cazia. Other
phylogenetic analyses (Norman and Egger 1999, Percudani et al 1999) have further shown Cazia to be
closely related to the truffle Terfeziaarenaria (Moris)
Trappe. Cazia and Terfeziaproduce stereothecia and
both have non-amyloid asci (type iv) (TABLEI). As in
the branches leading to Marcelleina (in group I) and
Terfezia terfezioides (possibly in group IV based on
Norman and Egger 1999), the amyloid reaction
might be lost along a branch leading to Cazia flexiascus and Terfezia arenaria. This suggests that the
amyloid reaction has been lost at least three times
within the Pezizaceae. Cazia flexiascus has ellipsoid,
hyaline, incompletely warty-reticulate spores, with
one guttule (Trappe 1989), much like the spores of
P. whitei and P ellipsospora.Two species of Tirmania,
T nivea and T pinoyi, also producing stereothecia
(Trappe 1971, Moreno et al 2000), are nested within
group VI. They group strongly together (bootstrap
MPT 99%, MLT 93%) as a sister group to most of
the epigeous and the hypogeous Peziza species and
Cazia (FIGS. 23-24). The similarity in excipulum
structure between Cazia, Terfeziaand Tirmania adds
support to the close relationship between these taxa
(TABLEI).
Ruhlandiella berolinensisproduces exothecia (FIG.
19) (Dissing and Korf 1980, Warcup and Talbot 1989
as Muciturbo, Galan and Moreno 1998). The excipulum is composed entirely of globose to angular
cells. Asci are evanescent at maturity. The paraphyses
in both R. berolinensisand P. whitei exceed the asci
and are embedded in a gelatinous sheath. Spores in
R. berolinensisare probably dispersed when the hymenium and upper part of the ascoma break down
to form a mucilaginous mass. Ruhlandiella berolinensis groups with P. ostracodermain all trees (FIGS.2224), but with low bootstrap support.
Few anamorphs have been reported for the group
VI taxa, but where reported they are referable to
Chromelosporium(Peziza ostracoderma,Plicaria endocarpoides, P. trachycarpa), Chromelosporium-likestates
(Ruhlandiella), or are unnamed states, producing hyaline to colored, 0-3 septate resting spores [P. saniosa, P phyllogena as Galactinia badioconfusa (Korf)
Svrcek] (Berthet 1964a, b, Paden 1972, Hennebert
1973, Hennebert and Korf 1975, Warcup and Talbot
1989). Most of the epigeous Peziza species occur on
bare, often disturbed soil, under ectomycorrhizaforming trees. Plicaria species and Peziza vacinii are
pyrophilous, while P ostracodermaalso occurs on other types of sterilized soil and P griseoroseaon very
degraded wood or soil mixed with wood.
Group VII. Two highly supported clades, VIIlaand
VIIb (bootstrap 96% to 100%) are united in group
VII, but with only weak support in the MLT and the
MPT (bootstrap <50%) (FIG. 22, 24). Clade VIIlaincludes three species of Peziza, P. polaripapulata, P. sp.
4 and P luteoloflavida. The apothecial colors of these
species are in the range of pale yellow, ochre-yellow,
warm yellow, brownish yellow to golden brown, dark
brown with an olivaceous tinge, or olive yellow to olive (FIG. 20) (Kristiansen and Schumacher 1993,
Hansen et al 1998). They are united by asci being
evenly amyloid over the entire length (type iii). The
spores contain two small guttules or are eguttulate.
Peziza sp. 4 has smooth spores (in SEM), whereas P
luteoloflavida has spores with a fine, irregular, undulating reticulum, and P. polaripapulata has spores
with isolated, rounded to elongated warts, becoming
more or less fused at the poles to a cap-like thickening. Based on the spore ornamentation P. polaripapulata has been placed with the "pseudoapiculate
Peziza species" in subgenus Phaeopezia (Sacc.) J. Moravec (Haffner 1995), along with P phyllogena and
the apiculate-spored P. apiculata. A close relationship
of these taxa is not supported in our analyses, which
places P. phyllogena in group VI and P apiculata either as a sister lineage to group II or III. However, to
test the monophyly of taxa with pseudoapiculate and
apiculate spores, more sampling is necessary. Peziza
HANSENETAL:PHYLOGENY
OF PEZIZACEAE
polaripapulata (FIG. 20) occurs on very degraded
hardwood, sawdust or soil in calcareous forests and
P. luteoloflavida on sand on river banks. Peziza sp. 4
was collected among mossess, on a forest road surfaced with cinders.
Clade VIIb consists of two collections of the strictly
subtropical or tropical genus, Iodowynnea. Our results support the segregation of Iodowynneafrom the
main groups of Peziza, but a close relationship to the
Peziza species in clade VIIla is possible. The asci in
Iodowynnea auriformis are evenly and strongly amyloid over the entire length (type iiib), and the apothecial colors have red tones. It fruits on soil or vegetable debris in forests with ectomyccorhizal trees
(Medel et al 1996).
Taxa not supportedin group I-VII. Peziza natrophila
and P. quelepidotia form a highly supported lineage
(bootstrap 100%, FIGS. 22-24), the placement of
which is unresolved. But, in our analyses this group
never falls within the main groups of Peziza. This observation is supported by the ascus amyloid reaction
in P natrophila, which is weak over the entire length
(type iiia). Peziza quelepidotia was placed in its own
genus Lepidotia, recognized by ellipsoid, eguttulate
spores and distinctly stipitate or obconical apothecia
with triangular, submembranaceous scales (Boudier
1885). On morphological
grounds Korf and
O'Donnell (Korf 1973c) did not consider Lepidotia
generically different from Peziza s.l. In contrast Norman and Egger (1999), on molecular phylogenetic
grounds, stated that P. quelepidotia is not a member
of Peziza, and should be maintained in Lepidotia. Although, in their analysis P quelepidotiaforms a clade
with P subisabellina (in Norman and Egger misnamed as Pachyella clypeata). In our MLT (FIG. 24)
the lineage of P natrophila and P quelepidotiais the
sister group to the clade of group II (including P
subisabellina) and group III. Peziza natrophila is mostly known from experimental plots treated with sodium- and potassium carbonate (Petersen 1970, Khan
1976), but one of our specimens, P natrophila (2)
(JHP 93.021), occurred on soil in an old peat bog.
This is similar to the substrate reported for P. quelepidotia, Sphagnum-pots and peat-moss agar ("Jiffy-7
Pellet infusion agar") (Korf 1973c, O'Donnell and
Beneke 1973). The apothecia of P natrophila are olivaceous brown, subsessile or stalked (2-10 mm
high), with an outer surface covered by short branching hyphae, much as described for P quelepidotiaby
Korf (1973c). Peziza natrophila has distinctly ornamented spores, while P. quelepidotiais illustrated with
less dense markings. Until further comparative studies have been carried out we cannot exclude the possibility that these species are con-specific.
985
Taxonomic conclusions.-The 14 genera sampled by
us, and currently accepted in the Pezizaceae (Eriksson 2000), are confirmed to be members of the family. The analyses, however, suggest that the Pezizaceae
should be emended to include the non-amyloid genus Marcelleina. The Ascobolaceae is confirmed to be
the sister group to the Pezizaceae, which supports the
general view that the amyloid reaction of the ascus is
phylogenetically informative in the Pezizales. Peziza
and most other genera of the Pezizaceae, as they are
currently delimited, cannot be maintained in a
monophyletic classification system. Peziza is composed of at least 6 major lineages, most of which include other genera of the Pezizaceae, and 4 species
of Peziza occur in separate lineages of yet uncertain
placement. Peziza should be disassembled into
groups that more closely reflect the phylogeny, and
several genera now recognized in the family require
emendation or change in status. Group IV includes
the type species of Peziza, P. vesiculosa, and should
serve as a core group for a future circumscription of
Peziza. The genera Kimbropeziaand Pfisterashould be
included in Peziza. A close relationship between Sarcosphaera and the truffle Hydnotryopsis is suggested
for the first time. Likewise, a close relationship between Scabropeziaand the truffle Amylascus is strongly indicated.
Overall, these LSU analyses imply that there has
been extensive homoplasy in some of the morphological characters that previously have been used to
delimit taxa. Hypogeous ascomata with concomitant
loss of active spore discharge have evolved independently several times, suggesting that their derivation
from apothecial Pezizaceae may involve relatively simple genetic changes. For example, stereothecia have
been derived within group III, V and VI. In addition,
ptychothecia and exothecia have evolved within
group VI. Derivation of ptychothecia and stereothecia from apothecia involves the evolution of enclosed
hymenia or asci, while the derivation of exothecia
involves the evolution of extremely convex hymenia.
Spore shape has been emphasized in the delimitation
of genera, but our results indicate that species with
divergent spore form may be closely related. Globosespored species occur in group I, III and VI, along
with ellipsoid- or fusiform-spored species. The amyloid reaction of the ascus appears to be a variable
character that has been lost in some lineages. On the
other hand, different types of ascus amyloid reactions
correspond to different lineages and seem to provide
clues to relationships within the Pezizaceae. Other
characters, such as apothecial colors, spore surface
relief, guttulation, excipulum structure and anamorph morphologies, seem to be phylogenetically informative, although considerable variation within
986
MYCOLOGIA
and between groups occur. For example, apothecial
colors are purplish in group I, in the range of yellowish, rose, reddish to purplish (brown) in group III,
paler yellowish-brown in group IVa, violaceous to vinaceous in group IVb, and dark brownish in group
VI. Smooth or finely verrucose spores, without or
with two small guttules, are mostly present in group
IV species, while highly ornamented spores with two
large guttules occur mostly in group VI species. Excipulum structure, although not clear-cut, seem to be
fairly uniform within most groups. Oedocephalum anamorphs are produced by taxa in group III and IV,
and by species of Iodophanus, while Chromelosporium
anamorphs are produced mainly by group VI taxa.
It is still premature to propose a phylogenetic classification of the Pezizaceae. For example, the monophyly of some of the genera, e.g., Boudiera and Scabropezia,has not been tested and six genera (TABLE
I) are not represented in this study. Most importantly,
an additional dataset is needed to provide a more
robust estimate of the phylogeny. Further studies of
morphological characters, especially of pigment composition and excipulum structure, and of anamorph
type and trophic status in the context of a robust
molecular phylogeny, that will facilitate refining hypotheses of homology, may be useful in understanding relationships within the Pezizaceae.
Note on P vesiculosa added after acceptance of the
manuscript.
In a detailed and large scale study now underway of
the group IVa Peziza species using ITS rDNA se-
quences it was discovered that the published voucher
specimen for P vesiculosa (ALTA 9066, GenBank
LSU: AF133170, SSU: AF133155, ITS: AF133182)
(Norman and Egger 1999) was misidentified. The
specimen (ALTA 9066) has been examined morphologically by K. Hansen and has been determined to
be Peziza fimeti. A comparison of the ITS1 sequence
of ALTA 9066 with the larger ITS data set confirms
this finding. In this paper ALTA 9066 has been renamed P. fimeti in FIGS. 22, 23 AND 24, and in TABLE
II. Unfortunately, this misidentification has resulted
in P vesiculosa, the type species of Peziza, being omitted from the study. However, we have now sequenced
the LSU rDNA (Dl and D2) from a collection of P.
vesiculosa (TL-6398, C) (GenBank AF378367), and
analyses of our LSU dataset including this sequence
suggest that group IVa does in fact include P vesiculosa (bootstrap 86%). The presence of a distinct
amyloid ascus ring zone in P. vesiculosa also supports
this placement. The placement of P vesiculosa within
group IVa is unresolved in the strict consensus tree
of the MPT's based on LSU, but ITS sequences suggest a possible relationship with P. ammophila.
ACKNOWLEDGEMENTS
We are grateful to David S. Hibbett and Henning Knudsen
for valuable discussions and encouragement throughout,
and for making helpful comments on the manuscript, and
all members of the Donoghue lab (Harvard Herbaria) for
advice and support. We wish to thank the curators of B, C,
CUP, FH, G, GOET, H, K, L, PC, PRM, S, TRTC, TUR,
TURA, for arranging loans of material, Betty Klug-Andersen, Henry Dissing, Steen A. Elborne, Jakob HeilmannClausen, Sven-Ake Hanson, Roy Kristiansen, Christian
Lange, Jens H. Petersen, Trond Schumacher, Anne Storgaard, James Trappe, Jukka Vauras, Nancy Weber and Wulfard Winterhoff for providing specimens, Keith Egger for
sharing data and results before publication, Niels Daugbjerg and Ole Seeberg for sharing knowledge and assisting in part of the phylogenetic analysis, Leif Bolding for
help with the graphics in FIGS.22-24, Steen A. Elborne,
David S. Hibbett, Roy Kristiansen, and Jens H. Petersen for
supplying photographs, J. H. Petersen for scanning the color photographs, and two anonymous reviewers for their insightful comments on the paper. We thank Michael Castellano, Daniel Luoma and the US Forest Service, Oregon
State University for permission to reproduce the photographs in FIGS.14 AND17-19, and Sara Landvik, Novozymes
A/S, for doing the PCR and sequencing of the LSU of P
vesiculosa added after acceptance of the paper. Funding was
provided by an NSF grant (DEB-9521944) to DHP and by
a grant from the Faculty of Science, University of Copenhagen to KH.
LITERATURE CITED
Arpin N. 1969. Les carot6noides des Discomycetes: essai chimiotaxinomique. Bull Mens Soc Linn Lyon 38(suppl.):
1-169.
Auerswald B. 1869. SarcosphaeraAwd., novum genus Discomycetum. Hedwigia 6:82-83.
Beaton G, Weste G. 1982. Australian hypogaean Ascomycetes. Trans Brit Mycol Soc 79:455-468.
Berthet P. 1964a. Essai biotaxinomique sur les Discomycetes
[Theses]. Lyon: Joanny Lorge. 157 p.
. 1964b. Formes conidiennes de divers Discomycetes.
Bull Soc Mycol France 80:125-149.
. 1970. Les ornementations sporales meconnues de
cinq especes de Discomycetes opercules. Extrait du bulletin mensuel de la Societe linn6enne de lyon 39:289292.
Boudier JLE. 1879. On the importance that should be attached to the dehiscence of asci in the classification of
the discomycetes. Grevillea 8:45-49.
. 1885. Nouvelle classification naturelle des Discomycetes charnus connus generalement sous le nom de
Pezizes. Bull Soc Mycol France 1:91-120.
. 1899. Note sur quelques champignons nouveaux
des environs de Paris. Bull Soc Mycol France 15:49-54.
1905-1910. Icones Mycologicae. Vols. 1-4. Paris.
362 p., 600 pl.
. 1907. Histoire et classification des Discomycetes
d'Europe. Paris: Klincksieck. 221 p.
HANSEN ET AL: PHYLOGENY
OF PEZIZACEAE
Brummelen J van. 1967. A world-monograph of the genera
Ascobolusand Saccobolus (Ascomycetes, Pezizales). Persoonia suppl. 1. 260 p.
. 1978. The operculate ascus and allied forms. Persoonia 10:113-128.
. 1998. Two bizarre ascus apices of Pezizales revealed
by their ultrastructure. Cryptogamie Bryol Lichen 19:
257-265
Bulliard JBF. 1791. Historie des champignons de la France.
1. Paris. 504 p.
Burdsall HH. 1968. A revision of the genus Hydnocystis
(Tuberales) and of the hypogeous species of Geopora
(Pezizales). Mycologia 60:496-525.
Chatin A. 1891. Contribution a l'histoire naturelle de la
truffe. Compt Rend Acad Sci. Paris 111:947-953.
Cooke MC. 1877. New British fungi. Grevillae 6:71-76.
Corda ACJ. 1854. Icones fungorum hucusque cognitorum.
In: ZobelJB, ed. Praha 6:1-86.
Curry KJ,KimbroughJW. 1983. Septal structures in apothecial tissues of the Pezizaceae (Pezizales, Ascomycetes).
Mycologia 75:781-794.
Dennis RWG. 1981. British Ascomycetes. 4th ed. Vaduz: Cramer. 585 p.
. 1983. Typification of Peziza (Ascomycetes: Pezizales). Kew. Bull. 37:643-652.
DilleniusJJ. 1719. Cathalogus plantarum circa Gissam sponte nascentium. Cum appendice. Francofurti ad Moenum. 480 p., 16 pl.
Dissing H. 2000. Pezizales. In: Hansen L, Knudsen H, eds.
Nordic macromycetes 1. Copenhagen: Nordsvamp. 309 p.
, Korf RP. 1980. Preliminary studies in the genera
Ruhlandiella, Sphaerosoma,and Sphaerozone(order Pezizales). Mycotaxon 12:287-306.
, Pfister DH. 1981. Scabropezia,a new genus of Pezizaceae (Pezizales). NordicJ Bot 1:102-108.
, Schumacher T. 1979. Preliminary studies in the genus Boudiera, taxonomy and ecology. Norw J Bot 26:
99-109.
Donadini JC. 1977. Le genre Peziza L. per Saint-Amans (I).
Bull Soc Linn Provence 30:37-92.
. 1978. Le genre Peziza L. per Saint-Amans (II). Les
Pezizes de Haute-Provence et de Dauphine-Savoie. Bull
Soc Linn Provence 31:9-36.
. 1979a. Le genre Peziza Linn6 per Saint-Amans (lere
Partie). Documents mycologiques 9:1-42.
. 1979b. Le genre Peziza Linn. per St. Amans (groupe
de Peziza badia). Documents mycologiques 10:48-58.
. 1979c. Un genre nouveau: Greletia nov. gen. (ex
Pulparia Karsten emend. Korf pro parte. Pezizales).
Bull Soc Mycol France 95:181-184.
. 1980a. Le genre Peziza. III. Sous-genre Galactinia.
Bull Soc Mycol France 96:239-246.
. 1980b. Le genre Peziza (Dill.) Linn6 per Saint
Amans sous-genre Pachyella (Boudier) nov. comb. Documents mycologiques 11:25-26.
. 1985. Le genre Peziza dans le sud-est de la France
complements (1) avec extension a l'Europe. Bull Soc
Linn Provence 35:153-166.
Dumortier BCJ. 1829. Analyse des familles des plantes, avec
987
l'indication des principaux genres qui s'y rattachent.
Tournay, Belgium: J. Casterman, aine. 104 p.
Eckblad F-E. 1968. The genera of the operculate Discomycetes. A re-evaluation of their taxonomy, phylogeny and
nomenclature. Nytt Mag Bot 15:1-191.
Egger KN. 1987. The taxonomic value of phenoloxidase
tests for separating Peziza and Plicaria (Pezizales). Mycotaxon 29:183-188.
Elliott ME, Kaufert M. 1974. Peziza badia and Peziza badioconfusa. Can J Bot 52:467-472.
Eriksson OE. 2000. Outline of the Ascomycota 2000. Myconet. http://www.umu.se/myconet/Myconet.html
, Hawksworth DL. 1995. Notes on ascomycete systematics-Nos 1885-2023. Syst Ascomycetum 14:41-77.
Felsenstein J. 1984. Distance methods for inferring phylogenies: a justification. Evolution 38:16-24.
Fischer E. 1938. Tuberineae. In: Engler A, Harms H. eds.
Nat. Pflanzenfam. 2nd. ed. 5b(8):1-42. Leipzig: Engelmann W.
Fries EM. 1822. Systema mycologicum. II. Lundae. 621 p.
Fuckel L. 1870. Symbolae mycologicae. Beitrage zur kenntnis der rheinischen pilze. Jb Nassau Ver Naturk 23-24:
1-459.
Galan R, Moreno G. 1998. Ruhlandiella berolinensis,an exotic species in Europe. Mycotaxon 68:265-271.
Gamundi IJ, Ranalli ME. 1964. Estudio sistematico y biol6gico de las Ascobolaceas de Argentina. I. Nova Hedwigia 7:517-533.
Gilkey HM. 1916. A revision of the Tuberales of California.
University of California publications in Botany 6:275356.
. 1954a. Taxonomic notes on Tuberales. Mycologia
46:783-793.
. 1954b. Tuberales. N Am Flora. II 1:1-36.
GinnsJ. 1980. Peziza badioconfusa.Fungi Canadensis 168. 2 p.
Graybeal A. 1998. Is it better to add taxa or characters to a
difficult phylogenetic problem? Syst Biol 47:9-17.
Hansen K, Sandal SK, Dissing H. 1998. New and rare species
of Pezizales from calcareous woodlands in Denmark.
Nordic J Bot 18:611-626.
, Pfister DH, Hibbett S. 1999. Phylogenetic relationships among species of Phillipsia inferred from molecular and morphological data. Mycologia 91:299-314.
Harmaja H. 1986. Studies on the Pezizales. Karstenia 26:4148.
Harrington FA, Pfister DH, Potter D, Donoghue MJ. 1999.
Phylogenetic studies within the Pezizales I. 18S rRNA
sequence data and classification. Mycologia 91:41-50.
Hawker L. 1954. British hypogeous fungi. Philos Trans R
Soc London 237:429-546.
Hawksworth DL, David JC. 1989. Family names. Index of
fungi supplement. Wallingford, UK: International Mycological Institute. 75 p.
, Kirk PM, Sutton BC, Pegler DN. 1995. Ainsworth
and Bisby's dictionary of the fungi. 8th ed. Wallingford,
UK: CAB International. 616 p.
Hennebert GL. 1973. Botrytis and Botrytis-likegenera. Persoonia 7:183-204.
, Korf RP. 1975. The peat mould, Chromelosporium
ollare, conidial state of Peziza ostracoderma,and its mis-
988
MYCOLOGIA
applied name, Botrytis crystallina, Botrytis spectabilis,
Ostracodermaepigaeum and Peziza atrovinosa. Mycologia 67:214-240.
Hennings P. 1903. Ruhlandiella berolinensisP. Henn. n. gen.
et n. sp., eine neue deutsche Rhizinacee. Hedwigia 42:
22-24.
Hirsch G. 1980. Beitrage zur Kenntnis der Gattung Boudiera
CKE. (Pezizales, Ascomycetes). I. Eine Boudiera-Kollektion aus der DDR und ihre Stellung zu den bisher bekannten Arten. Wiss. Z. Friedrich Schiller-Univ. Jena
Math.-nat. Reihe 29:649-655.
. 1984. Studies in the Pezizaceae. 1. Introduction. 2.
Peziza apiculata and its relatives. Mycotaxon 19:57-69.
. 1985. The genera Scabropeziaand Plicaria in the
German Democratic Republic. Agarica 6:241-258.
. 1992. Uber einige Peziza-arten mit r6tlichen Pigmenten. Boletus 16:3-9.
Hohmeyer H. 1986. Ein Schlfissel zu den europaischen Arten der Gattung Peziza L. Z Mykol 52:161-188.
Haffner J. 1995. Pseudoapiculate und apiculate Becherlinge-Emendation. (Rezente Ascomycetenfunde 16).
Rheinland-Pfalzisches Pilzjournal 5:4-31.
Karsten PA. 1869. Monographia Pezizarum fennicarum. Notiser ur Sallskapets pro Fauna et Flora Fennica F6rhandlingar 10:99-206.
Khan AZMNA. 1976. Peziza natrophila sp. nov. Trans Brit
Mycol Soc 67:540-543.
KimbroughJW. 1970. Current trends in the classification of
Discomycetes. Bot Rev 36:91-161.
. 1994. Septal ultrastructure and ascomycete systematics. In: Hawksworth DL, ed. Ascomycete systematics:
problems and perspectives in the nineties. New York:
Plenum Press. p 127-141.
, Curry KJ. 1985. Septal ultrastructure in the Ascobolaceae (Pezizales, Discomycetes). Mycologia 77:219229.
, Korf RP. 1967. A synopsis of the genera and species
of the tribe Theleboleae (= Pseudoascoboleae). Amer
J Bot 54:9-23.
, Wu C-G, Gibson JL. 1991. Ultrastructural evidence
for a phylogenetic linkage of the truffle genus Hydnobolites to the Pezizaceae (Pezizales, Ascomycetes). Bot
Gaz 152:408-420.
Kishino H, Hasegawa M. 1989. Evaluation of the maximum
likelihood estimate of the evolutionary tree topologies
from DNA sequence data, and the branching order in
Hominoidea. J Mol Evol 29:170-179.
Korf RP. 1954. Discomyceteae exsiccatae, Fasc. I. Mycologia
46:873-841.
. 1960. Nomenclatural notes. IV. The generic name
Plicaria. Mycologia 52:648-651.
. 1972. Synoptic key to the genera of the Pezizales.
Mycologia 64:937-994.
. 1973a. Sparassoid ascocarps in Pezizales and Tuberales. Rept Tottori Mycol Inst 10:389-403.
. 1973b. Discomycetes and Tuberales. In: Ainsworth
GC, Sparrow FK, Sussmann AS, eds. The fungi, an advanced treatise 4. New York: Academic Press. p 249319.
. 1973c. On Boudier's genus Lepidotia (Pezizaceae).
Persoonia 7:205-212.
, Zhuang W-Y. 1991. Kimbropeziaand Pfistera, two
new genera with bizarre ascus apices (Pezizales). Mycotaxon 40:269-279.
Kotlaba F, Pouzar Z. 1963. Two rare arenicolous fungi in
Czechoslovakia: Psathyrella ammophila (Dur. et Lev.) P.
D. Orton and Sarcosphaeraammophila (Dur. et Mont.)
Moesz. Ceska Mykol 17:71-76.
Kristiansen R, Schumacher T. 1993. Nye operkulate begersopper i Norges flora. Blyttia 51:131-140.
Krug JC, Jeng RS. 1984. Hapsidomyces,a new genus of the
Pezizaceae with ornamented ascospores. Mycologia 76:
748-751.
Landvik S. 1996. Neolecta, a fruit-body-producing genus of
the basal ascomycetes, as shown by SSU and LSU rDNA
sequences. Mycol Res 100:199-202.
, Egger KN, Schumacher T. 1997. Towards a subordinal classification of the Pezizales (Ascomycota): phylogenetic analysis of SSU rDNA sequences. NordicJ Bot
17:403-418.
, Eriksson OE, Gargas A, Gustafsson P. 1993. Relationship of the genus Neolecta (Neolectales ordo nov.,
Ascomycotina) inferred from 18S rDNA sequences.
Syst Ascomycetum 11:107-118.
, Kristiansen R, Schumacher T. 1998. Phylogenetic
and structural studies in the Thelebolaceae (Ascomycota). Mycoscience 39:49-56.
Le Gal M. 1937. Florule mycologique des Bois de la Grange
et de l'Etoile. Discomycetes. Rev Mycol 2:197-222.
. 1941. Les Aleuria et les Galactinia. Paris. Rev Mycol
Suppl 6:56-82.
. 1947(reprint 1970). Recherches sur les ornamentations sporales des discomycetes opercules. Bibliotheca mycologica 28:73-297.
1953a. Les Discomycetes de Madagascar. Prodr
Flore Mycol Madagascar 4:1-465.
. 1953b. Les Discomycetes de l'herbier Crouan. Rev
Mycol 18:73-132.
. 1960. Flore iconographique des champignons du
Congo. 9 fasc: Discomycetes. Bruxelles. p 167-183, pl
29-31.
. 1962. Combinaisons nouvelles concernant les
genres Galactinia (Cooke) Boud. emend. Le Gal, Scutellinia (Cooke) Lamb. emend. Le Gal et Sarcosoma
Casp. Bull Soc Mycol France 78:204-216.
Linnaeus C. 1753. Species plantarum. Holmiae. 1200 p.
Maas Geesteranus RA. 1967. De Fungi van Nederland II.
Pezizales-deel I. Wetenschap Mededel Konin Nederl
Natuurhist Veren 69:1-84.
Maddison DR, Ruvolo M, Swofford DL. 1992. Geographic
origins of human mitochondrial DNA: phylogenetic evidence from control region sequences. Syst Biol 41:
111-124.
Maddison WP. 1993. Missing data versus missing characters
in phylogenetic analysis. Syst Biol 42:576-581.
, Maddison DR. 1992. MacClade: analysis of phylogeny and character evolution. Version 3. Sinauer Associates, Sunderland, Massachusetts.
Maia LC, Yano AM, Kimbrough JW. 1996. Species of Asco-
HANSEN ET AL: PHYLOGENY
OF PEZIZACEAE
mycota forming ectomycorrhizae. Mycotaxon 57:371390.
Medel R, Guzman G, Chac6n S, Korf RP. 1996. Iodowynnea,
a new genus of the Pezizales known from Africa and
tropical America. Mycotaxon 59:127-135.
Moncalvo J-M, Lutzoni FM, Rehner SA, Johnson J, Vilgalys
R. 2000. Phylogenetic relationships of agaric fungi
based on nuclear large subunit ribosomal DNA sequences. Syst Biol 49:278-305.
Moravec J. 1985. A taxonomic revision of species related to
Peziza apiculata. Agarica 6:56-66.
. 1987. A taxonomic revision of the genus Marcelleina. Mycotaxon 30:473-499.
. 1994. Some new taxa and combinations in the Pezizales. Czech Mycol 47:261-269.
, Spooner BM. 1988. Peziza vacinii (Pezizales), with
notes on taxonomy of related brown-spored species.
Trans Brit Mycol Soc 90:43-48.
Moreno G, Diez J, Manj6n JL. 2000. Picoa lefebvreiand Tirmania nivea, two rare hypogeous fungi from Spain. Mycol Res 104:378-381.
Moser H. 1963. Ascomyceten. In: Gams IH, ed. Kleine Kryptogamenflora 2a. Innsbruck. 147 p.
Nei M. 1987. Molecular evolutionary genetics. New York:
Columbia University press. 512 p.
Norman JE, Egger KN. 1996. Phylogeny of the genus Plicaria and its relationship to Peziza inferred from ribosomal DNA sequence analysis. Mycologia 88:986-995.
. 1999. Molecular phylogenetic analysis of Pe,
ziza and related genera. Mycologia 91:820-829.
Nylander W. 1868. Observationes circa Pezizas Fennicae.
Notiser ur Sallsk pro fauna et flora Fennica Forh 9:197.
O'Donnell K, Beneke S. 1973. Apothecial formation by Peziza quelepidotiain pure culture. Mycologia 65:913-915.
, Cigelnik E, Weber NS, Trappe JM. 1997. Phylogenetic relationships among ascomycetous truffles and
the true and false morels inferred from 18S and 28S
ribosomal DNA sequence analysis. Mycologia 89:48-65.
Olmstead RG, Bremer B, Scott KM, Palmer JD. 1993. A molecular systematic analysis of the Asteridae sensu lato
based on rbcL sequences. Ann Missouri Bot Gard 80:
700-722.
Olsen GJ, Matsuda H, Hagstrom R, Overbeek R. 1994.
fastDNAml: a tool for construction of phylogenetic
trees of DNA sequences using maximum likelihood.
CABIOS 10:41-48.
Paden JW. 1972. Imperfect states and the taxonomy of the
Pezizales. Persoonia 6:405-414.
. 1973. The conidial state of Peziza ammophila. Can
J Bot 51:2251-2252.
Percudani R, Trevisi A, Zambonelli A, Ottonello S. 1999.
Molecular phylogeny of truffles (Pezizales: Terfeziaceae, Tuberaceae) derived from nuclear rDNA sequence analysis. Mol Phylogenet Evol 13:169-180.
Persoon CH. 1801. Synopsis Methodica Fungorum. Gottingae. 706 p.
Petersen PM. 1967. Studies on ecology of some species of
Pezizales. Bot Tidsskr 62:312-322.
989
. 1970. Changes of the fungus flora after treatment
with various chemicals. Bot Tidssk 65:264-280.
. 1985. The ecology of Danish soil inhabiting Pezizales with emphasis on edaphic conditions. Opera Bot
77:1-38.
Pfister DH. 1973. The psilopezioid fungi. IV. The genus
Pachyella (Pezizales). Can J Bot 51:2009-2023.
. 1974. Notes on Caribbean Discomycetes. V. A preliminary annotated checklist of the Caribbean Pezizales. Jour Agric Univ Puerto Rico 58:358-378.
. 1979a. Type studies in the genus Peziza V. Species
described by Rehm. Mycotaxon 8:187-192.
. 1979b. Type studies in the genus Peziza VIII-X.
Species described by G. Massee, E. K. Cash and F. J.
Seaver. Mycotaxon 9:501-504.
. 1991. A redescription of Peziza bananicola and comments on some similar tropical species. Mycotaxon 41:
505-507.
Platt JL, Spatafora JW. 2000. Evolutionary relationships of
nonsexual lichenized fungi: molecular phylogenetic hypotheses for the genera Siphula and Thamnolia from
SSU and LSU rDNA. Mycologia 92:475-487.
Pouzar Z. 1972. Sarcosphaeracrassa (Santi ex Steud.) Pouz.,
the correct name for Sarcosphaeracoronaria (Jacq. ex
M. C. Cooke) J. Schroet. (Pezizaceae). Ceska Mykol 26:
32-36.
Rehm H. 1894. Ascomyceten: Hysteriaceen und Discomyceten. In: Rabenhorst GL, ed. Kryptogamen-Flora von
Deutschland, Oesterreich und der Schweiz, I-III. Leipzig: Eduard Kummer. p 913-1040.
Rifai MA. 1968. The Australasian Pezizales in the herbarium
of the Royal Botanic Gardens Kew.Verh Kon Ned Akad
Wetensch, Afd Natuurk, Tweede Sect 57:1-295.
Romagnesi H. 1978. Les especes du genre Peziza St-Am. (=
Aleuria ss. Boud. et Galactinia Cke ss. Boud.). Bull Trimestriel F6d Mycol Dauphin6-Savoie 18:19-23.
Samuelson DA. 1978. Asci of the Pezizales. I. The apical
apparatus of iodine-positive species. Can J Bot 56:18601875.
Schumacher T, Jenssen KM. 1992. Discomycetes from the
Dovre mountains, Central South Norway. In: Gulden
G, Jenssen KM, Stordal T. eds. Arctic and Alpine Fungi
4. Oslo: Soppkonsulenten A/S. 66 p.
Seaver FJ. 1928. The North American cup-fungi (operculates). New York: Hafner Publishing Co, Inc. 377 p.
Svrcek M. 1969. Neue Gattungen operculater Discomyceten. CeskaiMykol 23:90-96.
. 1970. Uber einige arten der Discomycetengattung
Peziza [Dill.] L. ex St-Amans. Ceska Mykol 24:57-76.
Swofford DL. 1998. PAUP*. Phylogenetic Analysis Using
Parsimony (* and Other Methods). Version 4. Sinauer
Associates, Sunderland, Massachusetts.
Thiers HD. 1984. The secotioid syndrome. Mycologia 76:1-8.
Trappe JM. 1971. A synopsis of the Carbomycetaceae and
Terfeziaceae (Tuberales). Trans Brit Mycol Soc 57:8592.
. 1975a. Generic synonyms in the Tuberales. Mycotaxon 2:109-122.
. 1975b. The genus Amylascus (Tuberales). Trans
Brit Mycol Soc 65:496-499.
990
MYCOLOGIA
. 1979. The orders, families, and genera of hypogeous Ascomycotina (truffles and their relatives). Mycotaxon 9:297-340.
. 1989. Cazia flexiascus gen. et sp. nov., a hypogeous
fungus in the Helvellaceae. Mem New York Bot Gard
49:336-338.
, Beaton G. 1984. Mycoclelandia nom. nov. (hypogeous Ascomycotina), a replacement for the pre-empted
generic name Clelandia. Trans Brit Mycol Soc 83:535536.
, Maser C. 1977. Ectomycorrhizal fungi: interactions
of their research by mushrooms and truffles with beast
and trees. In: Walters T, ed. Mushrooms and man, an
interdisciplinary approach to mycology. Albany,
Oregon: Linn-Benton Community College. p 165-179.
Trimbach J. 1999. Novitates. Documents Mycologiques
29(113):8.
Tulasne L-R, Tulasne C. 1843. Champignons hypoges de la
famille des Lycoperdacees, observes dans les environs
de Paris et les departemens de la Vienne et d'Indre-etLoire. Annales des Sciences Naturelles Serie 2, 19:373381.
. 1844. Fungi nonnulli hypogaei, novi v. mi,
nus cogniti. Giornale Botanico Italiano parte 1 (2):5564.
. 1851. Fungi hypogaei. Paris: Friedrich
,
Klincksieck. 222 p.
Wakefield EM. 1939. Nomina generica conservada, contributions from the Nomenclature Committee of the British Mycological Society. II. Trans Brit Mycol Soc 23:
281-292.
Warcup JH. 1990. Occurrence of ectomycorrhizal and saprophytic discomycetes after a wild fire in a eucalypt forest. Mycol Res 94:1065-1069.
, Talbot PHB. 1989. Muciturbo: a new genus of hypogeous ectomycorrhizal Ascomycetes. Mycol Res 92:
95-100.
Weber NS, TrappeJM, Denison WC. 1997. Studies on Western American Pezizales. Collecting and describing ascomata-macroscopic features. Mycotaxon 61:153-176.
Webster J, Rifai MA, Samy El-Abyad S. 1964. Culture observations on some discomycetes from burnt ground.
Trans Brit Mycol Soc 47:445-454.
Wells WL, Kempton PE. 1967. Studies on the fleshy fungi
of Alaska. I. Lloydia 30:258-268.
Wiens JJ. 1998. Does adding characters with missing data
increase or decrease phylogenetic accuracy? Syst Biol
47:625-640.
Yao YJ,Spooner BM, Hawksworth DL. 1995. Author citation
of the generic name Peziza (Pezizales, Pezizaceae). Syst
Ascomycetum 14:17-24.
Zhuang W-Y, Korf RP. 1986. A monograph of the genus
Aleurina Massee (= Jafneadelphus Rifai). Mycotaxon
26:361-400.