Content uploaded by Gonzalo Platas
Author content
All content in this area was uploaded by Gonzalo Platas
Content may be subject to copyright.
249
Mycologia,
96(2), 2004, pp. 249–259.
q2004 by The Mycological Society of America, Lawrence, KS 66044-8897
Molecular phylogenetic studies on the Diatrypaceae based on
rDNA-ITS sequences
Francisco Javier Acero
Vicente Gonza´lez
Javier Sa´nchez-Ballesteros
Vı´ctor Rubio
Departamento de Biotecnologı´a Microbiana, Centro
Nacional de Biotecnologı´a (CNB-CSIC), Campus
Cantoblanco, Universidad Auto´noma de Madrid,
Madrid 28049, Spain
Julia Checa
Departamento de Biologı´a Vegetal, Universidad de
Alcala´ de Henares, Madrid 28871, Spain
Gerald F. Bills
Oscar Salazar
Gonzalo Platas
Fernando Pela´ez
1
Centro de Investigacio´n Ba´sica, Merck Sharp and
Dohme de Espan˜a S. A., Josefa Valca´ rcel 38, Madrid
28027, Spain
Abstract:
The order Diatrypales (Ascomycota) con-
tains one single family, the Diatrypaceae. To obtain
insight in the phylogenetic relationships within this
family, the complete sequences of the ITS region
(ITS1, 5.8S rRNA gene and ITS2) of 53 isolates from
the five main genera in the family (
Diatrype, Diatr y-
pella, Cryptosphaeria, Eutypa
and
Eutypella
) were de-
termined and aligned for phylogenetic reconstruc-
tion. Sequence analysis revealed the presence of tan-
dem repeated motifs 11 nucleotides-long, placed in
homologous positions along the ITS1 region. Parsi-
mony analysis established the existence of nine
monophyletic groups and one branch with a single
isolate of
Eutypella quaternata.
The phylogenetic re-
lationships established by parsimony analysis did not
correlate well with classical taxonomic schemes.
None of the five genera included in this study was
found to be monophyletic. The genera
Diatrype, Eu-
typa
and
Cryptosphaeria
each were divided into two
groups. Isolates of
Diatrype flavovirens
appeared in a
clade separated from the one that grouped
Diatrype
disciformis
and the rest of
Diatrype
species. The
Eu-
typa
strains appeared distributed into two clades, one
grouping
Eutypa lata
and related species (
Eutypa ar-
meniacae, Eutypa laevata, Eutypa petrakii
), and anoth-
Accepted for publication September 22, 2003.
1
Corresponding author. E-mail: fernandoppelaez@merck.com
er with the remaining species of the genus.
Eutypella
(excluding
Eutypella quaternaria
) appeared as an un-
stable monophyletic group, which was lost when the
sequence alignment was subjected to neighbor-join-
ing analysis. The genus
Diatrypella
was not associated
with any monophyletic group, suggesting that the
multisporate asci character has appeared several
times during the evolution of the group. Overall, our
study suggests the need to revise many of the con-
cepts usually applied to the classification of members
of the family.
Key words: Creosphaeria, Cryptosphaeria, Diatry-
paceae, Diatrype, Diatrypella, Eutypa, Eutypella,
ITS se-
quencing, phylogeny, rDNA sequencing, tandem re-
peat sequences
INTRODUCTION
The Diatrypaceae is considered the only family of the
Diatrypales (Ascomycotina) and currently includes
nine accepted genera (Kirk et al 2001). Members of
this family are common worldwide, typically occur-
ring on a broad range of dead or declining woody
angiosperms. Host specificity is variable within the
group, with some species apparently being associated
with one plant genus. For instance,
Diatrypella betu-
lina
(Peck) Sacc. is known only from
Betula,
whereas
others, such as
Diatrype flavovirens
(Pers. : Fr.) Fr.,
have a broad host range. Although some members of
the Diatrypales are considered parasitic, most are ac-
cepted to be saprobic. Species like
Eutypella parasitica
Davidson & Lorenz, and especially
Eutypa lata
(Pers. :
Fr.) Tul. & C. Tul., are known to cause severe dis-
eases on economically important plants (Carter et al
1983).
The selection of morphological traits used to dis-
criminate between genera and species within the Dia-
trypaceae has varied over time. Earlier systematic ar-
rangements of the family (Fries 1823) were based on
stromatal features, and most of the taxa included un-
der the modern concept of the group were recog-
nized primarily as different sections of the genus
Sphaeria
Haller. The heterogeneity of the group was
suggested first by Tulasne and Tulasne (1863) and
Currey (1858). An increasing number of diagnostic
characters were added subsequently to delimitate
taxa within the Diatrypaceae. Features such as mor-
250 M
YCOLOGIA
T
ABLE
I. Isolates used in this study
Code* Species Strain no. Host plant Origin Collector
GeneBank
accession
code
C1C
Cryptosphaeria eunomia
var.
eunomia
(Fr : Fr)
Fuckel.
CBS 216.87
Fraxinus excelsior
Switzerland F. Rappaz AJ302417
C2C
Cryptosphaeria lignyota
(Fr : Fr) Auersw. CBS 273.87
Populus tremula
Switzerland F. Rappaz AJ302418
C3A
Cryptosphaeria pullmanensis
Glawe.
ATCC 52655
Populus trichocarpa
Washington, USA D.A. Glawe AJ302419
C4C***
Cryptosphaeria subcutanea
(Wahl. : Fr.) F. Rap-
paz.
CBS 240.87
Salix borealis
Norway F. Rappaz AJ302420
C5C***
Cryptosphaeria eunomia
(Fr : Fr) Fuckel. var.
fraxini
(Richon) F. Rappaz.
CBS 223.87
Fraxinus excelsior
Switzerland F. Rappaz AJ302421
D6C
Diatrype bullata
(Hoffm. : Fr.) Fr. CBS 215.87
Salix
sp. Switzerland F. Rappaz AJ302422
D7M
Diatrype disciformis
(Hoffm. : Fr.) Fr. GB 5815
Fagus grandifolia
New Jersey, USA G.F. Bills AJ302423
D8M
Diatrype disciformis
(Hoffm. : Fr.) Fr. F-091,971
Fagus sylvatica
Segovia, Spain J. Checa AJ302424
D9M***
Diatrype disciformis
(Hoffm. : Fr.) Fr. F-091,972
Fagus sylvatica
Segovia, Spain J. Checa AJ302425
D10M
Diatrype flavovirens
(Pers. : Fr.) Fr. F-091,973
Cytisus purgans
Madrid, Spain J. Checa AJ302426
D11M
Diatrype flavovirens
(Pers. : Fr.) Fr. F-091,975
Fagus sylvatica
Segovia, Spain J. Checa AJ302427
D12M***
Diatrype flavovirens
(Pers. : Fr.) Fr. F-091,976
Cytisus purgans
Madrid, Spain J. Checa AJ302428
D13M
Diatrype flavovirens
(Pers. : Fr.) Fr. F-093,581
Castanea sativa
Huelva, Spain J. Checa AJ302429
D14M
Diatrype flavovirens
(Pers. : Fr.) Fr. F-093,582
Eucalyptus globulus
Huelva, Spain J. Checa AJ302430
D15C
Diatrype macowaniana
Thu¨m CBS 214.87
Chaenomeles japonica
Australia F. Rappaz AJ302431
D16C
Diatrype polycocca
Fuckel. CBS 213.87
Acer opalus
Switzerland F. Rappaz AJ302432
D17C
Diatrype spilomea
H. Syd. CBS 212.87
Acer campestre
Switzerland F. Rappaz AJ302433
D18M
Diatrype stigma
(Hoffm. : Fr.) Fr. GB 5814
Fagus grandifolia
New Jersey, USA G.F. Bills AJ302434
D19M
Diatrype stigma
(Hoffm. : Fr.) Fr. F-091,970
Fagus sylvatica
Segovia, Spain J. Checa AJ302435
D20C
Diatrype undulata
(Pers. : Fr.) Fr. CBS 271.87
Betula
sp. Switzerland F. Rappaz AJ302436
D21C
Diatrype disciformis
(Hoffm. : Fr.) Fr. CBS 205.87
Fagus sylvatica
Switzerland F. Rappaz AJ302437
D22C***
Diatrype stigma
(Hoffm. : Fr.) Fr. CBS 211.87
Quercus
sp. Irish Republic F. Rappaz AJ302438
D23M
Diatrype stigma
(Hoffm. : Fr.) Fr. F-101,130
Quercus agrifolia
Ensenada, Mexico J. Checa AJ302439
DL26C***
Diatrypella favacea
(Fr.) Cesati & De Not. CBS 527.82
Betula
sp. (dead limb) Netherlands H.A. van der Aa AJ302440
DL27A
Diatrypella frostii
Peck ATCC 52484
Acer
sp. Unknown D.A. Glawe AJ302441
DL28A***
Diatrypella prominens
(Howe) Ell. & Everh. ATCC 64182
Plantanus
sp. Illinois, USA D.A. Glawe AJ302442
DL29C
Diatrypella pulvinata
Nits. CBS 181.97
Quercus robur
Netherlands H.A. van der Aa AJ302443
DL30M
Diatrypella quercina
(Pers. ex.Fr.) De Not.
ex.Cke.
F-091,966
Quercus faginea
Guadalajara, Spain J. Checa AJ302444
251A
CERO ET AL
:M
OLECULAR PHYLOGENY OF THE
D
IATRYPACEAE
T
ABLE
I. Continued
Code* Species Strain no. Host plant Origin Collector
GeneBank
accession
code
E36A
Eutypa armeniacae
Hansford & Carter. ATCC 28120
Prunus avium
Australia M.V. Carter AJ302445
E37C
Eutypa armeniacae
Hansford & Carter. CBS 622.84
Vitis vinifera
Italy H.A. van der Aa AJ302446
E38M
Eutypa consobrina
(Mont.) F. Rappaz. F-091,961
Arundo donax
Almerı´a, Spain J. Checa AJ302447
E39C
Eutypa crustata
(Fr : Fr) Sacc. CBS 210.87
Ulmus
sp. France F. Rappaz AJ302448
E40C***
Eutypa laevata
(Nitschke) Sacc. CBS 291.87
Salix
sp. Switzerland F. Rappaz AJ302449
E41T
Eutypa lata
(Pers. : Fr.) Tul. & C. Tul. CECT 20118
Tilia
sp. Switzerland F. Rappaz AJ302450
E42C
Eutypa lata
(Pers.) Tul. & C. Tul. var.
aceris
F.
Rappaz
CBS 217.87
Acer campestre
France F. Rappaz AJ302451
E43M***
Eutypa lata
(Pers. : Fr.) Tul. & C. Tul. F-093,584
Cistus ladanifer
Huelva, Spain J. Checa AJ302452
E44C
Eutypa leptoplaca
(Mont.) F. Rappaz. CBS 286.87
Arundo donax
France F. Rappaz AJ302453
E45C***
Eutypa maura
(Fr : Fr) Fuckel. CBS 219.87
Acer pseudoplatanus
Switzerland F. Rappaz AJ302454
E46C
Eutypa petrakii
var.
petrakii
F. Rappaz. CBS 244.87
Prunus spinosa
Switzerland F. Rappaz AJ302455
E47C
Eutypa petrakii
var.
petrakii
F. Rappaz. CBS 245.87
Salix borealis
Norway F. Rappaz AJ302456
D48C
Diatrype flavovirens
(Pers. : Fr.) Fr. CBS 272.87
Quercus ilex
France F. Rappaz AJ302457
E49C
Eutypa astroidea
(Fr : Fr) F. Rappaz. CBS 292.87
Fraxinus excelsior
Switzerland F. Rappaz AJ302458
E50C
Eutypa lata
(Pers. : Fr.) Tul. & C. Tul. CBS 209.87
Lonicera xylosteum
Switzerland F. Rappaz AJ302459
EL51C
Eutypella caricae
(De Not.) Berl. CBS 274.87
Ficus carica
France F. Rappaz AJ302460
EL52M***
Eutypella cerviculata
(Fr : Fr) Sacc. F-092,374
Alnus glutinosa
Guadalajara, Spain J. Checa AJ302461
EL53M***
Eutypella kochiana
Rehm. F-092,373
Atriplex halimus
Almerı´a, Spain J. Checa AJ302462
EL54C
Eutypella leprosa
(Pers. ex.Fr. : Fr.) Berl. CBS 276.87
Tilia
sp. Switzerland F. Rappaz AJ302463
EL55C
Eutypella prunastri
(Pers. : Fr.) Sacc. CBS 277.87
Prunus avium
Switzerland F. Rappaz AJ302464
EL56C
Eutypella scoparia
(Schwein. : Fr.) Ellis & Ev-
erh.
CBS 242.87
Robinia pseudacacia
France F. Rappaz AJ302465
EL57A
Eutypella vitis
(Schwein. : Fr.) Ellis & Everh. ATCC 64171
Vitus labrusca
Illinois, USA D.A. Glawe AJ302466
EL58C
Eutypella alsophila
(Mont.) Berl. CBS 250.87
Arthrocnemum fruticosum
France F. Rappaz AJ302467
EL59C
Eutypella cerviculata
(Fr : Fr) Sacc. CBS 221.87
Alnus glutinosa
Switzerland F. Rappaz AJ302468
EL60C
Eutypella quaternata
(Pers. : Fr.) F. Rappaz. CBS 278.87
Fagus sulvatica
Switzerland F. Rappaz AJ302469
Cr90M***
Creosphaeria sassafras
(Schweinitz) Ju, Martı´n,
& Rogers
GB 4588
Lindera benzoin
New Jersey, USA G.F. Bills AJ390424
Cr91M
Creosphaeria sassafras
(Schweinitz) Ju, Martı´n,
& Rogers
GB 4591
Lindera benzoin
New Jersey, USA G.F. Bills AJ390425
* First letter in the strain code refers to the genus, last letter to the source of the isolate: A 5ATCC; C 5CBS; T 5CECT and M 5Merck, Sharp & Dohme).
** Originally labeled in CBS catalog as
Eutypa flavovirens,
considered to be a synonym of
Diatrype flavovirens
(Rappaz 1987a).
*** Strains for which the D1–D2 region of the 28S rRNA was sequenced.
252 M
YCOLOGIA
phology and disposition of perithecia (Nitschke
1867), type of anamorph (Winter 1887) or asci and
ascospore morphology (Wehmeyer 1926) were incor-
porated into the descriptions of these organisms.
However, stromatal configuration remains an impor-
tant diagnostic feature to distinguish among the gen-
era within the family (Glawe and Rogers 1984). Thus,
valsoid configuration, with perithecia converging at
the same point in a poorly developed stroma made
up of mixed tissue or fungal hyphae only, usually is
ascribed to the genus
Eutypella
(Nitschke) Sacc.
Eu-
typa
Tul. & C. Tul. and
Cryptosphaeria
Ces. & De Not.
show eutypoid stromata, characterized by perithecia
separately reaching the surface of a flat stroma con-
sisting of mixed tissues from the host and the fungus.
These two genera are distinguished by the degree of
immersion of the stroma, which is cortical in
Cryp-
tosphaeria,
whereas in
Eutypa
it develops in the wood.
Finally, in
Diatrype
Fr. and
Diatrypella
(Ces. & De
Not.) De Not. the stroma is diatrypoid, consisting of
perithecia with short necks that separately reach the
surface of a well-developed stroma made up mostly
of fungal tissues. These two genera are differentiated
by the number of ascospores per ascus; eight in
Dia-
trype
and more than eight in
Diatrypella.
Unlike other groups of Ascomycetes, anamorph
morphology is almost useless when differentiating
taxa in the Diatrypaceae, either at the genus or at
the species level, because the conidial states in the
Diatrypaceae are indistinguishable relatively (Glawe
and Rogers 1984, Rappaz 1987a). Three form-genera
have been applied to diatrypaceous anamorphs, i.e.,
Cytosporina
Sacc., for fungi with enclosed (pycnidial)
conidiomata and filiform conidia;
Libertella
Desm.,
for those with unenclosed (acervular) conidiomata
and filiform conidia and
Naemospora
Sacc., for fungi
with unenclosed conidiomata and allantoid conidia
(Glawe and Rogers 1984). However, there has been
a reluctance to assign names to the anamorphs found
in culture (e.g., Glawe and Rogers 1984, Rappaz
1987a) because of the unclear limits among those
form-genera and because many species produce an-
amorphs of different types. For instance,
Eutypella
parasitica
produces both pycnidia and acervuli on
both natural substrata and agar media (Glawe 1983).
Likewise, conidial ontogenesis varies highly in the
group and different types of conidiogenesis (e.g.,
sympodial and percurrent) have been reported in
the same strain (Glawe and Rogers 1982a, b). Finally,
conidial morphology relatively is indistinct, ranging
from allantoid to cylindrical or filiform and from
nearly straight to strongly curved.
In this study, the phylogenetic relationships among
53 isolates of the Diatrypaceae were explored based
on the comparison of the sequences of the internal-
transcribed spacer regions ITS1 and ITS2 (including
the 5.8S rRNA gene). The selected isolates repre-
sented 35 species from five of the nine accepted gen-
era (Kirk et al 2001) of the Diatrypaceae:
Diatrype,
Eutypa, Eutypella, Diatrypella
and
Cryptosphaeria.
Echinomyces
Rappaz,
Fassia
Dennis,
Leptoperidia
Rap-
paz and
Cryptovalsa
Ces. and De Not. ex Fuckel are
not considered here. This was the first time that this
fungal group was subjected to a molecular phyloge-
netic analysis. The results were compared with mor-
phology-based classification schemes, with the objec-
tive of evaluating the phylogenetic significance of
characters such as stromatal morphology, ascospore
number, anamorph and host. In addition, the phy-
logenetic relationships between
Creosphaeria,
a mem-
ber of the Xylariaceae with a
Libertella
-like anamorph
(Bills and Pela´ez 1996), and members of the Diatry-
paceae were assessed.
MATERIALS AND METHODS
Fungal isolates and culture conditions.
—The isolates used in
this work were isolated either by the authors or purchased
from the American Type Culture Collection (ATCC, Rock-
ville, Maryland), the Centraalbureau voor Schimmelcultu-
res (CBS, Utrecht, Netherlands) or the Coleccio´n Espan˜ola
de Cultivos Tipo (CECT, Valencia, Spain). Care was taken
to ensure that the strains obtained from collections had
been deposited or identified by well-known specialists in
this fungal group to minimize the risk of including mis-
identified strains in the analysis. The isolates, original sub-
strates, geographical origins and collectors are listed in T
A
-
BLE
I. Isolates were grown on liquid complete media (5 g
of each malt extract, yeast extract and glucose L
2
1
) in Petri
dishes at 26 C for up to 3 wk and maintained on plates at
4 C on potato-dextrose agar (Oxoid, CM139, Basingstoke,
Hampshire, U.K.).
DNA sequencing.
—All procedures used in this study for
DNA purification and ITS amplification have been de-
scribed previously (Sa´ nchez-Ballesteros et al 2000). Asym-
metric PCR amplification was done with a 50:1 molar ratio
between the two primers (Gyllenstein and Erlich 1988).
The primers used for amplification of the D1 and D2 do-
mains of 28S rRNA gene were LR1 (59GTAGGAA-
TACCCGCTG AACT39) as concentrated primer and LR4
(based on primer NL4, O’Donnell 1992) for one strand and
LR4 as concentrated primer and LR1 for the other strand.
The cycling parameters were the same as previously de-
scribed (Sa´nchez-Ballesteros et al 2000). PCR products were
analyzed by electrophoresis on 1% agarose gels on TBE
buffer (Sambrook et al 1989) and visualized by staining with
ethidium bromide. The amplified products were sequenced
with an ABI PRISMyDye Terminator Cycle sequencing Kit
(Perkin Elmer). All samples were sequenced in both direc-
tions, using primer LR3 (59TGACCATTACGCCAGCATCC
39), when LR1/LR4 were used for amplification, and LR2
(based on NL1 primer; O’Donnell 1992), when LR4/LR1
253A
CERO ET AL
:M
OLECULAR PHYLOGENY OF THE
D
IATRYPACEAE
were used for amplification. Sequences from each strain
were assembled to obtain the sequence of the entire ITS1-
5.8S-ITS2 region and the 59region of 28S rRNA gene using
the GCG Fragment Assembly System (Program Manual for
the Wisconsin Package, version 8). All sequences were de-
posited in GenBank (T
ABLE
I). Alignments were performed
using the CLUSTALW program (Thompson et al 1994) and
deposited in TreeBASE (SN734).
Phylogenetic analysis.
—Phylogenetic analysis of the aligned
sequences was performed by the maximum-parsimony
method using the heuristic search algorithm of the Phylog-
eny Analysis Using Parsimony (PAUP*) program version 4.0
(Swofford 1998). Heuristic search was performed with sim-
ple addition of sequences and TBR branch swapping, with
MaxTrees set to 100. All characters were unordered and
equally weighted, with gaps treated as missing data. The
trees were rooted with the ITS sequence of a
Neurospora
crassa
Shear and B.O. Dodge isolate as outgroup. The data
were resampled with 1000 bootstrap replicates (Felsenstein
1985). To complement the analysis of branch support we
also calculated the decay indexes (Bremer 1994), using the
application SEPAL version 1.01 (Salisbury 1999). Neighbor-
joining analysis also was applied to the same sequence align-
ment, using the options DNADIST and NEIGHBOR from
PHYLIP 3.5c package (Felsenstein 1993). The Jukes and
Cantor algorithm was used to estimate the distances be-
tween the sequences.
Tandem repeat motifs.
—The repeated motifs in the ITS1 re-
gion were found using the FINDPATTERNS application
from GCG software Wisconsin Package version 10.0.
RESULTS
The ITS region in the
Diatrypaceae
were relatively
similar in length across the isolates studied, ranging
from 503 to 521 bp, except five isolates,
Diatrypella
prominens, Eutypella kochiana
and
Eutypella quater-
nata,
which had much longer sequences (540, 539
and 539 respectively), and
Eutypella leprosa
and
Eu-
typella vitis,
which had shorter sequences (491 and
493 respectively). Except those five isolates, the
length of ITS1 ranged from 188 to 199 bp. The size
of the ITS2 ranged from 158 to 168 bp, always short-
er than the ITS1.
The analysis of the ITS1 sequences of all the iso-
lates studied of the Diatrypaceae revealed the pres-
ence of DNA motifs repeated in tandem. These were
modifications of the 11-nucleotides motif CTACCCT-
GTAG, found in pure tandem or interspersed in the
ITS1 region, in a number ranging from four to seven
(data not shown). We detected five repetitions in all
the isolates studied, except
Diatrypella prominens
and
Eutypella quaternata,
which had seven repetitions and
Eutypella leprosa
and
Eutypella vitis
with four. The dif-
ference in the number of repetitions would account
for the different ITS length of these four isolates.
The different ITS length of
E. kochiana
is not due to
a different number of tandem repeat motifs. This iso-
late also had five repetitions, and in this case the larg-
er size is explained by insertions of one or more nu-
cleotides along the whole ITS1 region.
The aligned sequences showed a percentage of nu-
cleotide divergence of up to 22.1% for the complete
ITS region, with up to 30.5% and 31% divergence in
the ITS1 and the ITS2, respectively. The 5.8 S rRNA
gene was conserved among all the strains, except
E.
kochiana,
which had a C/T transition at position 120.
One of the 70 most-parsimonious trees derived
from the analysis of the whole ITS1-5.8S-ITS2 region
is shown in F
IG
. 1. The complete alignment was 616
bp, with 304 constant characters, 205 parsimony-in-
formative positions and 107 parsimony-uninformative
positions. The length of the tree was 1006 steps, with
CI
5
0.489, RI
5
0.681, and RC
5
0.333.
The most basal branch in the tree separated the
Creosphaeria
isolates from a monophyletic group that
included all the Diatrypaceae sensu stricto isolates
studied, although the bootstrap support for this main
branch was only moderate (70%). The next division
in the tree left
Eutypella quaternata
alone in a branch.
Above this clade it was possible to distinguish nine
groups, seven of them supported by high bootstrap
and decay indexes (groups 1–5, 7 and 8). All the
groups consisted of members from the same genus,
except groups 1, 3 and 8, which contained strains
from different genera. The tree did not allow resolv-
ing the relationships further among those nine
groups, because none of the larger clades observed
were supported by bootstrap analysis.
Group 1 was taxonomically the most heteroge-
neous, although it had one of the highest bootstrap
values. It contained two branches, one with
Diatrype
macowaniana
and
Eutypella caricae
and another with
Diatrypella frostii
and
Diatrypella prominens,
and both
branches were supported by high bootstrap values.
Group 2 clustered all the isolates belonging to
Dia-
trype flavovirens.
Group 3 included the
Eutypa
species
analyzed more distant to
Eutypa lata
(
Eutypa leptopla-
ca, Eutypa consobrina, Eutypa maura, Eutypa crustata
and
Eutypa astroidea
), together with
Diatrype polycoc-
ca
and
Eutypella prunastri.
Group 4 clustered three
of the five strains analyzed of genus
Cryptosphaeria
(
Cryptosphaeria lygniota, Cr yptosphaeria pullmanensis
and
Cryptosphaeria subcutanea
). Group 5 included
two
Diatrypella
species,
Diatrypella pulvinata
and
Dia-
trypella favacea.
Group 6 contained sequences of the
strains of
Eutypa lata
and related taxa, but this group
was not supported by bootstrap analysis. Two
Eutypa
armeniacae
isolates and one
Eutypa lata
isolate were
in this group with identical sequences along the
whole region. The next two branches included
Eu-
254 M
YCOLOGIA
F
IG
. 1. One of the 70 most equally parsimonious phylogenetic trees generated from the alignment of the ITS1-5.8S-ITS2
region of 55 isolates from the Diatrypaceae. Bootstrap support values are indicated (when more than 50%) at the base of
the corresponding clade (above the line), together with decay indexes (below the line). Codes used for character mapping.
Number of ascospores per ascus: Ceight, ●more than eight. Type of stroma: meutypoid, Mdiatrypoid, qvalsoid. Type of
anamorph: C,
Cytosporina
;L,
Libertella
; AA, conidiomata acer vular, conidia allantoid (sensu Rappaz 1987a); AC, conidiomata
acervular, conidia straight to moderately curved (sensu Rappaz 1987a); PC, conidiomata pycnidial, conidia straight to mod-
erately curved (sensu Rappaz 1987a); S, sterile in cultures, no anamorph described (Rappaz 1987a); U, anamorph unknown,
species not studied in Rappaz (1987a). Host: ANG Angiosperms (broad host range), I Aceraceae, II Poaceae, III Platanaceae,
IV Oleaceae, V Moraceae, VI Rosaceae, VII Salicaceae, VIII Fagaceae, IX Betulaceae, X Chenopodiaceae.
255A
CERO ET AL
:M
OLECULAR PHYLOGENY OF THE
D
IATRYPACEAE
typa laevata
and
Eutypa lata
var.
aceris.
The clade also
included other two
Eutypa lata
and two
Eutypa pe-
trakii
var.
petrakii
isolates. Group 7 clustered the re-
maining two isolates of genus
Cryptosphaeria
(two va-
rieties of
Cryptosphaeria eunomia
) not included in
Group 4. The most populated clade was Group 8,
which clustered 11 isolates of genus
Diatrype
and
Dia-
trypella quercina.
All
Diatrype disciformis
isolates ap-
peared together in a cluster, with identical ITS se-
quences. The nearest isolate to the
Diatrype discifor-
mis
group was
Diatrype quercina.
A second branch
clustered three
Diatrype stigma
isolates (D23M, D22C
and D19M) and another branch clustered
Diatrype
spilomea, Diatrype bullata
and another
Diatrype stigma
strain (D18M).
Diatrype undulata
appeared in a basal
branch as the most external isolate. Most
Eutypella
species were clustered in Group 9, but genetic dis-
tances in this group were much higher than in the
other groups. This was not considered a reliable
monophyletic group because it was not supported by
bootstrap analysis. Three main branches were in this
clade. One included two
Eutypella cerviculata
isolates.
A second cluster grouped
Eutypella alsophila
and
Eu-
typella scoparia,
with
Eutypella kochiana
next to them,
alone in a single branch. The last branch grouped
Eutypella leprosa
and
Eutypella vitis.
In summary, parsimony analysis of the whole ITS1-
5.8S-ITS2 region revealed little correlation between
the molecular data and the morphological criteria
classically used for delimiting genera within the Dia-
trypaceae because no genus could be shown to be
monophyletic. Group 1 contained members of
Dia-
trype, Eutypella
and
Diatrypella
as a well supported
monophyletic group just like Group 8, which con-
tained taxa from
Diatrype
and
Diatrypella.
The mod-
erately well-supported Group 3 contained species of
Eutypa, Diatrype
and
Eutypella.
The
Cryptosphaeria
species were placed in two separate groups. Also, the
Diatrype flavovirens
isolates formed a well-supported
group, separate from the remaining species of
Dia-
trype.
Finally,
Eutypella quaternata
was excluded from
the clade containing the remaining
Eutypella
species
(Group 9), although this group was not supported by
bootstrap analysis.
Neighbor-joining analysis of the same alignment
resulted in a tree that upheld the topology of the
nine main clusters resolved in the parsimony tree ex-
cept Group 9, whose members were segregated in
two groups. The internal topology of the strongly
supported branches resolved by parsimony analysis
also was maintained (data not shown).
Sequences from the D1-D2 region of the 28S rRNA
gene from a subgroup of representative species also
were obtained to complement the phylogenetic anal-
ysis. The size of this region was 556 bp for the 12
isolates analyzed except
Eutypella kochiana,
with 561
bp. The alignment of these sequences revealed that
this region was conserved highly. Only 33 positions
from the alignment were parsimony informative,
making impossible any reliable reconstruction of the
phylogenetic relationships from these data.
DISCUSSION
One of the most interesting findings in this work is
the detection of tandem-repeat sequence patterns in
the ITS1 region. The presence of this type of se-
quences in fungal genomes is well documented (An-
dersen and Torsten 1997, Giraud et al 1998, Ze´ze´et
al 1999), but its occurrence in the ITS regions has
been reported only recently. These motifs previously
have been reported for
Eutypa lata
by DeScenzo et
al (1999), although in this work the number of rep-
etitions varied among isolates, whereas in our study
the number of motifs was consistent among strains
from the same species. These tandem-repeat se-
quences have been found in the ITS region of many
members of the Xylariales but not in other fungal
groups (Platas et al 2001). In the Diatrypaceae they
were located in the ITS1 region at homologous po-
sitions, between positions 55–65 and 111–155. Such
repeats are lost easily or incorporated by mechanisms
of slipping strand mispairing (Platas et al 2001).
Our molecular data poorly correlated with the
morphological criteria used for delimiting genera
and species within the Diatrypaceae, suggesting that
the current taxonomical schemes in the Diatrypaceae
might not reflect the natural relationships and limits
of the genera traditionally placed in this group. Par-
simony analysis of the ITS sequences seem to support
a monophyletic origin for the family, although the
bootstrap support is weak.
Genus
Eutypa.—Our molecular data suggested that
genus
Eutypa
is polyphyletic. The species analyzed ap-
peared distributed in two separate groups. This is
consistent with the heterogeneity of the genus hy-
pothesized by many authors, most likely as a conse-
quence of the low number of diagnostic features ex-
hibited. Although without bootstrap support, each
Eutypa
clade associated with some
Cryptosphaeria
spe-
cies, in agreement with the presumed relationship
between the two genera suggested by their similarity
in stromatal morphology (Wehmeyer 1975, Glawe
and Rogers 1984).
The distribution of taxa within Group 6 in the phy-
logenetic trees suggests a large sequence variability
among the strains belonging to
Eutypa lata
and re-
lated taxa. This taxon could be regarded as a species
complex, where delimitation of individuals repre-
256 M
YCOLOGIA
senting
Eutypa lata sensu stricto
could be difficult, giv-
en that this species is reported commonly from a
large number of plant hosts and old descriptions of
the species often lack enough diagnostic characters.
Within this group we found a robust subclade con-
taining one strain of
Eutypa lata
and two strains of
Eutypa armeniacae
showing identical sequences. Al-
though this could support the hypothesis that both
species are synonyms, as suggested by several authors
(Glawe 1992, Rappaz 1987a), two other
Eutypa lata
isolates are peripheral to this subclade. DeScenzo et
al (1999) assessed the genetic diversity in a group of
Eutypa lata
-like isolates from California using ITS se-
quencing and AFLP fingerprint and their results sup-
ported the separation of the two species. The next
two branches in Group 6 included
Eutypa lata
var.
aceris,
which differs from
Eutypa lata
only in cultural
features and
Eutypa laevata,
a taxon considered by
Rappaz (1987a) as a possible variant of
Eutypa lata
with smaller ascospores and habitat restricted to
Salix
spp. The high homology between one of the
Eutypa
lata
(E43M) and one of the
Eutypa petrakii
isolates
(E46C) is remarkable.
Group 3 contained the
Eutypa
species less related
to the
Eutypa lata
complex. The inclusion of
Diatrype
polycocca
in this clade is intriguing. Our molecular
data suggest that
Diatrype polycocca
is highly related
to species of
Eutypa,
but Rappaz (1987a) described
Diatrype polycocca
unambiguously as a member of ge-
nus
Diatrype,
with a diatrypoid stroma. It is interest-
ing to note that this species shows a pycnidial ana-
morph, similar to the typical
Eutypa
anamorphs,
whereas the species of
Diatrype
in Group 8 usually
produce a
Libertella
-like anamorph (i.e., with unen-
closed conidiomata). Another species in this clade
not ascribed to
Eutypa
is
Eutypella prunastri.
Tiffany
and Gilman (1965) have suggested that
Eutypella pru-
nastri
should be considered as belonging to genus
Eutypa,
and our data provide additional support for
that suggestion.
Genus
Cryptosphaeria.—Our molecular analysis sug-
gests a polyphyletic origin for genus
Cryptosphaeria.
The five species analyzed appear distributed in two
separate clades (groups 4 and 7), each related to one
of the two
Eutypa
clades. For the species studies,
there is an apparent correlation between this segre-
gation and their host plant range. Thus, the three
species included in Group 4 commonly are recorded
from members of the Salicaceae, whereas the two
taxa included in Group 7 are typical from the Ole-
aceae. Within Group 4,
Cryptosphaeria subcutanea
and
Cryptosphaeria lignyota
were grouped together.
They are closely related species, according to Rappaz
(1987a). The two varieties of
Cryptosphaeria eunomia
included in Group 7 showed enough genetic vari-
ability to be distinguished from each other. They
have been reported to be macroscopically identical,
except that var.
fraxini
posses a distinctive ascospore
septum (Rappaz 1987a). Although they appeared
grouped, the nucleotide divergence rate between
them was relatively high (4.8%), compared with oth-
er groups in the study. This would support maintain-
ing these two varieties as distinct taxa.
Genus
Diatrype.—Parsimony and neighbor-joining
analyses suggests a polyphyletic origin for the genus,
or at least that
Diatrype flavovirens
should be segre-
gated from the rest of the species of the genus ana-
lyzed. Thus, the strains studied here grouped in two
distinct clades, one of them containing sequences
from
Diatrype flavovirens
strains and the other in-
cluding the remaining
Diatrype
spp. studied (except
Diatrype polycocca
and
Diatrype macowaniana
). The
systematic position of
Diatrype flavovirens
(Group 2)
has been reported to be unclear, because it exhibits
morphological characters intermediate between
Dia-
trype
and
Eutypa.
Rappaz (1987a) considered
Diatry-
pe flavovirens
difficult to delimitate because of the
limits between well-developed and poorly developed
diatrypoid stromata. Our data suggests that it could
be considered a taxon different from both
Diatrype
and
Eutypa,
but further molecular analyses involving
other genes is required to assign the members of this
taxon to an independent genus.
Group 8 contained the remaining species of
Dia-
trype,
including
Diatrype disciformis,
the type species
of the genus. The sequence of
Diatrypella quercina
also was included in this group. This taxon has been
considered a
Diatrypella
because of its multisporate
asci, but Wehmeyer (1926) discussed the conve-
nience of including this species in
Diatrype.
In addi-
tion, Ruhland (1900) pointed out that
Diatrypella
quercina
could be considered under the concept of
Diatrype
because of the strongly developed ectostro-
mata and Croxall (1950) later distinguished it from
other
Diatrypella
species because of its strongly
curved ascospores. The molecular data presented
here suggest that
Diatrypella quercina
should be con-
sidered a member of
Diatrype
despite its multisporate
asci.
All species included in Group 8 have been report-
ed as related to some degree. In addition,
Diatrype
disciformis
clearly was separated from the remaining
Diatrype
species. All isolates belonging to this taxon
showed identical ITS sequences, despite their differ-
ent geographic origins, and they were arranged to-
gether in a monophyletic group with a high boot-
strap index. In contrast, the isolates of
Diatrype stigma
did not cluster in a monophyletic group, suggesting
257A
CERO ET AL
:M
OLECULAR PHYLOGENY OF THE
D
IATRYPACEAE
that this might be a species complex as hypothesized
by several authors. Thus, Wehmeyer (1926) and Nit-
schke (1867) found differences in conidial sizes in
different collections of
Diatrype stigma,
suggesting
that more than one species were included under this
epithet. Likewise, Glawe and Rogers (1984) consid-
ered five groups for
Diatrype stigma
based on asco-
spore size, conidial size and stromatal features. Rap-
paz (1987b) considered three taxa for
Diatrype stig-
ma
:
Diatrype stigma sensu stricto, Diatrype decorticata
and
Diatrype undulata.
Our study does not include
Diatrype decorticata,
but F
IG
. 1 suggests a clear dis-
tinction between
Diatrype stigma sensu stricto
and
Dia-
trype undulata.
On the other hand,
Diatrype bullata
and
Diatrype disciformis
also were seen as similar to
Diatrype stigma
by Wehmeyer (1926) and Rappaz
(1987b) because of their similar stromatic develop-
ment. Nevertheless, our analysis did not resolve the
relationships among these species.
Genus
Diatrypella.—The five isolates of
Diatrypella
in-
cluded in this study were distributed into three dif-
ferent clades with high statistical support, in some
cases (groups 1 and 8) together with members of oth-
er genera. This would suggest that the multisporate
ascus trait might have appeared independently sev-
eral times during the evolution of the Diatrypaceae.
Group 5, containing
Diatrypella pulvinata
and
Diatry-
pella favacea,
the type species of genus
Diatrypella,
could be the representative group of the genus, pro-
vided that any character, other than the number of
spores per ascus, were used to define this genus. The
small nucleotide divergence between these two iso-
lates (1.1%) would suggest that they are related close-
ly or even conspecific. The sequencing of additional
genes and isolates from these species would be re-
quired to confirm this possibility. As already men-
tioned,
Diatrypella quercina
should be considered a
member of genus
Diatrype.
Finally, the two
Diatrypel-
la
species in Group 1 probably should be considered
out of the concept of the genus, according to its rel-
ative position in the tree. It is interesting to note that
Group 1 includes species from three different gen-
era, with very low percentages of divergence, sug-
gesting close affinities among the four taxa. The sim-
ilarity between the sequences of
Diatrype macowani-
ana
and
Eutypella caricae
is particularly striking
(0.2%). Although we cannot rule out the possibility
that the nomenclatural heterogeneity in this clade
could be due to strain misidentifications, our data
suggest at least the need of a taxonomic revision of
these species.
Genus
Eutypella.—The clade containing most of the
species from genus
Eutypella
(Group 9) appeared as
an unstable monophyletic group in the analysis of the
entire ITS region, with low bootstrap values (F
IG
. 1).
Moreover, such arrangement was not conserved in
the neighbor-joining tree (data not shown). It also is
interesting to note that several of the strains analyzed
showed large differences in length in the ITS1 re-
gion, in some cases with a different distribution of
the tandem-repeat sequences. This was the most het-
erogeneous group at the sequence level, with nucle-
otide divergence percentages ranging between 3.2
and 22.1%. In fact, the appearance of monophyly
could be caused by a ‘‘long-branch attraction’’ phe-
nomenon (Maley and Marshall 1998). This group
also included species from other ancient genera
(e.g.,
Quaternaria
Tul. and C. Tul,
Scoptria
Nitschke)
lately synonymized under
Eutypella
to maintain name
stability (Rappaz 1987a, 1989). However, some of the
relationships among the isolates analyzed are con-
served and well supported. Thus, the two strains of
Eutypella cerviculata
clustered together,
Eutypella le-
prosa
and
Eutypella vitis
also were grouped, and there
was a third group with
Eutypella alsophila, Eutypella
scoparia
and
Eutypella kochiana.
Rappaz (1987a) re-
ported that
Eutypella kochiana
was close to
Eutypella
alsophila,
although the former had smaller asco-
spores. However,
Eutypella alsophila
appeared more
related to
Eutypella scoparia
than to
Eutypella kochi-
ana
in the phylogenetic trees. Furthermore, both
Eu-
typella scoparia
and
Eutypella alsophila
showed a sim-
ilar distribution pattern of tandem-repeat motifs
(data not shown). Moreover,
Eutypella kochiana
was
the only isolate in this study with a different nucleo-
tide in the 5.8S rRNA gene and with five additional
nucleotides in the 5
9
region of 28S rRNA. To get in-
sight to the implications of such observations, the se-
quencing of other isolates of
Eutypella kochiana
would be desirable.
Our analysis suggests that
Eutypella quaternata
(EL60C) should be considered a member of a differ-
ent genus. This taxon was described as the type spe-
cies of the genus
Quaternaria
(as
Quaternaria quater-
nata
) and later proposed to be included in genus
Eutypella
(Rappaz 1987a, Eriksson 1988). Molecular
data support maintaining
Quaternaria
as an indepen-
dent genus, as proposed by Gams (1994).
Genus
Creosphaeria.—One of the goals of this work
was to assess the phylogenetic relationships between
Creosphaeria
and members of the Diatrypaceae.
Creos-
phaeria
is classified within the Xylariaceae, but its
Lib-
ertella
-like anamorph (Bills and Pela´ez 1996) suggests
that it could have affinities with the Diatrypaceae
(Rappaz 1987a). A previous study based on ITS se-
quences revealed that
Creosphaeria sassafras
was pe-
ripheral to other members of the Xylariaceae (Sa´n-
chez-Ballesteros et al 2000). However, our phyloge-
258 M
YCOLOGIA
netic reconstruction did not reflect a clear link be-
tween this genus and the Diatrypaceae. In the
parsimony analysis the two
Creosphaeria sassafras,
se-
quences appeared in a basal node, out of the main
clade that included all the sequences of the Diatry-
paceae. Although based on the number and position
of the tandem repeat motifs in the ITS1 region,
Creos-
phaeria sassafras
would be closer to the Diatrypaceae
than to the Xylariaceae (data not shown), the phy-
logenetic relevance of this finding is unknown. In any
case, and although more sequence-based work would
be desirable to clarify the systematic placement and
evolutionary affinities of the genus, our results sug-
gest maintaining
Creosphaeria
out of the Diatrypa-
ceae.
Molecular phylogeny and morphological traits.
—Most of
the monophyletic clades identified in the phyloge-
netic trees were homogeneous with respect to the
type of stromata of the species clustered within. The
exceptions were groups 1 and 3, which contained
taxa showing different stromatal types. However, the
groups containing taxa with diatrypoid or eutypoid
configuration were intermingled along the clado-
gram. The valsoid type was apparently more homo-
geneous because all of the
Eutypella
species (except
Eutypella prunastri
and
Eutypella caricae
) were ar-
ranged in a group, although without bootstrap sup-
port. Likewise, species of
Eutypa
and
Cryptosphaeria,
which share a similar type of stromata, were grouped
together but also lacking bootstrap support. Howev-
er, the data presented here are insufficient to draw
any conclusions about the evolutionary relationships
among the stromatal configurations used to define
genera in the Diatrypaceae. Our results suggest a pos-
sible polyphyletic origin for these stromatal types; the
three main types seem to have appeared several times
along the natural history of the group. Likewise, the
number of ascospores per ascus is not a character
associated with any monophyletic group.
In the phylogenetic trees presented here, we have
mapped the anamorphs for the species studied based
on two sources. The database ANATELEO (http://
www.cbs.knaw.nl/databases/anateleo.html) provides
anamorph epithets for some of these species. These
have been incorporated in the trees and referred to
as
Cytosporina
or
Libertella.
However, the anamorphs
for most of the species studied have not been named
in the literature. In those cases we have assigned a
code defining the type of conidiomata and conidia,
as described by Rappaz (1987a). The distribution of
species in F
IG
. 1 does not reveal any apparent overall
correlation with their anamorphs. The segregation of
Diatrype flavovirens
from other
Diatrype
spp. corre-
lates with its different anamorph
Cytosporina
-like,
with pycnidial conidiomata, compared with the ana-
morphs with conidiomata unenclosed produced by
the members of Group 8. However, this apparent cor-
relation is weakened by the observation that
Diatrype
flavovirens
is known to produce pycnidia on the host
but unenclosed conidiomata in agar media (Glawe
1983).
Like morphological characters, host range did not
show any apparent correlation with the distribution
of species in the phylogenetic trees. Species with
broad host range appeared distributed across the
cladogram and those with restricted host range were
not clustered.
In summary, this work presents a preliminary as-
sessment of the phylogenetic relationships among
genera of the family Diatrypaceae by sequencing of
the ITS region. Our molecular phylogenetic analysis
shows little correlation with the current morpholog-
ical concepts used for delimiting genera in the family.
The current generic divisions within this family might
not reflect the natural relationships among different
taxa.
In addition to its contribution to the understand-
ing of the systematics of the Diatrypaceae, our work
may be a useful tool for the identification of diatry-
paceous fungi in culture, which is hampered by the
fact that anamorphs are almost indistinguishable and
not always produced in culture. PCR primers have
been designed recently that are useful in the rapid
identification of
Eutypa lata
in culture (Lecomte et
al 2000). Although the design of primers for the
identification of other species would require se-
quencing more isolates from diverse geographic lo-
cations, our work provides a foundational database
that can be used as a reference to compare sequences
of unknown isolates of this important family.
LITERATURE CITED
Andersen TH, Torsten NT. 1997. A fungal minisatellite. Na-
ture 386:771.
Bills GF, Pela´ez F. 1996. Endophytic isolates of
Creosphaeria
sassafras.
Mycotaxon 57:471–477.
Bremer K. 1994. Branch support and tree stability. Cladistics
10:295–304.
Carter MV, Bolay A, Rappaz F. 1983. An annotated host list
and bibliography of
Eutypa armeniacae.
Rev Plant
Pathol 62:251–258.
Croxall HE. 1950. Studies on british pyrenomycetes III. The
British species of the genus
Diatrypella
Cesati and de
Notaris. Trans Br Mycol Soc 33:45–72.
Currey F. 1858. Synopsis of the fructification of the com-
pound
Sphaeriae
of the Hookeria herbarium. Trans
Linn Soc London 22:313–335.
DeScenzo RA, Engel SR, Gomez G, Jackson EL, Munkvold
GP, Weller J, Irelan NA. 1999. Genetic analysis of
Eu-
259A
CERO ET AL
:M
OLECULAR PHYLOGENY OF THE
D
IATRYPACEAE
typa
strains from California supports the presence of
two pathogenic species. Phytopathology 89:884–893.
Eriksson OE, Hawksworth DL. 1988. Notes on ascomycete
systematics-N. 552–732. Systema Ascomycetum 7:59–
101.
Felsenstein J. 1985. Confidence intervals on phylogenies: an
approach using the bootstrap. Evolution 39:783–791.
. 1993. PHYLIP manual version 3.5. Seattle: Univer-
sity of Washington.
Fries EM. 1823.
Systema mycologicum
3, Index: 1–202. Greifs-
wald.
Gams W. 1994. Report of the committee for fungi: 4. Taxon
43:265–267.
Giraud T, Fortini D, Levis C, Brygoo Y. 1998. The minisa-
tellite MSB1, in the fungus
Botrytis cinerea,
probably
mutates by slippage. Mol Biol Evol 15:1524–1531.
Glawe DA. 1983. Observations on the anamorph of
Diatry-
pella frostii.
Mycologia 75:913–915.
. 1992. What’s in a name?
Eutypa armeniacae
vs.
Eu-
typa lata.
Phytopathology 82:992.
, Rogers JD. 1982a. Observations on the anamorphs
of six species of
Diatrype
and
Diatrypella.
Can J Bot 60:
245–251.
, . 1982b. Observations on the anamorphs of
six species of
Eutypa
and
Eutypella.
Mycotaxon 14:334–
346.
, . 1984. Diatr ypaceae in the Pacific North-
west. Mycotaxon 20:401–460.
Gyllenstein UB, Erlich HA. 1988. Generation of single-
stranded DNA by the polymerase chain reaction and
its application to direct sequencing of the HLA-DQA
locus. Proc Natl Acad Sci USA 85:7652–7656.
Kirk PM, Cannon PF, David JC, Stalpers JA. 2001. Ainsworth
and Bisby9s Dictionary of the Fungi. 9th ed. CAB In-
ternational, Wallingford, UK. 655 p.
Lecomte P, Pe´ros J-P, Blancard D, Bastien N, De´lye C. 2000.
PCR assays that identify the grapevine dieback fungus
Eutypa lata.
Appl Environ Microbiol 66:447–4480.
Maley LE, Marshall CR. 1998. The coming of age of molec-
ular systematics. Science 279:505–506.
Nitschke T. 1867. Pyrenomycetes Germanici. Die Kernpilze
Deutschlands. Breslau, 1:1–160.
O’Donnell K. 1992. Ribosomal DNA internal transcribed
spacers are highly divergent in the phytopathogenic as-
comycete
Fusarium sambucinum
(
Gibberella pulicaris
).
Curr Genet 22:213–220.
Platas G, Salazar O, Acero J, Sa´ nchez-Ballesteros J, Rubio V,
Gonza´lez V, Portal MA, Borkowski J, Pela´ez F. 2001.
Presence of a simple Tandem Repeat DNA sequence
in the ITS1 region of the Xylariales. Curr Microbiol 43:
43–50.
Rappaz F. 1987a. Taxonomie et nomenclature des Diatry-
pace´es a´ asques octospore´s. Mycologia Helvetica 2:285–
648.
. 1987b. Taxonomie et nomenclature de
Diatrype
stigma, D. decorticata
et
D. undulata
(Diatrypaceae, As-
comycetes). Mycotaxon 30:209–219.
. 1989. Proposal to conserve
Eutypella
(Nitschke)
Sacc. (1875) against
Quaternaria
Tul and Tul (1863)
and
Scoptria
Nitschke (1867) (Ascomycotina). Taxon
38:665.
Ruhland W. 1900. Untersuchungen zu einer Morphologie
der stromabildenden Sphaeriales. Hedwigia 39:1–79.
Salisbury BA. 1999. Strongest evidence: maximum apparent
phylogenetic signal as a new cladistic optimality crite-
rion. Cladistics 15:137–149.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular clon-
ing: a laboratory manual. 2nd ed. Cold Spring Harbor,
New York: Cold Spring Harbor Press. 1592 p.
Sa´nchez-Ballesteros J, Gonzalez V, Salazar O, Acero J, Portal
MA, Julia´n M, Rubio V, Bills GF, Polishook JD, Platas
G, Mochales S, Pela´ez F. 2000. Phylogenetic study of
Hypoxylon
and related genera based on ribosomal ITS
sequences. Mycologia 92:964–977.
Swofford DL. 1998. PAUP*. Phylogenetic Analysis using Par-
simony (* and other methods). Version 4. Sunderland,
MA: Sinauer Associates.
Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTALW:
improving the sensitivity of progressive multiple se-
quence alignment through sequence weighting, posi-
tion-specific gap penalties and weight matrix choice.
Nucl Acid Res 22:4673–4680.
Tiffany LH, Gilman JC. 1965. Iowa ascomycetes 4: Diatry-
paceae. Iowa St J Sci 40:121–161.
Tulasne L-R, Tulasne C. 1863. Selecta Fungorum carpologia
2: 1–319. Paris.
Wehmeyer LE. 1926. A biologic and phylogenetic study of
the stromatic sphaeriales. Amer J Bot 13:574–645.
Wehmeyer LE. 1975. The pyrenomycetous fungi. Mycol
Mem 6:1–250.
Winter G. 1884–1887. Ascomyceten, in
Rabenhorst’s Kryp-
togamen-Flora von Deutschland, O
¨sterreich und der
Schweiz
1. Abt. 1–2:1–928. Leipzig.
Ze´ze´ A, Hosny M, van Tuinen D, Gianinazzi-Pearson V, Du-
lieu H. 1999. MYCDIRE, a dispersed repetitive DNA
element in arbuscular mycorrhizal fungi. Mycol Res
103:572–576.
