Mycosphaerella Eucalyptus S

Mycosphaerella Eucalyptus S
STUDIES IN MYCOLOGY 55: 147–161. 2006.
A multi-gene phylogeny for species of Mycosphaerella occurring
on Eucalyptus leaves
Gavin C. Hunter1*, Brenda D. Wingfield2, Pedro W. Crous3 and Michael J. Wingfield1
1Department
of Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa;
of Genetics, University of Pretoria, Pretoria, 0002, South Africa; 3Centraalbureau voor Schimmelcultures, Fungal Biodiversity
Centre, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands
2Department
*Correspondence: Gavin C. Hunter, [email protected]
Abstract: Species of the ascomycete genus Mycosphaerella are regarded as some of the most destructive leaf pathogens of a large number of
economically important crop plants. Amongst these, approximately 60 Mycosphaerella spp. have been identified from various Eucalyptus spp.
where they cause leaf diseases collectively known as Mycosphaerella Leaf Disease (MLD). Species concepts for this group of fungi remain
confused, and hence their species identification is notoriously difficult. Thus, the introduction of DNA sequence comparisons has become
the definitive characteristic used to distinguish species of Mycosphaerella. Sequences of the Internal Transcribed Spacer (ITS) region of the
ribosomal RNA operon have most commonly been used to consider species boundaries in Mycosphaerella. However, sequences for this gene
region do not always provide sufficient resolution for cryptic taxa. The aim of this study was, therefore, to use DNA sequences for three loci, ITS,
Elongation factor 1-alpha (EF-1α) and Actin (ACT) to reconsider species boundaries for Mycosphaerella spp. from Eucalyptus. A further aim
was to study the anamorph concepts and resolve the deeper nodes of Mycosphaerella, for which part of the Large Subunit (LSU) of the nuclear
rRNA operon was sequenced. The ITS and EF-1α gene regions were found to be useful, but the ACT gene region did not provide species-level
resolution in Mycosphaerella. A phylogeny of the combined DNA datasets showed that species of Mycosphaerella from Eucalyptus cluster in two
distinct groups, which might ultimately represent discrete genera.
Key words: Actin, Ascomycetes, Translation Elongation factor 1-alpha, Multi-gene phylogeny, Mycosphaerella, Mycosphaerella Leaf Disease,
ribosomal RNA operon.
INTRODUCTION
Species of Eucalyptus are native to Australia with isolated pockets of native Eucalyptus forests also occurring
in Papua New Guinea and the Philippines (Turnbull
2000). Many Eucalyptus spp. have been removed from
these centres of origin to new environments where they
are typically propagated in plantations for the production
of paper, pulp and other wood products (Wingfield 1999,
Turnbull 2000, Wingfield et al. 2001). In these nonnative environments, Eucalyptus trees are susceptible
to many pests and diseases including those known in
their areas of origin and others that have undergone
host shifts (Wingfield 2003, Slippers et al. 2005). These
pests and diseases cause significant annual losses to
Eucalyptus plantations resulting in decreased revenue
for commercial forestry companies.
Mycosphaerella Johanson is one of the largest
genera of the ascomycetes, accommodating more
than 2000 species. Approximately 60 Mycosphaerella
spp. have been associated with leaf diseases of many
Eucalyptus spp., and these are collectively referred to
as Mycosphaerella Leaf Disease (MLD) (Crous 1998,
Maxwell et al. 2003, Crous et al. 2004a). The disease
is particularly prevalent on the juvenile leaves and
shoots of Eucalyptus trees, where infection results in
premature defoliation, twig cankers and stunting of
tree growth (Lundquist & Purnell 1987, Crous 1998,
Park et al. 2000). However, several Mycosphaerella
spp. can also infect adult Eucalyptus foliage, and this
has been attributed to their ability to produce a protoappressorium that enables direct cuticle penetration
(Ganapathi 1979, Park & Keane 1982b). In some
situations, trees may thus be subjected to infection by
a suite of different Mycosphaerella spp.
Identification of Mycosphaerella spp. based on
morphology is known to be difficult. This is because
these fungi tend to produce very small fruiting
structures with highly conserved morphology, and
they are host-specific pathogens that grow poorly in
culture. Traditionally, morphological characters of the
teleomorph and anamorph have been used in species
delimitation (Crous 1998). Park & Keane (1982a)
introduced ascospore germination patterns as an
additional characteristic to identify Mycosphaerella spp.,
and Crous (1998) subsequently identified 14 different
ascospore germination patterns for the Mycosphaerella
spp. occurring on Eucalyptus. Crous (1998) and Crous
et al. (2000) also introduced features of these fungi
growing in culture and especially anamorph morphology
as important and useful characteristics on which to
base species delimitation. DNA-based methods such
as RAPDs and species-specific primers have also
been employed to distinguish between Mycosphaerella
species occurring on Eucalyptus (Carnegie et al. 2001,
Maxwell et al. 2005).
Comparisons of DNA sequence data have emerged
as the most reliable technique to identify Mycosphaerella
spp. The majority of studies employing DNA sequence
data for species identification have relied on sequence
data from the Internal Transcribed Spacer (ITS) region
of the ribosomal RNA operon (Crous et al. 1999,
2001, 2004a, b, Hunter et al. 2004a, b). Although
comparisons of gene sequences for this region have
been useful, the resolution provided by this region is not
uniformly adequate to discriminate between individuals
147
HUNTER ET AL.
of a species complex or to effectively detect cryptic
species (Crous et al. 2004b). Thus, recent studies
have shown the importance of employing Multi-Locus
Sequence Typing (MLST) to effectively identify cryptic
fungal species and to study species concepts (Taylor &
Fischer 2003).
A single morphological species does not always
reflect a single phylogenetic unit (Taylor et al. 2000).
Within Mycosphaerella, teleomorph morphology is
conserved and the anamorph morphology provides
additional characteristics to discriminate between taxa
(Crous et al. 2000). Yet the collective teleomorph and
anamorph morphology is often not congruent with
phylogenetic data. Thus, recent phylogenetic studies
have led to the recognition of several species complexes
within Mycosphaerella (Crous et al. 2001, 2004b, Braun
et al. 2003). Most of these studies have been based
on comparisons of sequences for the ITS regions of
the ribosomal DNA operon. Given the important data
that have emerged from them, it is well recognised
that greater phylogenetic resolution will be required for
future taxonomic studies on Mycosphaerella species.
The aim of this study was to use MLST to consider
species and anamorph concepts in Mycosphaerella
spp. occurring on Eucalyptus. This was achieved by
sequencing four nuclear gene regions, namely part of
the Large Subunit (D1−D3 of LSU) and ITS region of
the nuclear rRNA operon, and a portion of the Actin
(ACT) and Elongation Factor 1-alpha (EF-1α) gene
regions.
MATERIALS AND METHODS
Mycosphaerella isolates
For this study, an attempt was made to obtain cultures
of as many Mycosphaerella spp. known to infect
Eucalyptus leaves as possible. All cultures used in
the investigation are housed in culture collections of
the Forestry and Agricultural Biotechnology Institute
(FABI), University of Pretoria, South Africa and the
Centraalbureau voor Schimmelcultures (CBS), Utrecht,
The Netherlands (Table 1). All cultures were grown on 2
% (w/v) malt extract agar (MEA) (Biolab, South Africa),
at 25 ºC for approximately 2–3 mo to obtain sufficient
mycelial growth for DNA extraction.
DNA isolation
Mycelium from actively growing cultures was scraped
from the surface of cultures, freeze-dried for 24 h and
then ground to a fine powder using liquid nitrogen.
DNA was isolated using the phenol : chloroform (1:
1) extraction protocol as described in Hunter et al.
(2004a, b). DNA was precipitated by the addition of
absolute ethanol (100 % EtOH). Isolated DNA was
cleaned by washing with 70 % Ethanol (70 % EtOH)
and dried under vacuum. SABAX water was used to
resuspend the isolated DNA. RNaseA (10 µg/µL) was
added to the resuspended DNA and incubated at 37
°C for approximately 2 h to digest any residual RNA.
Isolated DNA was visualised in a 1 % agarose gel (w/v)
(Roche Diagnostics, Mannheim), stained with ethidium
148
bromide and visualised under ultra-violet light.
PCR amplification and purification
DNA (ca. 20 ng) isolated from the Mycosphaerella
spp. used in this study was used as a template for
amplification using the Polymerase Chain Reaction
(PCR). All PCR reactions were mixed in a total volume
of 25 µL containing 10× PCR Buffer (5 mM TrisHCl, 0.75 mM MgCl2, 25 mM KCl, pH 8.3) (Roche
Diagnostics, South Africa), 2.5 mM of each dNTP
(dATP, dTTP, dCTP, dGTP) (Roche Diagnostics, South
Africa), 0.2 µM of forward and reverse primers (Inqaba
Biotech, South Africa) and 1.25 U Taq DNA Polymerase
(Roche Diagnostics, South Africa) and DNA (20 ng/µL).
Sterilised distilled water was added to obtain a final
volume of 25 µL.
The ITS-1, ITS-2 and the 5.8 S gene regions of the
ITS region of the rRNA operon were amplified using
primers ITS-1 (5`− TCC GTA GGT GAA CCT GCG G
–3`) and LR-1 (5`- GGT TGG TTT CTT TTC CT – 3`)
(Vilgalys & Hester 1990, White et al. 1990). Reaction
conditions for the ITS gene regions followed those of
Crous et al. (2004a) and Hunter et al. (2004a, b).
A portion of the LSU (including domains D1−D3) of
the rRNA operon was amplified using primers LR0R (5’ACC CGC TGA ACT TAA GC-3’) (Moncalvo et al. 1995)
and LR7 (5’-TAC TAC CAC CAA GAT CT-3’) (Vilgalys &
Hester 1990). PCR cycling conditions were as follows:
an initial denaturation step of 96 °C for 2 min, followed
by 35 cycles of denaturation at 94 °C for 30 s, primer
annealing at 62 °C for 30 s, primer extension at 72°C for
1 min and a final elongation step at 72 °C for 7 min.
A portion of the EF-1α was amplified using the
primers EF1-728F (5’-CAT CGA GAA GTT CGA GAA
GG-3’) and EF1-986R (5’-TAC TTG AAG GAA CCC TTA
CC-3’) (Carbone & Kohn 1999). Reaction conditions
were: an initial denaturation step of 96 °C for 2 min,
followed by 35 cycles of denaturation at 94 °C for 30 s,
primer annealing at 56 °C for 30 s and primer extension
at 72 °C for 30 s. The reaction was completed with a
final extension at 72 °C for 7 min.
A portion of the ACT gene was amplified using the
primers ACT-512F (5’-ATG TGC AAG GCC GGT TTC
GC-3’) and ACT-783R (5’-TAC GAG TCC TTC TGG
CCC AT-3’) (Carbone & Kohn 1999). PCR reaction
conditions were: an initial denaturation step at 96 °C for
2 min, followed by 10 cycles of denaturation at 94 °C for
30 s, primer annealing at 61 °C for 45 s and extension
at 72 °C for 45 s. This was followed by 25 cycles of
denaturation at 94 °C for 30 s, primer annealing at 61
°C and elongation at 72 °C for 45 s with an increase of
5 s per cycle. The reaction was completed with a final
elongation step at 72 °C for 7 min.
All PCR products were visualised in 1.5 % agarose
gels (wt/v) stained with ethidium bromide and viewed
under ultra-violet light. Sizes of PCR amplicons were
estimated by comparison against a 100 bp molecular
weight marker (O’ RangeRulerTM 100 bp DNA ladder)
(Fermentas Life Sciences, U.S.A.). Prior to DNA
sequencing, PCR products were purified through Centrisep spin columns (Princeton Separations, Adelphia,
NJ) containing Sephadex G-50 (Sigma Aldrich, St.
Louis, MO) as outlined by the manufacturer.
A MULTI-GENE PHYLOGENY OF MYCOSPHAERELLA
DNA sequencing and phylogenetic analysis
Purified PCR products were used as template DNA
for sequencing reactions on an ABI PRISMTM 3100
Automated DNA sequencer (Applied Biosystems,
Foster City, CA). The ABI Prism Big Dye Terminator
Cycle sequencing reaction kit v. 3.1 (Applied
Biosystems, Foster City, CA) was used for sequencing
reactions following the manufacturer’s instructions.
Most sequencing reactions were performed with the
same primers used for PCR reactions. Exceptions
were in the case of the ITS region where two internal
primers ITS-2 (5’-GCT GCG TTC TTC ATC GAT GC-3’)
and ITS-3 (5’-GCA TCG ATG AAG AAC GCA GC-3’)
(White et al. 1990) were included for the sequencing
reactions. Similarly, for the LSU region two internal
primers LR3R (5’-GTC TTG AAA CAC GGA CC-3’) and
LR-16 (5’-TTC CAC CCA AAC ACT CG-3’) were used
for the sequencing reactions.
All resulting sequences were analysed with
Sequence Navigator v. 1.0.1 (Applied Biosystems,
Foster City, CA). Sequence alignments were done
using MAFFT (Multiple alignment program for amino
acid or nucleotide sequences) v. 5.667 (Katoh et
al. 2005) and manually adjusted where necessary.
Phylogenetic analyses and most parsimonious trees
were generated in PAUP v. 4.0b10 (Swofford 2002) by
heuristic searches with starting trees obtained through
stepwise addition with simple addition sequence and
with the MULPAR function enabled. Tree Bisection
Reconnection (TBR) was employed as the swapping
algorithm. All gaps were coded as missing data and
characters were assigned equal weight. Branch support
for nodes was obtained by performing 1000 bootstrap
replicates of the aligned sequences. For parsimony
analyses, measures that were calculated include tree
length (TL), retention index (RI), consistency index
(CI), rescaled consistency index (RC) and homoplasy
index (HI). Botryosphaeria ribis Grossenb. & Duggar
was used as the outgroup to root all trees.
A Partition Homogeneity Test (Farris et al. 1994), of
all possible combinations, consisting of 1000 replicates
on all informative characters was conducted in PAUP
to determine if the LSU, ITS and EF-1α data sets were
combinable. All sequences of Mycosphaerella spp.
used in this study have been deposited in GenBank
(Table. 1). Sequence alignments and trees of the LSU,
ITS, EF-1α and ACT have been deposited in TreeBASE
(accession numbers: LSU = SN2535, ITS = SN2534,
EF-1α = SN2536, ACT = SN2537).
Parsimony and distance analyses of combined
DNA sequence alignments were conducted in PAUP.
Parsimony analyses of all DNA sequence alignments
were identical to those described earlier. For distance
analyses, Modeltest v. 3.04 (Posada & Crandall
1998) was used to determine the best evolutionary
model to fit the combined DNA sequence alignment. A
neighbour-joining analysis with an evolutionary model
was conducted in PAUP. Here, the distance measure
was a general time-reversible (GTR) and the proportion
of sites assumed to be invariable (I) was 0.4919,
identical sites were removed proportionally to base
frequencies estimated from all sites, rates of variable
sites assumed to follow a gamma distribution (G) with
shape parameter of 0.6198. Ties (if encountered) were
broken randomly.
RESULTS
DNA sequencing and phylogenetic analysis
Large Subunit (LSU) phylogeny: The LSU alignment
had a total length of 1714 characters. An indel of 383
bp present in M. ohnowa Crous & M.J. Wingf. (CBS
112973) and Mycosphaerella mexicana Crous (CBS
110502) was excluded from the analyses. In the LSU
data set, 1075 characters were constant while 77
characters were parsimony-uninformative and 179
characters were parsimony-informative. Parsimony
analysis of the LSU data set resulted in the retention
of thirty most parsimonious trees (TL = 663, CI =
0.519, RI = 0.878, RC = 0.456). One of these trees
(Fig. 1) could be resolved into two clades (Clades
1−2). Clade 1, supported with a bootstrap value of 70
%, included Mycosphaerella isolates characterised by
Phaeophleospora Rangel (M. ambiphylla A. Maxwell,
M. suttoniae Crous & M.J. Wingf.), Colletogloeopsis
Crous & M.J. Wingf. [M. molleriana (Thüm.) Lindau, M.
vespa Carnegie & Keane, M. cryptica (Cooke) Hansf.],
Uwebraunia Crous & M.J. Wingf. [M. nubilosa (Cooke)
Hansf.], M. ohnowa, Readeriella Syd. & P. Syd. (M.
readeriellophora Crous & J.P. Mansilla), and Passalora
Fr. (M. tasmaniensis Crous & M.J. Wingf.) anamorphs.
The second major clade (Clade 2) resolved in the
LSU tree was well-supported with a bootstrap value
of 98 %. Mycosphaerella species in this clade also
grouped strongly following their anamorph associations.
Here Mycosphaerella isolates could be resolved
into several sub-clades also characterised by their
anamorph associations. These were Sonderhenia (M.
walkeri R.F. Park & Keane.), Pseudocercospora Speg.
[M. heimioides Crous & M.J. Wingf., M. heimii Crous,
M. crystallina Crous & M.J. Wingf., M. irregulariramosa
Crous & M.J. Wingf., M. colombiensis Crous & M.J.
Wingf., M. gracilis Crous & Alfenas, Pseudocercospora
robusta Crous & M.J. Wingf., Ps. natalensis Crous & T.
Coutinho, M. fori G.C. Hunter, Crous & M.J. Wingf., Ps.
basitruncata Crous, Ps. pseudoeucalyptorum Crous,
Ps. eucalyptorum Crous, M.J. Wingf., Marasas & B.
Sutton., Ps. paraguayensis (Koboyashi) Crous, Ps.
basiramifera Crous] Passalora [Pass. eucalypti (Crous
& Alfenas) Crous & U. Braun, Pass. zambiae Crous &
T. Coutinho], and Dissoconium (M. lateralis Crous &
M.J. Wingf., M. communis Crous & J.P. Mansilla).
Internal Transcribed Spacer Region (ITS) phylogeny:
The ITS sequence alignment consisted of a total of 793
characters. Of these 499 characters were constant, 62
characters were variable and parsimony-uninformative
and 232 characters were parsimony-informative. A
185 bp indel was observed in isolates of M. gregaria
Carnegie & Keane (CBS 110501), M. endophytica
Crous & H. Smith (CBS 111519) and M. endophytica
(CMW 5225) and was excluded in the phylogenetic
analysis.
149
Teleomorph
Anamorph
M. africana
Unknown
M. ambiphylla
Phaeophleospora sp.
M. aurantia
M. colombiensis
M. communis
M. cryptica
M. crystallina
M. ellipsoidea
M. endophytica
Isolate No.a
Host
Country
Collector
GenBank Accession No.
CMW
CBS
STEU
LSU
ITS
ACT
EF-1a
3026
116155
795
E. viminalis
South Africa
P.W. Crous
DQ246258
DQ267577
DQ147608
DQ235098
4945
116154
794
E. viminalis
South Africa
P.W. Crous
DQ246257
AF309602
DQ147609
DQ235099
14180
110499
N/A
E. globulus
Australia
A. Maxwell
DQ246219
AY725530
DQ147669
DQ235103
Unknown
14460
110500
N/A
E. globulus
Australia
A. Maxwell
DQ246256
AY725531
DQ147610
DQ235097
Pseudocercospora colombiensis
4944
110969
1106
E. urophylla
Colombia
M.J. Wingfield
DQ204744
AY752149
DQ147639
DQ211660
11255
110967
1104
E. urophylla
Colombia
M.J. Wingfield
DQ204745
AY752147
DQ147640
DQ211661
14672
114238
10440
E. globulus
Spain
J.P. Mansilla
DQ246262
AY725541
DQ147655
DQ235141
14673
110976
849
E. cladocalyx
South Africa
P.W. Crous
DQ246261
AY725537
DQ147654
DQ235140
Dissoconium commune
Colletogloeopsis nubilosum
Pseudocercospora crystallina
Uwebraunia ellipsoidea
Pseudocercosporella endophytica
3279
110975
936
E. globulus
Australia
A.J. Carnegie
DQ246222
AF309623
DQ147674
DQ235119
2732
N/A
355
Eucalyptus sp.
Chile
M.J. Wingfield
N/A
AF309622
N/A
N/A
3042
N/A
800
E. bicostata
South Africa
M.J. Wingfield
DQ204746
DQ267578
DQ147637
DQ211662
3033
681.95
802
E. bicostata
South Africa
M.J. Wingfield
DQ204747
AY490757
DQ147636
DQ211663
4934
N/A
1224
Eucalyptus sp.
South Africa
Unknown
DQ246253
AF309592
DQ147647
DQ235129
5166
N/A
1225
Eucalyptus sp.
South Africa
Unknown
DQ246254
AF309593
DQ147648
DQ235127
14912
111519
1191
Eucalyptus sp.
South Africa
P.W. Crous
DQ246255
DQ267579
DQ147646
DQ235131
5225
N/A
1192
Eucalyptus sp.
South Africa
P.W. Crous
DQ246252
DQ267580
DQ147649
DQ235128
M. flexuosa
Unknown
5224
111012
1109
E. globulus
Colombia
M.J. Wingfield
DQ246232
AF309603
DQ147653
DQ235126
M. fori
Pseudocercospora sp.
9095
N/A
N/A
E. grandis
South Africa
G.C. Hunter
DQ204748
AF468869
DQ147618
DQ211664
9096
N/A
N/A
E. grandis
South Africa
G.C. Hunter
DQ204749
DQ267581
DQ147619
DQ211665
M. gracilis
Pseudocercospora gracilis
14455
243.94
730
E. urophylla
Indonesia
A.C. Alfenas
DQ204750
DQ267582
DQ147616
DQ211666
M. grandis
Unknown
8557
N/A
N/A
E. globulus
Chile
A.Rotella
DQ246241
DQ267583
DQ147644
DQ235108
8554
N/A
N/A
E. globulus
Chile
M.J. Wingfield
DQ246240
DQ267584
DQ147643
DQ235107
M. gregaria
Unknown
14462
110501
N/A
E. globulus
Australia
A. Maxwell
DQ246251
DQ267585
DQ147650
DQ235130
M. heimii
Pseudocercospora heimii
4942
110682
760
Eucalyptus sp.
Madagascar
P.W. Crous
DQ204751
AF309606
DQ147638
DQ211667
M. heimioides
Pseudocercospora heimioides
14776
111364
N/A
Eucalyptus sp.
Indonesia
M.J. Wingfield
DQ204752
DQ267586
DQ147632
DQ211668
3046
111190
1312
Eucalyptus sp.
Indonesia
M.J. Wingfield
DQ204753
AF309609
DQ147633
DQ211669
7163
114356
10902
E. saligna
New Zealand
K. Dobbie
DQ246247
AY725546
N/A
N/A
7164
114415
10922
E. saligna
New Zealand
K. Dobbie
DQ246248
AY725547
DQ147627
DQ235132
M. intermedia
M. irregulariramosa
M. ohnowa
M. keniensis
Unknown
Pseudocercospora irregulariramosa
Unknown
Unknown
4943
114774
1360
E. saligna
South Africa
M.J. Wingfield
DQ204754
AF309607
DQ147634
DQ211670
5223
N/A
1362
E. saligna
South Africa
M.J. Wingfield
DQ204755
AF309608
DQ147635
DQ211671
4937
112896
1004
E. grandis
South Africa
M.J. Wingfield
N/A
AF309604
DQ147662
DQ235125
4936
112973
1005
E. grandis
South Africa
M.J. Wingfield
DQ246231
AF309605
DQ147661
DQ235124
5147
111001
1084
E. grandis
Kenya
T. Coutinho
DQ246259
AF309601
DQ147611
DQ235100
HUNTER ET AL.
150
Table 1. Isolates of Mycosphaerella used in this study for DNA sequencing and phylogenetic analysis.
Table 1. (Continued).
Teleomorph
Anamorph
M. lateralis
Dissoconium dekkeri
Isolate No.a
Host
Country
Collector
GenBank Accession No.
CMW
CBS
STEU
LSU
ITS
ACT
EF-1a
14906
110748
825
E. grandis ×
E. saligna
South Africa
G. Kemp
DQ204768
AF173315
DQ147651
DQ211684
5164
111169
1232
E. globulus
Zambia
T. Coutinho
DQ246260
AY25550
DQ147652
DQ235139
M. madeirae
Pseudocercospora sp.
14458
112895
3745
E. globulus
Madeira
S. Denman
DQ204756
AY725553
DQ147641
DQ211672
M. marksii
Unknown
14781
682.95
842
E. grandis
South Africa
G. Kemp
DQ246249
DQ267587
DQ147624
DQ235133
5150
110920
935
E. botryoides
Australia
A.J. Carnegie
DQ246250
AF309588
DQ147625
DQ235134
5230
N/A
782
E. botryoides
Australia
A.J. Carnegie
DQ246246
DQ267588
DQ147626
DQ235135
14461
110502
N/A
E. globulus
Australia
A. Maxwell
DQ246237
AY725558
DQ147660
DQ235123
M. mexicana
Unknown
M. readeriellophora
Readeriella readeriellophora
14233
114240
10375
E. globulus
Spain
J.P. Mansilla
DQ246238
AY725577
DQ147658
DQ235117
M. molleriana
Colletogloeopsis molleriana
4940
111164
1214
E. globulus
Portugal
S. McCrae
DQ246220
AF309620
DQ147671
DQ235104
2734
111132
784
E. globulus
U. S. A.
M.J. Wingfield
DQ246223
AF309619
DQ147670
DQ235105
M. nubilosa
Uwebraunia juvenis
3282
116005
937
E. globulus
Australia
A.J. Carnegie
DQ246228
AF309618
DQ147666
DQ235111
9003
114708
N/A
E. nitens
South Africa
G.C. Hunter
DQ246229
AF449099
DQ147667
DQ235112
M. parkii
Stenella parkii
14775
387.92
353
E. grandis
Brazil
M.J. Wingfield
DQ246245
AY626979
DQ147612
DQ235137
M. parva
Unknown
14459
110503
N/A
E. globulus
Australia
A. Maxwell
DQ246243
AY626980
DQ147645
DQ235110
14917
116289
10935
Eucalyptus sp.
South Africa
P.W. Crous
DQ246242
AY725576
DQ147642
DQ235109
M. suberosa
Unknown
5226
436.92
515
E. dunnii
Brazil
M.J. Wingfield
DQ246235
AY626985
DQ147656
DQ235101
7165
N/A
N/A
E. muelleriana
New Zealand
Unknown
DQ246236
DQ267589
DQ147657
DQ235102
N/A
1346
Eucalyptus sp.
Indonesia
M.J. Wingfield
DQ246227
AF309621
DQ147673
DQ235116
Phaeophleospora epicoccoides
5348
M. vespa
Colletogloeopsis sp.
11558
117924
N/A
E. globulus
Tasmania
Unknown
DQ246221
DQ267590
DQ147668
DQ235106
M. tasmaniensis
Passalora tasmaniensis
14780
111687
1555
E. nitens
Tasmania
M.J. Wingfield
DQ246233
DQ267591
DQ147676
DQ235121
14663
114556
N/A
E. nitens
Tasmania
M.J. Wingfield
DQ246234
DQ267592
DQ147677
DQ235122
M. toledana
Phaeophleospora toledana
14457
113313
N/A
Eucalyptus sp.
Spain
P.W. Crous
DQ246230
AY725580
DQ147672
DQ235120
M. walkerii
Sonderhenia eucalypticola
20333
N/A
N/A
E. globulus
Chile
M.J. Wingfield
DQ267574
DQ267593
DQ147630
DQ235095
20334
N/A
N/A
E. globulus
Chile
M.J. Wingfield
DQ267575
DQ267594
DQ147631
DQ235096
Unknown
Passalora eucalypti
14907
111306
1457
E. saligna
Brazil
P.W. Crous
DQ246244
AF309617
DQ147678
DQ235138
Unknown
Passalora zambiae
14782
112971
1227
E. globulus
Zambia
T. Coutinho
DQ246264
AF725523
DQ147675
DQ235136
Unknown
Pseudocercospora epispermogonia
14778
110750
822
E. grandis ×
E. saligna
South Africa
G. Kemp
DQ204757
DQ267596
DQ147629
DQ211673
14786
110693
823
E. grandis ×
E. saligna
South Africa
G. Kemp
DQ204758
DQ267597
DQ147628
DQ211674
11687
113992
N/A
E. nitens
New Zealand
M. Dick
DQ246225
DQ267598
DQ147664
DQ235115
Unknown
Phaeophleospora eucalypti
151
A MULTI-GENE PHYLOGENY OF MYCOSPHAERELLA
M. suttonii
Teleomorph
Isolate No.a
Anamorph
Host
Country
CMW
CBS
STEU
14910
111692
1582
Eucalyptus sp.
New Zealand
14914
114664
1202
E. grandis
Colombia
Collector
GenBank Accession No.
LSU
ITS
ACT
EF-1a
M.J. Wingfield
DQ246224
DQ267599
DQ147663
DQ235114
M.J. Wingfield
DQ204759
DQ267600
DQ147622
DQ211675
Unknown
Pseudocercospora basitruncata
14785
111280
1203
E. grandis
Colombia
M.J. Wingfield
DQ204760
DQ267601
DQ147621
DQ211676
Unknown
Pseudocercospora basiramifera
5148
N/A
N/A
E. pellita
Thailand
M.J. Wingfield
DQ204761
AF309595
DQ147607
DQ211677
Unknown
Pseudocercospora eucalyptorum
5228
110777
16
E. nitens
South Africa
P.W. Crous
DQ204762
AF309598
DQ147614
DQ211678
Unknown
Pseudocercospora natalensis
14777
111069
1263
E. nitens
South Africa
T. Coutinho
DQ267576
N/A
DQ147620
N/A
14784
111070
1264
E. nitens
South Africa
T. Coutinho
DQ204763
AF309594
DQ147623
DQ211679
Unknown
Pseudocercospora paraguayensis
14779
111286
1459
E. nitens
Brazil
P.W. Crous
DQ204764
DQ267602
DQ147606
DQ211680
Unknown
Pseudocercospora
pseudoeucalyptorum
14908
114242
10390
E. globulus
Spain
J.P. Mansilla
DQ204765
AY725526
DQ147613
DQ211681
14911
114243
10500
E. nitens
New Zealand
W. Gams
DQ204766
AY725527
DQ147615
DQ211682
Unknown
Pseudocercospora robusta
5151
111175
1269
E. robusta
Malaysia
M.J. Wingfield
DQ204767
AF309597
DQ147617
DQ211683
Unknown
Readeriella novaezelandiae
14913
114357
10895
E. botryoides
New Zealand
M.A. Dick
DQ246239
DQ267603
DQ147659
DQ235118
Botryosphaeria ribis
Fusicoccum ribis
7773
N/A
N/A
Ribus sp.
U. S. A.
G. Hudler.
DQ246263
DQ267604
DQ267605
DQ235142
aCMW:
Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.
CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
STEU: Culture collection of Stellenbosch University, South Africa. Isolate numbers from Crous (1998).
N/A: Not available
HUNTER ET AL.
152
Table 1. (Continued).
A MULTI-GENE PHYLOGENY OF MYCOSPHAERELLA
A heuristic search of the ITS data set resulted in
the retention of four most parsimonious trees (TL =
871, RI = 0.782, CI = 0.358, RC = 0.280). One of these
phylogenetic trees (Fig. 2) generated by parsimony
analysis of the ITS alignment could be resolved into
two monophyletic clades (Clades 1−2). Clade 1 was
only weakly supported with a bootstrap value of 50 %
after 1000 bootstrap replicates. Clade 1 could be further
resolved into several smaller sub-clades where isolates
grouped strongly based on their anamorph affiliations.
These included Sonderhenia, Pseudocercospora,
Passalora, Uwebraunia/Pseudocercosporella, Stenella,
Readeriella, Phaeophleospora and Colletogloeopsis.
The second monophyletic clade (Clade 2) grouped
sister to the first larger monophyletic clade and
contained isolates of M. lateralis and M. communis
(Dissoconium anamorphs). This clade was wellsupported with a bootstrap value of 100 % after 1000
bootstrap replicates.
Translation Elongation factor 1-alpha (EF -1α)
phylogeny: The EF-1α alignment contained 373
characters. Of these, 41 characters were constant, 23
characters were variable and parsimony-uninformative
and 309 characters were parsimony-informative.
Heuristic searches resulted in the retention of six most
parsimonious trees (TL = 3194, RI = 0.777, CI = 0.345,
RC = 0.268), one of which is shown (Fig. 3). Species
of Mycosphaerella could be resolved into three clades
(Clades 1−3).
M. ambiphylla CBS 110499
M. molleriana CBS 111164
M. vespa CBS 117924
M. molleriana CBS 111132
75
99 Phaeoph. eucalypti CBS 111692
Phaeoph. eucalypti CBS 113992
64
M. nubilosa CBS 116005
98
M. nubilosa CBS 114708
M. toledana CBS 113313
63
M. suttoniae CMW 5348
M. cryptica CBS 110975
100
M.
ohnowa
CBS
112973
Clade 1
M. flexuosa CBS 111012
M. suberosa CBS 436.92
98
M. suberosa CMW 7165
61
70
M. mexicana CBS 110502
100
M. readeriellophora CBS 114240
Read. novaezelandiae CBS 114357
100
M. tasmaniensis CBS 111687
M. tasmaniensis CBS 114556
M. grandis CMW 8554
M. parva CBS 116289
100
M. grandis CMW 8557
M. parva CBS 110503
M. walkeri CMW 20333
M. walkeri CMW 20334
M. heimioides CBS 111190
M. heimii CBS 110682
79
M. heimioides CBS 111364
M. crystallina CBS 681.95
82 M. crystallina CMW 3042
M. irregulariramosa CBS 114774
99
M. irregulariramosa CMW 5223
M. colombiensis CBS 110969
M. colombiensis CBS 110967
M. gracilis CBS 243.94
92
Ps. robusta CBS 111175
Ps. natalensis CBS 111069
M. fori CMW 9095
Ps. basitruncata CBS 111280
Ps. natalensis CBS 111070
83
Ps. basitruncata CBS 114664
Ps. pseudoeucalyptorum CBS 114242
61
Ps. eucalyptorum CBS 110777
Ps. pseudoeucalyptorum CBS 114243
M. fori CMW 9096
88
Ps. paraguayensis CBS 111286
Ps. basiramifera CMW 5148
Pass. eucalypti CBS 111306
M. lateralis CBS 110748
100
M. lateralis CBS 111169
M. communis CBS 110976
M. communis CBS 114238
54
M.
parkii
CBS
387.92
73
M. madeirae CBS 112895
M. marksii CMW 5230
M. intermedia CBS 114356
Clade 2
M. intermedia CBS 114415
62
M. marksii CBS 110920
M. marksii CBS 682.95
98
99
Ps. epispermogonia CBS 110750
Ps. epispermogonia CBS 110693
Pass. zambiae CBS 112971
M. gregaria CBS 110501
100 M. endophytica CMW 5225
M. endophytica CBS 111519
M. ellipsoidea CMW 4934
M. ellipsoidea CMW 5166
92 M. aurantia CBS 110500
M. africana CBS 116154
100
89
M. africana CBS 116155
M. keniensis CBS 111001
B. ribis CMW 7773
100
Phaeophleospora
Colletogloeopsis
Phaeophleospora
Uwebraunia
Phaeophleospora
Colletogloeopsis
Readeriella
Passalora
Sonderhenia
Pseudocercospora
Pseudocercospora
Passalora
Dissoconium
Stenella /
Pseudocercospora
Passalora
Pseudocercosporella /
Uwebraunia
5 changes
Fig. 1. Phylogram obtained from the Large Subunit (LSU) rDNA sequence alignment of Mycosphaerella spp. occurring on Eucalyptus leaves
showing two well-supported main clades (Clades 1−2). Tree length = 663, CI = 0.519, RI = 0.878, RC = 0.456. Bootstrap values based on 1000
replicates are indicated above branches. Anamorph affinities are indicated next to the vertical lines.
153
HUNTER ET AL.
Clade 1 was weakly supported with a bootstrap value
of 67 %. This clade contained Mycosphaerella isolates
represented by Pseudocercospora, Sonderhenia,
Phaeophleospora, Colletogloeopsis, Uwebraunia,
Readeriella and Passalora anamorphs. Clade 2 was
sister to Clade 1 and had a higher bootstrap support of
80 %. Within this clade, Mycosphaerella isolates could
be separated into three sub-clades that were wellsupported. These three sub-clades contained species
of Mycosphaerella that produced Pseudocercosporella,
Uwebraunia, Pseudocercospora, Passalora and
Stenella anamorphs. Clade 3 with bootstrap support of
80 % included isolates of M. lateralis and M. communis
and was basal to Clades 1 and 2.
Actin (ACT) phylogeny: The aligned ACT sequence
dataset contained a total of 294 characters. Of
these, 135 characters were constant, 30 characters
were variable and parsimony-uninformative and 129
characters were parsimony-informative. Heuristic
100
50
M. parva CBS 116289
M. grandis CMW 8554
M. grandis CMW 8557
M. parva CBS 110503
100
M. walkeri CMW 20333
M. walkeri CMW 20334
M. heimioides CBS 111364
M. heimioides CBS 111190
M. crystallina CBS 681.95
M. crystallina CMW 3042
M. irregulariramosa CBS 114774
88
M. irregulariramosa CMW 5223
M. heimii CBS 110682
M. colombiensis CBS 110969
M. colombiensis CBS 110967
M. gracilis CBS 243.94
Ps. pseudoeucalyptorum CBS 114242
Ps. pseudoeucalyptorum CBS 114243
M. fori CMW 9095
57
M. fori CMW 9096
Ps. robusta CBS 111175
Ps. eucalyptorum CBS 110777
80
Ps. paraguayensis CBS 111286
76
Ps. basiramifera CMW 5148
75
Ps natalensis CBS 111070
Ps. basitruncata CBS 114664
Ps. basitruncata CBS 111280
Pass. eucalypti CBS 111306
M. aurantia CBS 110500
100 M. africana CBS 116155
M. africana CBS 116154
M. keniensis CBS 111001
78
M. gregaria CBS 110501
M. endophytica CBS 111519
M. endophytica CMW 5225
100
M. ellipsoidea CMW 4934
M. ellipsoidea CMW 5166
M. marksii CBS 682.95
Ps. epispermogonia CBS 110693
Ps. epispermogonia CBS 110750
100
M. marksii CMW 5230
M intermedia CBS 114356
98
M. intermedia CBS 114415
M. marksii CBS 110920
M. parkii CBS 387.92
M. madeirae CBS 112895
100
M. suberosa CBS 436.92
M. suberosa CMW 7165
M. mexicana CBS 110502
M. readeriellophora CBS 114240
100
Read. novaezelandiae CBS 114357
M. ambiphylla CBS 110499
100 M. vespa CBS 117924
M. molleriana CBS 111132
M. molleriana CBS 111164
M. cryptica CBS 110975
100 M. nubilosa CBS 116005
59
59
M. nubilosa CBS 114708
72
Phaeoph. eucalypti CBS 111692
Phaeoph. eucalypti CBS 113992
M. toledana CBS 113313
M. suttoniae CMW 5348
100 M. ohnowa CBS 112973
100
M. ohnowa CBS 112896
M. flexuosa CBS 111012
100
M. tasmaniensis CBS 111687
M. tasmaniensis CBS 114556
Pass. zambiae CBS 112971
92 M. lateralis CBS 110748
M. lateralis CBS 111169
M. communis CBS 110976
85
M. communis CBS 114238
B. ribis CMW 7773
100
100
searches of the aligned ACT dataset resulted in the
retention of six most parsimonious trees (TL = 1007,
RI = 0.682, CI = 0.235, RC = 0.160). One of these
trees, shown in Fig. 4, was very poorly resolved and
all deeper nodes were present in a basal polytomy.
However, certain smaller clades were resolved and
these included a clade including M. fori, M. gracilis, Ps.
eucalyptorum, Ps. pseudoeucalyptorum, Ps. robusta,
Ps. basitruncata, Ps. natalensis, Ps. basiramifera and
Ps. paraguayensis. This clade was supported with a
bootstrap value of only 67 %. Another clade supported
with a bootstrap value of 100 % contained isolates of M.
ellipsoidea Crous & M.J. Wingf., M. endophytica and M.
gregaria. Isolates of M. ambiphylla, M. molleriana and
M. vespa also clustered together with 100 % bootstrap
support. Isolates of M. intermedia M.A. Dick & Dobbie,
M. marksii Carnegie & Keane and Pseudocercospora
epispermogonia Crous & M. J. Wingf. grouped together
in a clade that was supported with a bootstrap value of
Sonderhenia
Pseudocercospora
Pseudocercospora
Passalora
Pseudocercosporella /
Uwebraunia
Pseudocercospora /
Stenella
Readeriella
Phaeophleospora /
Colletogloeopsis /
Uwebraunia
Phaeophleospora
Passalora
Passalora
Dissoconium
5 changes
Fig. 2. Phylogram obtained from the Internal Transcribed Spacer (ITS) DNA sequence alignment of Mycosphaerella spp. occurring on Eucalyptus
leaves indicating two monophyletic clades (Clades 1−2). Tree length = 871, CI = 0.358, RI = 0.782, RC = 0.280.
154
A MULTI-GENE PHYLOGENY OF MYCOSPHAERELLA
84 %. Isolates of M. flexuosa Crous & M.J. Wingf., M.
lateralis and M. communis were also accommodated in
a well-supported clade with a bootstrap value of 99 %.
Isolates of M. grandis Carnegie & Keane and M. parva
R.F. Park & Keane were also resolved into a clade with
a bootstrap value of 99 %.
Phylogeny of combined data set: A partition
homogeneity test of the combined LSU, ITS and EF1α alignment conducted in PAUP resulted in a P-value
of 0.001 for all possible combinations of the LSU, ITS
and EF-1α DNA alignments. This value is less than the
conventionally accepted P-value of P > 0.05 required to
combine data. However, several studies have accepted
a P-value of 0.001 or greater and have further stated
that the conventional P-value of 0.005 is inordinately
conservative (Cunningham 1997, Darlu & Lecointre
2002, Dettman et al. 2003). Thus, the LSU, ITS and EF1α DNA sequence data sets were combined. The ACT
dataset was omitted from the combined data set due to
the lack of resolution among species of Mycosphaerella.
Therefore, the combined LSU, ITS and EF-1α data set
had a total length of 2880 characters. Of these, 1459
were constant, 150 were variable and parsimonyuninformative and 701 characters were parsimonyinformative. An indel of 382 bp was excluded for M.
ohnowa CBS 112973 and M. mexicana CBS 110502
and another indel of 186 bp was excluded for M.
gregaria CBS 110501 and M. endophytica CMW 5225
and CBS 111519. A parsimony analysis resulted in the
retention of ten most parsimonious trees (TL = 1677, CI
= 0.384, RI = 0.817, RC = 0.314, HI = 0.616). One of
these trees (Fig. 5) exhibited a similar topology to that
obtained from the LSU alignment. From the analysis
of the combined data set, isolates of Mycosphaerella
could again be resolved into two clades (Clades
1−2) (Fig. 5). Clade 1 was poorly supported with a
bootstrap value of only 66 % and the same isolates
were contained in this clade as in the LSU Clade 1
86
Ps. natalensis CBS 111070
Ps. basitruncata CBS 114664
Ps. basitruncata CBS 111280
Ps. pseudoeucalyptorum CBS 114242
97 Ps. pseudoeucalyptorum CBS 114243
98
78 Ps. eucalyptorum CBS 110777
M. gracilis CBS 243.94
76
Ps. robusta CBS 111175
M. fori CMW 9095
72
100 M. fori CMW 9096
Ps. paraguayensis CBS 111286
Ps. basiramifera CMW 5148
100 M. walkeri CMW 20333
M. walkeri CMW 20334
M. heimioides CBS 111364
60
M. heimioides CBS 111190
100 M. crystallina CBS 681.95
M. crystallina CMW 3042
M. heimii CBS 110682
100
M. irregulariramosa CBS 114774
70
M. irregulariramosa CMW 5223
M. colombiensis CBS 110969
M. colombiensis CBS 110967
M. aurantia CBS 110500
100 M. africana CBS 116155
Clade 1
M. africana CBS 116154
M. keniensis CBS 111001
100
M. suberosa CBS 436.92
M. suberosa CMW 7165
67
M. ambiphylla CBS 110499
100 M. molleriana CBS 111132
M. vespa CBS 117924
M. molleriana CBS 111164
M. grandis CMW 8554
M. grandis CMW 8557
100
M. parva CBS 116289
86
M. parva CBS 110503
87 100 M. nubilosa CBS 116005
M. nubilosa CBS 114708
100 Phaeoph. eucalypti CBS 111692
Phaeoph. eucalypti CMW 11687
M. suttoniae CMW 5348
81
M. cryptica CBS 110975
79
M. toledana CBS 113313
100
M. readeriellophora CBS 114240
Raed. novaezelandiae CBS 114357
M. tasmaniensis CBS 111687
65
M. tasmaniensis CBS 114556
M. mexicana CBS 110502
79
M. ohnowa CBS112973
M. ohnowa CBS 112896
100
Clade 2
M. flexuosa CBS 111012
Pass. eucalypti CBS 111306
M. ellipsoidea CMW 5166
75
100 M. endophytica CMW 5225
M. ellipsoidea CMW 4934
M. gregaria CBS 110501
M. endophytica CBS 111519
Ps. epispermogonia CBS 110693
80
M. intermedia CBS 114415
100 M. marksii CBS 682.95
Ps. epispermogonia CBS 110750
73
M. marksii CBS 110920
M. marksii CMW 5230
89
Pass. zambiae CBS 112971
Clade 3
M. parkii CBS 387.92
M. madeirae CBS 112895
100 M. lateralis CBS 111169
81
M. lateralis CBS 110748
M. communis CBS 110976
M. communis CBS 114238
B. ribis CMW 7773
100
Pseudocercospora
Sonderhenia
Pseudocercospora
Phaeophleospora /
Colletogloeopsis
Uwebraunia
Phaeophleospora
Phaeophleospora /
Colletogloeopsis
Readeriella
Passalora
Passalora
Pseudocercosporella /
Uwebraunia
Pseudocercospora
Passalora / Stenella
Pseudocercospora
Dissoconium
10 changes
Fig. 3. Phylogram obtained from the Elongation factor 1-alpha (EF-1α) DNA sequence alignment of Mycosphaerella spp. occurring on Eucalyptus
leaves showing three main clades. Tree length = 3194, CI = 0.345, RI = 0.777, RC = 0.268.
155
HUNTER ET AL.
(Fig. 1). Clade 2 of the combined phylogenetic tree was
well-supported with a bootstrap value of 81 %. This
clade could be further resolved into several smaller
well-supported sub-clades containing Mycosphaerella
isolates that grouped according to their anamorph
associations (Fig. 5). Neighbour-joining analysis
yielded a phylogenetic tree with the same topology as
the most parsimonious trees (data not shown). Here, all
Mycosphaerella spp. could be resolved into two main
clades (Clade 1−2), similar to those of the parsimony
analysis (Fig. 5). Mycosphaerella spp. could be further
sub-divided into several sub-clades corresponding to
their anamorph associations, similar to those observed
for the parsimony analysis.
97
DISCUSSION
Results of this study represent the first attempt to
employ DNA sequence data from a relatively large
number of nuclear gene regions in order to consider
the phylogenetic relationships for Mycosphaerella
spp. occurring on Eucalyptus leaves. Other similar
studies have relied entirely on sequence data of the
ITS region (Crous et al. 1999, 2001, 2004a, and 2006
– this volume, Hunter et al. 2004b). Although the ITS
region offers sufficient resolution to distinguish most
taxa, it has not been adequate to separate cryptic taxa
in Mycosphaerella (Crous et al. 2004b). Results of the
present study showed that combined DNA sequence
65
M. africana CBS 116154
M. africana CBS 116155
M. aurantia CBS 110500
M. keniensis CBS 111001
100 M. colombiensis CBS 110969
M. colombiensis CBS 110967
70 M. crystallina CMW 3042
M. heimii CBS 110682
M. crystallina CBS 681.95
51
M. irregulariramosa CBS 114774
M. heimioides CBS 111364
96
M. heimioides CBS 111190
M. irregulariramosa CMW 5223
100
M. walkeri CMW 20334
M. walkeri CMW 20333
88 M. fori CMW 9095
M. fori CMW 9096
96
M. gracilis CBS 243.94
72
Ps. eucalyptorum CBS 110777
96
Ps. pseudoeucalyptorum CBS 114242
70
Ps. pseudoeucalyptorum CBS 114243
Ps. robusta CBS 111175
Ps.
basitruncata
CBS 114664
67
100
Ps. basitruncata CBS 111280
Ps. natalensis CBS 111070
97
Ps. basiramifera CMW 5148
Ps. paraguayensis CBS 111286
Pass. eucalypti CBS 111306
M. ellipsoidea CMW 4934
M. endophytica CMW 5225
100
M. ellipsoidea CMW 5166
M. endophytica CBS 111519
M. gregaria CBS 110501
Pass. zambiae CBS 112971
M. ambiphylla CBS 110499
100
M. molleriana CBS 111132
M. vespa CBS 117924
M. molleriana CBS 111164
100
M. nubilosa CBS 116005
M. nubilosa CBS 114708
57
99 Phaeoph. eucalypti CBS 113992
Phaeoph. eucalypti CBS 111692
M. suttoniae CMW 5348
M. cryptica CBS 110975
M. toledana CBS 113313
M. intermedia CBS 114415
100 M. marksii CMW 5230
M. marksii CBS 682.95
84
M. marksii CBS 110920
99 Ps. epispermogonia CBS 110750
Ps. epispermogonia CBS 110693
M. madeirae CBS 112895
M. parkii CBS 387.92
M. mexicana CBS 110502
M. flexuosa CBS 111012
100
M. lateralis CBS 110748
99
M. lateralis CBS 111169
99
M. communis CBS 114238
M. communis CBS 110976
100
M. ohnowa CBS 112896
M. ohnowa CBS 112973
100
M. suberosa CMW 5226
M. suberosa CMW 7165
99
M. readeriellophora CBS 114240
Read. novaezelandiae CBS 114357
100 M. grandis CMW 8557
M. grandis CMW 8554
99
M. parva CBS 116289
M. parva CBS 110503
100
M. tasmaniensis CBS 111687
M. tasmaniensis CBS 114556
B. ribis CMW 7773
100
5 changes
Fig. 4. Phylogram obtained from the Actin (ACT) DNA sequence alignment of Mycosphaerella spp. occurring on Eucalyptus leaves. Tree length
= 1007, CI = 0.235, RI = 0.682, RC = 0.160.
156
A MULTI-GENE PHYLOGENY OF MYCOSPHAERELLA
data from the LSU, ITS, EF-1α gene regions offer
increased genetic resolution to study species concepts
in Mycosphaerella. However, genes such as the ACT,
did not support data emerging from the other loci
sequenced, and indicated variation within some clades
that were well supported by sequences of other loci
and morphological characteristics. These observations
led us to exclude ACT data from the final analyses. A
similar finding has also emerged from other studies
including greater numbers of Mycosphaerella species
(Crous & Groenewald, unpubl. data).
Mycosphaerella ambiphylla, M. molleriana and M.
vespa grouped together in a well-supported clade in the
phylogeny emerging from the combined alignment. This
was also true for the ITS, EF-1α and ACT phylogenies
where these isolates grouped in a distinct clade with a
100 % bootstrap support. Mycosphaerella molleriana
and M. vespa both have Colletogloeopsis anamorphs,
however, M. ambiphylla produces a Phaeophleospora
anamorph (Crous & Wingfield 1997a, Maxwell et al.
2003). Interestingly, the Phaeophleospora anamorph of
M. ambiphylla was differentiated from Colletogloeopsis
only by the fact that conidia are produced in a pycnidium
as opposed to an acervulus (Maxwell et al. 2003).
Application of conidiomatal structure to differentiate
anamorphs of Mycosphaerella has previously been
viewed with circumspection especially because
Mycosphaerella anamorphs can produce different
M. ambiphylla CBS 110499
M. molleriana CBS 111164
M. vespa CBS 117924
M. molleriana CBS 111132
Phaeoph. eucalypti CBS 111692
100 Phaeoph. eucalypti CBS 113992
89
73
100 M. nubilosa CBS 116005
M. nubilosa CBS 114708
M. toledana CBS 113313
M. cryptica CBS 110975
M. suttoniae CMW 5348
100
M. ohnowa CBS 112973
M. flexuosa CBS 111012
100 M. suberosa CBS 436.92
89
M. suberosa CMW 7165
M. mexicana CBS 110502
100
M. readeriellophora CBS 114240
Read. novaezelandiae CBS 114357
100
M. tasmaniensis CBS 111687
M. tasmaniensis CBS 114556
M. grandis CMW 8554
M. parva CBS 116289
100
M. grandis CMW 8557
M. parva CBS 110503
100 M. walkeri CMW 20333
M. walkeri CMW 20334
M. heimioides CBS 111190
88
97 M. heimioides CBS 111364
M. heimii CBS 110682
M. crystallina CBS 681.95
M. crystallina CMW 3042
99
M. irregulariramosa CBS 114774
M. irregulariramosa CMW 5223
100 M. colombiensis CBS 110969
M. colombiensis CBS 110967
M. gracilis CBS 243.94
96
Ps. robusta CBS 111175
M. fori CMW 9095
M. fori CMW 9096
Ps. pseudoeucalyptorum CBS 114242
Ps. pseudoeucalyptorum CBS 114243
Ps. eucalyptorum CBS 110777
99
Ps. basitruncata CBS 111280
Ps. natalensis CBS 111070
95
Ps. basitruncata CBS 114664
Ps. paraguayensis CBS 111286
Ps. basiramifera CMW 5148
Pass. eucalypti CBS 111306
58
57
M. parkii CBS 387.92
M. madeirae CBS 112895
M. marksii CMW 5230
99
M. intermedia CBS 114415
M. marksii CBS 110920
100 M. marksii CBS 682.95
Ps. epispermogonia CBS 110750
Ps. epispermogonia CBS 110693
M. gregaria CBS 110501
M. endophytica CMW 5225
100
M. endophytica CBS 111519
M. ellipsoidea CMW 4934
85
M. ellipsoidea CMW 5166
94 M. aurantia CBS 110500
100 M. africana CBS 116154
M. africana CBS 116155
M. keniensis CBS 111001
M. lateralis CBS 110748
100
M. lateralis CBS 111169
M. communis CBS 110976
72
M. communis CBS 114238
Pass. zambiae CBS 112971
100
Clade 1
66
Clade 2
81
B. ribis CMW 7773
Phaeophleospora /
Colletogloeopsis /
Uwebraunia
Readeriella
Passalora
Sonderhenia
Pseudocercospora
Pseudocercospora
Passalora
Stenella / Pseudocercospora
Pseudocercospora
Pseudocercosporella /
Uwebraunia
Dissoconium
10 changes
Fig. 5. Phylogram obtained from the combined LSU, ITS and EF-1α DNA sequence alignment of Mycosphaerella spp. occurring on Eucalyptus
leaves showing two main clades. Tree length = 1677, CI = 0.384, RI = 0.817, RC = 0.314.
157
HUNTER ET AL.
conidiomatal forms under differing environmental
conditions (Crous et al. 2000, Cortinas et al. 2006 – this
volume). Therefore, the placement of the M. ambiphylla
anamorph in Phaeophleospora is questioned and it
should be re-evaluated in terms of its morphological
similarities to Colletogloeopsis.
Ascospore germination patterns of M. ambiphylla,
M. molleriana and M. vespa are all similar, with germ
tubes that grow parallel to the long axis of the spore
and ascospores with a slight constriction at the median
septum, typical of a type C ascospore germination
pattern (Crous 1998, Carnegie & Keane 1998, Maxwell
et al. 2003). Furthermore, overlap is seen in ascospore
dimensions of the three species where those of M.
molleriana are (11−)12−14(−17) × (2.5−)3.5−4(−4.5)
µm, those of M. ambiphylla are (12−)14−15(−22)
×(3.5−)4.5−5(−6) µm and those of M. vespa 9.5−16.5
× 2.5−4 µm (Crous 1998, Carnegie & Keane 1998,
Maxwell et al. 2003). Leaf lesions of the three species
are also similar, pale brown to dark red-brown with
lesions of M. vespa and M. ambiphylla often producing
a red margin that was, however, not observed in M.
molleriana (Crous 1998, Carnegie & Keane 1998,
Maxwell et al. 2003). Morphological features of M.
ambiphylla, M. molleriana and M. vespa are also very
similar. This is supported in the DNA phylogeny of the
present study where these three species appear to
represent a single taxon and therefore suggest that
M. ambiphylla, M. molleriana and M. vespa should be
synonomised under M. molleriana, which is the oldest
epithet. We therefore reduce M. ambiphylla and M.
vespa to synonymy with M. molleriana as follows:
molleriana
(Thüm.)
Mycosphaerella
Natürliche Pfanzenfamilie, 1: 424. 1897.
Lindau,
≡ Sphaerella molleriana Thüm., Revista Inst. Sci. Lit. Coimbra
28: 31. 1881.
= Mycosphaerella vespa Carnegie & Keane, Mycol. Res. 102:
1275. 1998.
= Mycosphaerella ambiphylla A. Maxwell, Mycol. Res. 107: 354.
2003.
Anamorph: Colletogloeopsis molleriana Crous & M.J.
Wingf., Canad. J. Bot. 75: 670. 1997.
Mycosphaerella flexuosa has no known anamorph
(Crous 1998). An isolate of this fungus included in the
present study grouped together with isolates of M.
ohnowa in the LSU, ITS, EF-1α and combined data set
with high bootstrap support. This similarity was also
observed in a recent study of Mycosphaerella spp. on
Eucalyptus based on ITS sequence data (Crous et al.
2004a). Mycosphaerella ohnowa is also not known to
produce an anamorph (Crous et al. 2004a). Although
these two species are phylogenetically similar, they
can be distinguished from one another based on
different ascus and ascospore dimensions, ascospore
germination patterns and cultural characteristics (Crous
1998, Crous et al. 2004a). Although morphologically
distinct, it is interesting that these two taxa are
phylogenetically so closely related and might suggest
a recent speciation event.
Isolates of M. grandis and M. parva consistently
grouped together in a separate clade in all of the DNA
158
sequence data sets in this study. This has also been
shown by Crous et al. (2004a), where isolates of these
two species grouped together in a distinct clade based
on ITS DNA sequences. Mycosphaerella grandis was
originally described from E. grandis in Australia, and
recognised as a distinct species of Mycosphaerella due
to its lesion characteristics, and ascospore morphology
(Carnegie & Keane 1994). However, Crous (1998)
examined the type of M. grandis and M. parva and
found the two species to be congeneric, and reduced
them to synonymy under M. parva. Results from the
present study support the synonymy.
Mycosphaerella lateralis and M. communis, both
known to have Dissoconium anamorphs, showed
various phylogenetic placements in this study. From
the LSU phylogeny, M. lateralis and M. communis were
situated within a large Mycosphaerella clade sister to
a Pseudocercospora sub-clade. However, in the ITS
and EF-1α phylogenies the Dissoconium clade was
situated basal to the larger Mycosphaerella clade.
This is consistent with findings of Crous et al. (1999,
2000) where the Dissoconium clade also resided
outside the larger monophyletic Mycosphaerella clade.
The LSU gene region is well-known to be conserved
and to show less nucleotide differences than the ITS
and EF-1α gene regions. Although the house-keeping
genes investigated here lead to the conclusion that
Dissoconium could be different from Mycosphaerella s.
str., this proved not to be the case when LSU data were
considered. Dissoconium is morphologically identical to
Uwebraunia, and the separation of these two genera no
longer seems tenable. Only two species, M. ellipsoidea
and M. nubilosa, have Uwebraunia anamorphs (Crous
et al. 2004a). However, cultures of both species
produced these anamorphs only upon initial isolation,
and those that are currently available are sterile. In
contrast, strains with Dissoconium anamorphs readily
produce those in culture, and they usually sporulate
profusely. It appears that the status of Uwebraunia will
only be resolved once fresh, sporulating collections of
either M. ellipsoidea or M. nubilosa can be obtained.
Mycosphaerella spp. with Pseudocercospora
anamorphs grouped into three clades in all of the
phylogenies generated in this study. Species in the
Pseudocercospora clades have short branch lengths
arising from a common internode, suggesting that
they have speciated relatively recently from a common
ancestor (Ávila et al. 2005) and, most likely have coevolved with their Eucalyptus hosts as suggested by
Crous et al. (2000). Ávila et al. (2005) suggested that
Pseudocercospora may represent a monophyletic
lineage. But, results of this and other studies (AyalaEscobar et al. 2006) have shown that Pseudocercospora
is paraphyletic in Mycosphaerella and has evolved
more than once in the genus. The availability of new
DNA datasets for several gene regions are likely to
resolve cryptic species and species complexes within
Pseudocercospora, as has already been shown for the
M. heimii and the P. eucalyptorum species complexes
(Crous et al. 2000, 2004a).
Mycosphaerella heimioides, M. heimii, M. crystallina
and M. irregulariramosa are all morphologically similar
A MULTI-GENE PHYLOGENY OF MYCOSPHAERELLA
and are regarded as members of the M. heimii species
complex (Crous & Wingfield 1997b, Crous et al. 2001).
Previous studies based on ITS DNA sequence data have
demonstrated the phylogenetic relatedness of these
four species (Crous et al. 2001, Crous et al. 2004a).
However, bootstrap support for their phylogenetic
placement was low (Crous et al. 2004a). The phylogeny
of combined DNA sequence data in this study showed
that the four species in the M. heimii complex reside in
a well-supported clade (bootstrap support 97 %). The
short branch lengths indicate that the four species have
also recently diverged from a common ancestor.
In the phylogeny based on the combined sequence
data sets, M. gracilis grouped in a well-supported
Pseudocercospora clade that also included isolates
of Ps. robusta, M. fori, Ps. pseudoeucalyptorum, Ps.
eucalyptorum, Ps. basitruncata, Ps. natalensis, Ps.
paraguayensis and Ps. basiramifera. This is the first
study in which DNA sequence data for M. gracilis have
been incorporated into a phylogeny. In the ITS, EF-1α
and ACT phylogenies, M. gracilis was phylogenetically
most closely related to Ps. pseudoeucalyptorum.
However, M. gracilis (anamorph: Pseudocercospora
gracilis Crous & Alfenas) can be distinguished from
Ps. pseudoeucalyptorum by its single conidiophores
arising exclusively from secondary mycelium, which
is different to Ps. pseudoeucalyptorum in which
conidiophores arise from loose or dense fascicles of a
stroma (Crous 1998, Crous et al. 2004a). Furthermore,
conidia of Ps. gracilis are more septate, longer, and
more uniformly cylindrical in shape than those of Ps.
pseudoeucalyptorum (Crous 1998, Crous et al. 2004a).
Results of the present study clearly emphasise the fact
that species which are morphologically distinct, can be
very closely related.
An interesting result emerging from the
phylogenetic analyses in this study was the placement
of Pseudocercospora epispermogonia in relation
to Mycosphaerella marksii and Mycosphaerella
intermedia. Sequences for all but the ACT gene region
showed that these three taxa represent the same
phylogenetic species. Although it has previously been
suggested that M. marksii should have a Stenella
anamorph because of its proximity to M. parkii (Crous et
al. 2001), the current data suggest that this anamorph
could be Ps. epispermogonia. Crous & Wingfield (1996)
described Ps. epispermogonia from spermatogonia on
lesions colonised by M. marksii, but failed to link the two
states because single-ascospore cultures did not form
an anamorph in culture. Mycosphaerella intermedia
is morphologically similar to M. marksii, and probably
represents the same taxon. We therefore reduce M.
intermedia to synonymy with M. marksii as follows:
Mycosphaerella marksii Carnegie & Keane, Mycol.
Res. 98: 413−416. 1994.
= Mycosphaerella intermedia M. A. Dick & Dobbie, New Zealand
J. Bot. 39: 270. 2001.
Anamorph: Pseudocercospora epispermogonia Crous
& M.J. Wingf., Mycologia 88: 456. 1996.
Mycosphaerella africana, M. aurantia and M. keniensis
have no known anamorphs. Previous studies based on
ITS sequence data have suggested that M. africana
and M. keniensis grouped close to Mycosphaerella spp.
with Passalora anamorphs. It has thus been assumed
that M. africana and M. keniensis would have Passalora
anamorphs if they were to be found (Crous et al. 2000).
However, the phylogenies emerging from LSU, ITS
and EF-1α sequences and the combined data for the
three regions showed that M. africana, M. keniensis
and M. aurantia consistently group separately from the
Passalora anamorphs, close to a clade of isolates with
Uwebraunia and Pseudocercosporella anamorphs.
The association of these three taxa to Passalora is thus
doubted. Furthermore, the clade containing M. africana,
M. aurantia and M. keniensis is also well-supported and
seems to represent a single evolving lineage.
Moreover, results of the present study show that M.
aurantia and M. africana represent a single phylogenetic
species. These two species consistently grouped
together in all phylogenies with M. keniensis grouping
as a sister. Mycosphaerella aurantia was described
from leaves of E. globulus in south-western Australia
and is known only from this location (Maxwell et al.
2003). Morphologically, M. aurantia produces asci and
ascospores that are similar in size and morphology to
M. africana. However, the ascospores of M. aurantia are
not constricted at the median septum whereas those of
M. africana had such constrictions, and ascospores of
M. aurantia are longer (9−)11−12(−15) µm than those
of M. africana (7−)8−10(−11) µm (Crous 1998, Maxwell
et al. 2003). Furthermore, M. aurantia produces lateral
hyaline germ tubes that grow parallel to the long axis
of the ascospore and become slightly verrucose to
produce lateral branches upon prolonged incubation
(Maxwell et al. 2003). This is in contrast to ascospores
of M. africana that germinate in an irregular fashion
producing distinctly dark verrucose germ tubes from
different positions of the distorted ascospore (Crous
1998). It is intriguing that these two species, which
are morphologically quite distinct, would represent
a single phylogenetic species. Additional isolates of
these species are required to determine whether they
represent two distinct taxa or are conspecific.
Mycosphaerella gregaria was described from
leaves of E. grandis in Victoria, Australia (Carnegie
& Keane 1997). No anamorph has been observed
for this species (Carnegie & Keane 1997, Crous
1998). An isolate of M. gregaria, collected from E.
globulus in Australia, consistently grouped in a clade
with isolates of M. endophytica and M. ellipsoidea.
Mycosphaerella endophytica and M. ellipsoidea are
known to have Pseudocercosporella and Uwebraunia
anamorphs, respectively (Crous 1998). Based on
previous studies employing ITS sequence data,
isolates of M. endophytica grouped sister to isolates
of M. aurantia, M. ellipsoidea and M. africana (Crous
et al. 2004a). However, based on sequence data from
the four gene regions employed in this study, isolates
of M. endophytica grouped in a distinct well-supported
clade with M. ellipsoidea. This is interesting because
M. ellipsoidea has an Uwebraunia anamorph (Crous &
Wingfield 1996). Mycosphaerella endophytica and M.
159
HUNTER ET AL.
pseudoendophytica Crous & G. Hunter are the only
Mycosphaerella spp. occurring on Eucalyptus that
are known to have Pseudocercosporella anamorphs
(Crous 1998, Crous et al. 2006 – this volume).
Phylogenies emerging from analyses of sequences
for the four gene regions considered in this study
suggest that Mycosphaerella constitutes heterogenous
groups of which only a few are closely linked to
certain anamorph genera. It is evident that for the
larger part the evolution of the anamorph genera
within Mycosphaerella has been polyphyletic, and not
monophyletic as previously suggested. This can be seen
by the multiple evolution of anamorph genera such as
Passalora, Pseudocercospora, Phaeophleospora and
Stenella within Mycosphaerella (Crous et al. 2006).
It would thus not be advisable to predict anamorph
relationships based on the phylogenetic position within
Mycosphaerella. Not only has the same morphology
evolved more than once in the group, but disjunct
anamorph morphologies also frequently cluster
together (Crous et al. 2000, 2004a, 2006). This makes
the interpretation difficult, and predictions based on
position in clades unreliable.
The production of four nucleotide sequence data
sets for species of Mycosphaerella occurring on
Eucalyptus leaves should serve as a framework for
the more accurate taxonomic placement of these
fungi in future. The importance of species complexes
in Mycosphaerella has become more evident in this
genus in recent years (Crous et al. 2004a, b, 2006
– this volume). To study species complexes, variable
gene regions must be studied and the generation
of greater numbers of data sets should allow for
increased resolution at the species level. This in turn
will aid in the resolution of species complexes and
cryptic speciation. Studies of the deeper branches for
groups in Mycosphaerella can in future utilise sequence
data for the LSU region that have not previously
been available. These should provide a more lucid
indication and support for phenotypic characters that
are phylogenetically informative.
ACKNOWLEDGEMENTS
We thank the members of the Tree Protection Co-operative
Programme (TPCP), the National Research Foundation (NRF), the
Mellon Foundation and the THRIP initiative of the Department of
Trade and Industry, South Africa for financial support.
REFERENCES
Ayala-Escobar V, Yáñez-Morales M de Jesús, Braun U, Groenewald
JZ, Crous PW (2006). Pseudocercospora opuntiae sp. nov. the
causal organism of cactus leaf spot in Mexico. Fungal Diversity
21: 1–9.
Ávila A, Groenewald JZ, Trapero A, Crous PW (2005). Characterisation
and epitypification of Pseudocercospora cladosporioides, the
causal organism of Cercospora leaf spot of olives. Mycological
Research 109: 881−888.
Braun U, Crous PW, Dugan F, Groenewald JZ, De Hoog GS (2003)
Phylogeny and taxonomy of Cladosporium-like hyphomycetes,
including Davidiella gen. nov., the teleomorph of Cladosporium
s. str. Mycological Progress 2: 3−18.
160
Carbone I, Kohn LM (1999). A method for designing primer sets for
speciation studies in filamentous ascomycetes. Mycologia 91:
553–556.
Carnegie AJ, Ades PK, Ford R (2001). The use of RAPD-PCR
analysis for the differentiation of Mycosphaerella species from
Eucalyptus in Australia. Mycological Research 105: 1313–1320.
Carnegie AJ, Keane PJ (1994). Further Mycosphaerella species
associated with leaf diseases of Eucalyptus. Mycological
Research 98: 413−418.
Carnegie AJ, Keane PJ (1997). A revised Mycosphaerella gregaria
nom. nov. for M. aggregata on Eucalyptus. Mycological Research
101: 843−844.
Carnegie AJ, Keane PJ (1998). Mycosphaerella vespa sp. nov. from
diseased Eucalyptus leaves in Australia. Mycological Research
102: 1274−1276.
Cortinas MN, Crous PW, Wingfield BD, Wingfield MJ (2006). Multilocus
gene phylogenies and phenotypic characters distinguish two
fungi previously identified as Colletogloeopsis zuluensis causing
Eucalyptus cankers. Studies in Mycology 55: 133–146.
Crous PW (1998). Mycosphaerella spp. and their anamorphs
associated with leaf spot diseases of Eucalyptus. Mycologia
Memoir 21: 1−170.
Crous PW, Aptroot A, Kang JC, Braun U, Wingfield MJ (2000). The
genus Mycosphaerella and its anamorphs. Studies in Mycology
45: 107−121.
Crous PW, Groenewald JZ, Mansilla JP, Hunter GC, Wingfield MJ.
(2004a). Phylogenetic analysis of Mycosphaerella spp. and their
anamorphs occurring on Eucalyptus. Studies in Mycology 50:
195−214.
Crous PW, Groenewald JZ, Pongpanich K, Himaman W, Arzanlou
M, Wingfield MJ (2004b). Cryptic speciation and host specificity
among Mycosphaerella spp. occurring on Australian Acacia
species grown as exotics in the tropics. Studies in Mycology 50:
457−469.
Crous PW, Hong L, Wingfield BD, Wingfield, MJ (2001). ITS rDNA
phylogeny of selected Mycosphaerella species and their
anamorphs occurring on Myrtaceae. Mycological Research 105:
425−431.
Crous PW, Hong L, Wingfield MJ, Wingfield BD, Kang JC (1999).
Uwebraunia and Dissoconium, two morphologically similar
anamorph genera with different teleomorph affinity. Sydowia 51:
155−166.
Crous PW, Wingfield MJ (1996). Species of Mycosphaerella and their
anamorphs associated with leaf blotch disease of Eucalyptus in
South Africa. Mycologia 88: 441−458.
Crous PW, Wingfield MJ (1997a). Colletogloeopsis, a new
coelomycete genus to accommodate anamorphs of two species
of Mycosphaerella on Eucalyptus. Canadian Journal of Botany
75: 667−674.
Crous PW, Wingfield MJ (1997b). New species of Mycosphaerella
occurring on Eucalyptus leaves in Indonesia and Africa. Canadian
Journal of Botany 75: 781−790.
Crous PW, Wingfield MJ, Mansilla JP, Alfenas AC, Groenewald JZ
(2006). Phylogenetic reassessment of Mycosphaerella spp. and
their anamorphs occurring on Eucalyptus. II. Studies in Mycology
55: 99–131.
Cunningham CW (1997). Can three incongruence tests predict when
data should be combined? Molecular Biology and Evolution 14:
733−740.
Darlu P, Lecointre G (2002). When does the incongruence length
difference test fail?. Molecular Biology and Evolution 19:
432−437.
Dettman JR, Jacobson DJ, Taylor JW (2003). A multilocus
genealogical approach to phylogenetic species recognition in the
model eukaryote Neurospora. Evolution 57: 2703−2720.
Farris JS, Kallersjo M, Kluge AG, Bult C (1994). Testing significance
of Incongruence. Cladistics 10: 315−320.
Ganapathi A (1979). Studies on the etiology of the leaf blotch disease
of Eucalyptus spp. caused by Mycosphaerella nubilosa (Cke)
Hansf. PhD. dissertation. Department of Botany, University of
Auckland, New Zealand.
Hunter GC, Crous PW, Roux J, Wingfield BD, Wingfield MJ (2004a).
Identification of Mycosphaerella species associated with
Eucalyptus nitens leaf defoliation in South Africa. Australasian
Plant Pathology 33: 349−355.
Hunter GC, Roux J, Wingfield BD, Crous PW, Wingfield MJ (2004b).
Mycosphaerella species causing leaf disease in South African
A MULTI-GENE PHYLOGENY OF MYCOSPHAERELLA
Eucalyptus plantations. Mycological Research 108: 672−681.
Katoh K, Kuma K, Toh H, Miyata T (2005). MAFFT version 5:
Improvement in accuracy of multiple sequence alignment.
Nucleic Acid Research 33: 511−518.
Lundquist JE, Purnell RC (1987). Effects of Mycosphaerella leaf spot
on growth of Eucalyptus nitens. Plant Disease 71: 1025−1029.
Maxwell A, Dell B, Neumeister-Kemp HG, Hardy EStJ (2003).
Mycosphaerella species associated with Eucalyptus in
southwestern Australia: new species, new records and a key.
Mycological Research 107: 351−359.
Maxwell A, Jackson SL, Dell B, Hardy EStJ (2005). PCR-identification
of Mycosphaerella species associated with leaf diseases of
Eucalyptus. Mycological Research 109: 992−1004.
Moncalvo JM, Wamg HH, Hseu RS (1995). Phylogenetic relationships
in Ganoderma inferred from the internal transcribed spacers and
25S ribosomal DNA sequences. Mycologia 87: 223−238.
Park RF, Keane PJ (1982a). Three Mycosphaerella species from leaf
diseases of Eucalyptus. Transactions of the British Mycological
Society 79: 95−100.
Park RF, Keane PJ (1982b). Leaf diseases of Eucalyptus associated
with Mycosphaerella species. Transactions of the British
Mycological Society 79: 101−115.
Park RF, Keane PJ, Wingfield MJ, Crous PW (2000). Fungal diseases
of Eucalypt foliage. In Diseases and Pathogens of Eucalypts. (PJ
Keane, GA Kile, FD Podger, BN Brown, eds): 153−259. CSIRO
Publishing, Collingwood, Australia.
Posada D, Crandall KA (1998). Modeltest: testing the model of DNA
substitution. Bioinformatics 14: 817−818.
Slippers B, Stenlid J, Wingfield MJ (2005). Emerging pathogens:
fungal host jumps following anthropogenic introduction. Trends
in Ecology and Evolution 20: 420−421.
Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony
(*and other methods). Version 4.0b10. Sinauer Associates,
Sunderland, MA.
Taylor JW, Fischer MC (2003). Fungal multilocus sequence
typing−it’s not just for bacteria. Current Opinion in Microbiology
6: 351−356.
Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbet
DS, Fischer MC (2000). Phylogenetic species recognition and
species concepts in fungi. Fungal Genetics and Biology 31:
21−32.
Turnbull JW (2000). Economic and social importance of Eucalypts.
In: Diseases and pathogens of eucalypts. (Keane PJ, Kile GA,
Podger FD, Brown BN, eds) CSIRO Publishing, Australia: 1−7.
Vilgalys R, Hester M (1990). Rapid genetic identification and
mapping of enzymatically amplified ribosomal DNA from several
Cryptococcus species. Journal of Bacteriology 172: 4238−4246.
White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In
PCR protocols: a guide to methods and applications. (Innis MA,
Gelfand DH, Snisky JJ, White TJ, eds) Academic Press, U.S.A.:
282−287.
Wingfield MJ (1999). Pathogens in exotic plantation forestry.
International Forestry Review 1: 163−168.
Wingfield MJ (2003). Increasing threat of diseases to exotic plantation
forests in the Southern Hemisphere: lessons from Cryphonectria
canker. Australasian Plant Pathology 32: 133−139.
Wingfield MJ, Slippers B, Roux J, Wingfield BD (2001) Worldwide
movement of exotic forest fungi, especially in the tropics and the
Southern Hemisphere. Bioscience 51: 134−140.
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Mycologia, 98(6), 2006, pp. 1041–1052.
# 2006 by The Mycological Society of America, Lawrence, KS 66044-8897
A multigene phylogeny of the Dothideomycetes using four nuclear loci
Conrad L. Schoch1
INTRODUCTION
Department of Botany and Plant Pathology, Oregon
State University, Corvallis, Oregon 93133
Members of the Dothideomycetes often are found as
pathogens, endophytes or epiphytes of living plants
and also as saprobes degrading cellulose and other
complex carbohydrates in dead or partially digested
plant matter in leaf litter or dung. Combinations of
these niches can be occupied by a single fungus as it
passes through its life cycle; for example several fungi
initiate their life cycles on living plants and switch to
saprobic states when the plant dies or leaves are lost.
The nutritional modes are not limited to associations
with plants and several species are lichenized, while
others occur as parasites on fungi or members of the
kingdom animalia.
Although to a casual observer there is little to
distinguish the flask-, spherical- or disk-shaped fruiting bodies of the Dothideomycetes from several other
ascomycete groups, they share a distinctive pattern of
development. The asci bearing the sexual spores
develop in locules already formed lysigenously within
vegetative hyphae. This, defined as ascolocular development, is in contrast to ascohymenial development found in the majority of other fungal classes.
Ascohymenial development generates asci in a broad
hymenium interspersed with apically free paraphyses
and the reproductive structure is derived from cells
after fertilization.
Building on earlier descriptions of ascolocular
development Nannfeldt (1932) proposed the group
‘‘Ascoloculares’’ and in 1955 this was formally proposed as a class ‘‘Loculoascomycetes’’ by Luttrell
(1955). The importance of ascus morphology and
dehiscence, in addition to the presence of surrounding elements inside the ascostromata, was emphasized
(Luttrell 1951). The bitunicate ascus remains a defining character in modern dothideomycete taxonomy. It consists of a thick extensible inner layer
(endotunica) and a thin inextensible outer layer
(ectotunica). Most species release their ascospores by
the extension of the inner ascus wall and the rupture
of the outer wall, similar to a jack-in-the-box
(fissitunicate), but variations are numerous. Another
character of note, the centrum, defined as the tissues
and cells occupying the cavity of the sexual structure,
was expanded by Luttrell when he described three
different ascostromatal developmental types exemplified by the genera Dothidea, Pleospora and Elsinoë
forming part of the currently accepted orders,
Dothideales, Pleosporales and Myriangiales (see
tolweb.org/Dothideomycetes for details). The ha-
Robert A. Shoemaker
Keith A. Seifert
Sarah Hambleton
Biodiversity Theme (Mycology and Botany), Agriculture
and Agri-Food Canada, Ottawa, K1A 0C6 Canada
Joseph W. Spatafora
Department of Botany and Plant Pathology, Oregon
State University, Corvallis, Oregon 93133
Pedro W. Crous
Centraalbureau voor Schimmelcultures, Fungal
Biodiversity Centre, P.O. Box 85167, 3508 AD,
Utrecht, The Netherlands
Abstract: We present an expanded multigene phylogeny of the Dothideomycetes. The final data matrix
consisted of four loci (nuc SSU rDNA, nuc LSU
rDNA, TEF1, RPB2) for 96 taxa, representing five of
the seven orders in the current classification of
Dothideomycetes and several outgroup taxa representative of the major clades in the Pezizomycotina.
The resulting phylogeny differentiated two main
dothideomycete lineages comprising the pseudoparaphysate Pleosporales and aparaphysate Dothideales.
We propose the subclasses Pleosporomycetidae (order Pleosporales) and Dothideomycetidae (orders
Dothideales, Capnodiales and Myriangiales). Furthermore we provide strong molecular support for the
placement of Mycosphaerellaceae and Piedraiaceae
within the Capnodiales and introduce Davidiellaceae
as a new family to accommodate species of Davidiella
with Cladosporium anamorphs. Some taxa could not
be placed with certainty (e.g. Hysteriales), but there
was strong support for new groupings. The clade
containing members of the genera Botryosphaeria and
Guignardia resolved well but without support for any
relationship to any other described orders and we
hereby propose the new order Botryosphaeriales.
These data also are consistent with the removal of
Chaetothyriales and Coryneliales from the Dothideomycetes and strongly support their placement in the
Eurotiomycetes.
Key words: bitunicate asci, hamathecium,
loculoascomycetes, pseudoparaphyses
Accepted for publication 4 August 2006.
1
Corresponding author. E-mail: [email protected]
1041
1042
MYCOLOGIA
mathecium (Eriksson 1981) (i.e. the sterile centrum
tissues existing between the asci) is one of the most
reliable characters used to delineate ordinal classifications within the Dothideomycetes. The presence of
pseudoparaphyses (sterile cells extending down from
the upper portion of the ascoma, initially attached at
both ends, although the upper part may become free) is
a notable character for the Pleosporales, together with
mainly ostiolate flask-shaped pseudothecia. Conversely
the absence of pseudoparaphyses and the presence of
fascicles of asci are important in the Dothideales. The
Myriangiales also do not have pseudoparaphyses but
produce single globose asci in multiple locules. Several
additional orders currently accepted are defined by
combinations of centrum and ascomal characters. For
a summary of different centrum types and features see
Kirk et al (2001 p 224–225).
The different classification systems proposed thus
far exhibited an emphasis on varying characters. For
instance, the presence and morphology of characters
in the hamathecium, together with ascostroma shape
were used as the main characters to define ordinal
groups by Luttrell (1955), while von Arx and Müller
(1975) emphasized the form of the ascus and the
specific opening of the ascoma. Although basing her
classification on the work of Luttrell, Barr (1987)
employed additional characters such as the morphology of pseudoparaphyses.
The best studied species in this group tend to be
plant pathogens on important agricultural crops.
Therefore a large body of work in dothideomycete
taxonomy and systematics concerns descriptions of
anamorphs, the predominant morphological state
encountered on agricultural crops; in fact several
families in this class (e.g. Pleosporaceae, Mycosphaerellaceae, Tubeufiaceae) include a high proportion of
anamorphic species. These include both hyphomycetes and coelomycetes. Many of the hyphomycetes
have sympodially proliferating conidiogenous cells.
Phoma-like and other coelomycetes occur in several
families (e.g. Leptosphaeriaceae, Lophiostomataceae); these have ostiolate pycnidia lined with
phialidic, annellidic or holoblastic conidiogenous
cells and produce small, aseptate conidia in slime.
Other important species include the group now
informally referred to as the ‘‘black yeasts’’ (some
of which also belong to the Eurotiomycetes) characterized by the production of dark, slimy colonies and
sporulation patterns that resemble the budding of
true yeasts but actually are reduced versions of
phialidic, annellidic or sympodially proliferating
conidiogenous cells (de Hoog 1974). A selection of
the variety of morphological structures exhibited by
teleomorph and anamorph forms in the Dothideomycetes is shown (FIG. 1).
The refinement of character state homologies and
the development of morphology-based classifications
into a phylogenetic classification system are accelerating with the advent of molecular data. Initial
analyses using DNA sequence data from the small
subunit ribosomal RNA gene did not support the
monophyly of the Loculoascomycetes (Spatafora et al
1995, Berbee 1996). A more recent phylogeny produced from protein gene coding data (Liu and Hall
2004) was inferred as supporting the taxonomic
concepts for a monophyletic lineage for ascostromatic
taxa, but the ontogenetic designations were considered oversimplified by some (Lumbsch et al 2005).
Other studies combining data from protein-coding
genes and the ribosomal operon have shown the
paraphyly of ascostromatic, bitunicate lineages (Lutzoni et al 2004, Reeb et al 2004). An example is the
group of fungi that recently were transferred to the
Eurotiomycetes based on nuclear small subunit
ribosomal sequences, the ‘‘black yeasts’’ of the
Chaetothyriales (Winka et al 1998). Together with
the Verrucariales and Pyrenulales these bitunicate
taxa have been placed within a separate subclass, the
Chaetothyriomycetidae (Miadlikowska and Lutzoni
2004), which is sister of the Eurotiomycetidae
(Lutzoni et al 2004, Reeb et al 2004) in the class
Eurotiomycetes (also see Geiser et al in this issue).
Several studies provide the groundwork for a phylogenetically based classification for the Dothideomycetes. Most have used nuclear small subunit ribosomal
data, but nuclear large subunit ribosomal and
mitochondrial small subunit sequences also were
used (Lindemuth et al 2001, Lumbsch and Lindemuth 2001). This allowed for the reassessment of
specific morphological characters proposed in earlier
work. Specifically, poor support for phylogenetic
groups based on the morphology of pseudoparaphyses was found while phylogenetic correlation of
their presence or absence was well supported (Liew et
al 2000, Lumbsch and Lindemuth 2001), although
a single exception to this was noted (Silva-Hanlin and
Hanlin 2000). In spite of these recent examples of
interordinal, molecular-based phylogenetic studies,
a large number of species within the ascostromatic
Ascomycota remain listed as Dothideomycetes or
Chaetothyriomycetes incertae sedis (Eriksson 2006).
Furthermore the question of whether Dothideomycetes represents a natural group derived from a single
ancestor is not settled and the need to investigate its
relationships to a number of the bitunicate lichen
species such as the currently separate class Arthoniomycetes remains essential. The main focus of this
study however is to provide an extension of previous
ribosomal DNA-based phylogenetic studies and combine a number of smaller phylogenetic analyses
SCHOCH ET AL: DOTHIDEOMYCETES
PHYLOGENY
1043
FIG. 1. A selection of dothideomycete morphological forms. Teleomorphs, ascostromata: A. Light-colored, flask-shaped
pseudothecia of Tubeufia cerea (Tubeufiaceae) on wood. B. Dark pseudothecia of Cochliobolus heterostrophus (Pleosporales) on
corn leaf. C. Hysterothecia of Hysteropatella prostii (Hysteriales), with slit-like openings. Teleomorphs, asci and locules: D.
Stylodothis puccinioides (Dothideales), multiascus locules. E. Pyrenophora brizae (Pleosporales) bitunicate asci, one with broken
ectotunica. F. Guignardia magniferae (Botryosphaeriales) asci with ascospores. G. Bitunicate ascus of Davidiella tassiana
(Capnodiales). H. Phaeosphaeria avenaria, juvenile ascoma with pseudoparaphyses. I. Myriangium duriaei (Myriangiales),
monascus locules in stroma. Anamorphs: J. Conidia borne in pycnidium of Dothiorella sp. (Botryosphaeriales). K, L. Helical
conidia, in two different dimensions, of Helicoon and Helicoma spp. (Tubeufiaceae). M. Conidia and conidiophore of Bipolaris
sp. (Pleosporales). N. Stroma of Trimmatostroma abietis (Capnodiales) bearing conidia in culture. O. Chlamydospores of
Trimmatostroma abietis (Capnodiales). Scale bars are approximations obtained from published sources; the bar indicates
10 mm except in A, B, C and N where it indicates 200 mm. Photo credits, courtesy of: Jean-Paul Priou (A), B. Gillian Turgeon
(B), Hans-Otto Baral (C), Robert A. Shoemaker (D, E, H, I), Gary Samuels (F), Pedro W. Crous (G, J, N, O), Clement K.M.
Tsui (K, L), Keith A. Seifert (M).
within the framework of a multiple gene analysis
showing intraordinal relationships in the Dothideomycetes.
MATERIALS AND METHODS
Sampling and alignments.—Sequence data were obtained
from GenBank and the Assembling the Fungal Tree of Life
Project (AFTOL; http://ocid.nacse.org/research/aftol/).
All strains and sequences used in this study are listed
(SUPPLEMENTARY TABLE I). DNA alignments are available
from the AFTOL Web site and TreeBASE (SN2913-11828).
A number of sequences generated by the AFTOL project
and available from the AFTOL Web site as well as from
GenBank were used. Newly generated DNA sequences were
deposited at GenBank (TABLE I supplement). Genes used
were nuclear small subunit ribosomal RNA gene DNA (nuc
SSU), nuclear large subunit rDNA (nuc LSU), elongation
factor la gene (TEF1), and the second largest subunits of
RNA polymerase II gene (RPB2). Herbaria and culture
collections where strains and specimens used in this study
are deposited are listed (TABLE I supplement).
Phylogenetic analysis.—Maximum and weighted parsimony
(MP and WP) analyses were performed on a combined
dataset with a total of 117 taxa that included 96 Dothideomycetes. Nineteen taxa contained data for only three loci to
maximize taxon sampling. The majority of the missing data
were in the terminal branches of the tree, and care was
1044
MYCOLOGIA
taken to include complete data sampling for taxa on
branches underpinned by the more basal nodes. Two taxa
with only ribosomal data (AFTOL ID 1856 Phoma herbarum
and AFTOL ID 1864, Didymella cucurbitacearum) also were
included to clarify the position of the clade surrounding
Phoma herbarum. Removal of these taxa did not significantly
affect support values in other parts of the tree. Likewise
a comparison of a parsimony and Bayesian analysis with and
without complete sets of characters yielded trees with
congruent topologies. DNA sequences from a single strain
(Leptosphaeria maculans DAOM 229267) inadvertently were
included twice in the final analysis but were left in the final
tree to ensure correct comparison across all approaches. We
rooted the tree with three taxa from the class Pezizomycetes
as outgroups (Pyronema domesticum, Caloscypha fulgens
Gyromitra californica) (not shown in figure).
For the WP analyses the unambiguously aligned regions
were subjected to symmetric step matrices for eight
partitions (i.e. nuc SSU rDNA, nuc LSU rDNA and six
codon positions of TEF1 and RPB2) to incorporate the
differences in substitution rates and patterns as described in
Lutzoni et al (2004). MP and WP analyses were performed
with only parsimony informative characters with these
settings: 100 replicates of random sequence addition, TBR
branch swapping and MULTREES in effect. Maximum likelihood was performed with PHYML (Guindon and Gascuel
2003) using a GTR+I+C model of evolution. In all preceding cases nodal support was verified by nonparametric
bootstrapping under the conditions mentioned, using 500
replicates.
Initial incongruence in the single gene trees for the taxa
used was tested by examining single gene analyses with WP
under the conditions previously mentioned for a set of taxa
containing data for all four loci. A 70% majority rule
consensus tree was compared in each case. Phylogenetic
analysis using Bayesian inference of maximum likelihood
was performed with a parallelized version of MrBayes v 3.1.2
across four processors (Altekar et al 2004). MrBayes was run
with these parameters: a general time reversible model of
DNA substitution (GTR) with gamma-distributed rate
variation across sites (invariance, partitioning across genes
and codons). A Markov chain Monte Carlo (MCMC)
analysis with metropolis coupling was run starting from
a random tree for 5 3 106 generations, sampling every
100th cycle. Four chains were run simultaneously with the
initial 1000 cycles discarded as burn-in. Two additional runs
with 5 3 106 generations were compared to confirm that
stationarity in likelihood values was reached and compared.
The phylogenies obtained in all cases were congruent. A
50% majority rule tree from a total of 45 000 trees obtained
from a single run is presented (FIG. 2).
RESULTS AND DISCUSSION
Data analyses.—The alignment for the phylogenetic
analyses, after excluding introns and ambiguously
aligned regions, consisted of 5098 base pairs, 1882 of
which were parsimony informative. The reciprocal
comparisons of 70% bootstrap trees from each gene
with 61 core taxa did not reveal any incongruence
(data not shown). Therefore all of 109 taxa in the
current taxon sampling were used in the combined
analyses. The heuristic search in MP and WP analyses
yielded six MPTs with 20 917 steps (CI 5 0.204, RI 5
0.535) and three MPTs with 34 319.54 steps, respectively. In model-based methods, ML heuristic
search analysis resulted in a tree of 294 457.67 log
likelihood and resulted after the GTR model was
applied with a gamma value of 0.395 across four rate
categories with a proportion of invariant sites equal to
0.287. The Bayesian analysis converged on the plateau
of the log-likelihood on a mean value of 293 955. The
tree from Bayesian analyses is shown (FIG. 2) with all
of the bootstrap proportions as well as the Bayesian
posterior probabilities. Internodes were considered
strongly supported if they received all of bootstrap
proportions $ 70% and posterior probabilities $
95% (Lutzoni et al 2004).
Overview.—The tree (FIG. 2) contains representatives
of the major classes in the Ascomycota, as defined
previously (Eriksson 2001). The supraclass relationships in our analysis indicated no support for a close
relationship between the Dothideomycetes and Sordariomycetes, alluded to in an earlier study (Lutzoni
et al 2004) and the sister relationships of the
Sordariomycetes and Leotiomycetes are supported
in agreement with recent data (Lumbsch et al 2005).
A few taxon pairs containing isolates used in previous
works have remarkably high similarity to each other
over all four loci. Two examples noted in this analysis
were incorrectly identified strains, namely ‘‘Clathrospora diplospora’’ CBS 174.52 5 Alternaria alternata
and ‘‘Epipolaeum longisetosum5Raciborskiomyces longisetosus’’ CBS 180.53 5 Cladosporium herbarum.
Non-Dothideomycete bitunicate groups. Several
lineages historically associated with the loculoascomycetes, such as the two species representing the
Coryneliales, also were included. The placement of
Caliciopsis orientalis together with Caliciopsis pinea
(FIG. 2) indicates a close relationship with the Eurotiomycetidae (Geiser et al this issue). Other ordinal
groups traditionally associated with the Dothideomycetes and now placed in the Eurotiomycetes were
mentioned earlier. These groups share a number of
centrum characters with members of the Dothideomycetes, such as the presence of periphysoids (Verrucariales, Chaetothyriales) and periphysate ostioles (Verrucariales, Chaetothyriales, Pyrenulales). The phylogeny
(FIG. 2) confirms the separation of the Chaetothyriales
and Verrucariales from the Dothideomycetes.
Dothideomycetes-Arthoniomycetes clade. The relationship of the Dothideomycetes and Arthoniomycetes
(node A) is well supported by Bayesian and
SCHOCH ET AL: DOTHIDEOMYCETES
maximum likelihood but not parsimony, although in
an analysis without third codon positions, support by
MP bootstrap and WP bootstrap increased. The
internal node supporting the monophyly of the
Dothideomycetes (node B) also had higher support
in maximum likelihood and the two parsimony
processes when the more saturated third codon
positions were omitted. In more complete analyses
containing characters from the RPB1 locus, this node
was moderately supported and the Trypethelium strain
is shown inside the Dothideomycetes (see Spatafora
et al this issue).
Although taxon sampling for the Arthoniomycetes
is sparse in our dataset, these levels of support
(FIG. 2) largely agree with other recent large analyses
where the Dothideomycetes is resolved as monophyletic but with low statistical support (Lumbsch et
al 2005). A possible sister relationship of Dothideomycete/Arthoniomycetes has been proposed (Barr
1987, Tehler 1990) and there is some phylogenetic
support for this (Lumbsch et al 2005, Lutzoni et al
2004). Clear differences between the groups exist,
such as the ascohymenial type development of the
Arthoniomycetes apothecium (Henssen and Thor
1994). More thorough sampling of Arthoniomycetes
will test the monophyly of its relationship with the
Dothideomycetes. It is premature to comment on the
ultimate monophyly of the Dothideomycetes, but it
seems quite reasonable that increased sampling of
taxa and genes could increase support for this node.
As pointed out by Lumbsch et al (2005), most of the
large scale interclass relationships have been in
conflict in recent publications and taxon sampling
should be an important consideration before making
major classification changes.
Dothideomycetes. The addition of protein gene data
illustrates that the lineages clustering around the core
orders Pleosporales and Dothideales correlate with the
presence or absence of pseudoparaphyses and other
centrum characteristics. The node supporting the
Dothideales, Capnodiales, Myriangiales and Mycosphaerellaceae (C) is strongly supported. This node
was unaffected when third base codon positions were
removed, but a small increase in parsimony bootstrap
support was noted at node M, combining the Dothideales and Myriangiales, although ML bootstrap decreased. Saturation and the specific evolutionary model
applied might have influenced this. Node C might
indicate a single loss of pseudoparaphyses in all the
terminal clades. However previous molecular phylogenies based on nuc SSU rDNA data have shown the
presence of members of the aparaphysate genus
Leptosphaerulina nested within the Pleosporales (SilvaHanlin and Hanlin 2000), which could imply multiple,
isolated losses of this character in other parts of the tree.
PHYLOGENY
1045
Anamorphs play an important role in the life cycles
of many orders of Dothideomycetes. Many are coelomycetes, especially phialidic, Phoma-like anamorphs,
which may be a plesiomorphic anamorph character in
the class, perhaps serving some kind of spermatial
function. In the Pleosporaceae and Mycosphaerellaceae hyphomycetes with sympodially proliferating
conidiogenous cells with scars, and dry conidia, are
particularly common and strictly anamorphic species
may comprise the majority in these families. The
Capnodiales, with their multitude of hyphomycete and
coelomycete synanamorphs, and the helicoconidial
anamorphs of the Tubeufiaceae, contain particularly
distinctive anamorph groups. The anamorph genera
of both hyphomycetes and coelomycetes, lacking
teleomorph connections, continue to be examined
for their phylogenetic relationships, many of them
undoubtedly will be found to be associated with the
Dothideomycetes. Several clades are well supported
(FIG. 2) and will be discussed in more detail below.
Aparaphysate Dothideomycetes.—We hereby propose
an emendation of the subclass Dothideomycetidae
(nom. nud.) (Kirk et al 2001), which has been
superceded by the Dothideomycetes O.E. Erikss.
and Winka (2000). Dothideomycetidae sensu Lutzoni
et al (2004) also was included in the Sordariomycetes
as subclass Dothideomycetidae along with the subclass
Sordariomycetidae (syn. Sordariomycetes s. str.) and
Arthoniomycetidae (syn. Arthoniomycetes), although
there was no strong statistical support for this
broadened concept of Sordariomycetes. We validate
and emend the concept of Dothideomycetidae sensu
Kirk et al (2001) to include the bitunicate orders
Dothideales, Capnodiales and Myriangiales, which
lack paraphyses, pseudoparaphyses and paraphysoids.
This emended subclass overlaps with the Loculoparenchymatomycetidae (Barr 1983) but differs by
including the Myriangiales and excluding the Asterinales, now listed under its constituent families as
Dothideomycetes et Chaetothyriomycetes incertae
sedis by Eriksson (2006).
Dothideomycetidae P.M. Kirk, P.F. Cannon, J.C.
David & J.A. Stalpers, ex Schoch, Spatafora, Crous
et Shoemaker, subclass nov.
; Dothideomycetidae P.M. Kirk, P.F. Cannon & J.C. David
& J.A. Stalpers, in Kirk et al, Dictionary of Fungi, 9th
ed., p 165, 572. 2001 (nom. nud.).
Ascomata immersa vel erumpentia vel superficialia,
minuta vel magnitudine media, separata vel in
stromate basilari aggregata, unilocularia vel plurilocularia, ostiolata, nonnumquam periphysata. Pseudoparaphyses absentes, periphysoideae nonnumquam
praesentes. Asci globosi vel ellipsoidei vel clavati vel
1046
MYCOLOGIA
SCHOCH ET AL: DOTHIDEOMYCETES
subcylindrici. Ascosporae hyalinae vel subhyalinae vel
fuscae, unicellulares vel pluriseptatae vel muriformes.
Anamorphoses seu coelomycetes seu hyphomycetes.
Ascomata immersed, erumpent or sometimes superficial, minute, small or medium-sized; separate or
merged or grouped on basal stroma, uni- to multiloculate apical pore mostly present, when present
ostiolar canal at times periphysate, stromatic tissues
may contain pseudoparenchymatous cells. Pseudoparaphyses lacking, periphysoids may be present; Asci
globose, subglobose, ovoid to ellipsoid, saccate,
oblong, clavate or subcylindrical, Ascospores hyaline,
subhyaline or dark brown, variable in shape and size,
one celled or one to several septate or muriform.
Anamorphs coelomycetous and/or hyphomycetous.
Type order. Dothideales (1897) Lindau in Engler &
Prantl, Nat. Pflanzenfam. 1(1):373. 1897.
Represented orders. Dothideales Lindau 1897, Capnodiales Woron. 1925, Myriangiales Starbäck 1899.
Dothideales. Species from this order generally have
smaller ascomata and fewer asci than the pseudoparaphysate Pleosporales (node D) and traditionally have
been segregated because of the absence of pseudoparaphyses in their pseudothecia. The species included in
this order encompass saprotrophs, hemibiotrophs and
biotrophs. It is represented by eight species in our
analysis, including the recent epitype isolate of
Dothidea sambuci, the type of the genus Dothidea
(Shoemaker et al 2003). The family Dothideaceae
includes biotrophs, necrotrophs and saprobes on
plant tissue. Stylodothis puccinoides was redescribed as
a separate species from Dothidea but remains closely
associated with the genus in our phylogeny.
Three members of the Dothioraceae are polyphetic
in the tree. The so-called black yeast anamorphs
associated with Dothideomycetes tend to occur in this
family, with Aureobasidium pullulans (probably an
anamorph species complex based on the ITS
sequences deposited in GenBank), and the micromorphologically similar Hormonema dematioides (teleomorph Sydowia polyspora, perhaps also a complex of
anamorph species) (de Hoog 1974). These species
are found commonly on moist surfaces of plants and
can convert from yeast to meristematic growth under
PHYLOGENY
1047
nutritional stress. Some progress in the resolution of
the nature of Aureobasidium pullulans has been made
here with the linkage of Columnosphaeria fagi (H.J.
Hudson) M.E. Barr to a ‘‘neotype’’ culture CBS
584.75 of A. pullulans var. pullulans (SUPPLEMENTARY
TABLE I).
Capnodiales. The node I is well supported in this
multigene analysis. This same node is present in
a ribosomal rDNA phylogeny containing ‘‘Raciborskiomyces longisetosus’’ as erroneous name for a Cladosporium species with Capnodium citri (Lumbsch and
Lindemuth 2001). Synapomorphies are limited in this
expanded order and these taxa have not been
grouped together before. The presence of short,
periphyses-like cells in the ostiolar pore of some
genera of the Capnodiales such as Capnodium also
are reported from other families, including the Mycosphaerellaceae (von Arx and Müller 1975) and might
be a synapomorphy uniting these taxa. We hereby
propose an expansion of the current Capnodiales to
include the Mycosphaereallaceae and Piedraiaceae.
The constituent families are discussed below.
Capnodiaceae. An ascostromatal family without
pseudoparaphyses, the Capnodiaceae are leaf epiphytes associated with the honeydew of insects. Also
known as sooty molds, they tend to live in complex
communities, with multiple species, and often multiple fungal parasites of those species, inhabiting
a common, sooty mass. They are noted for the
production of darkly pigmented hyphae, often of very
characteristic morphology (Hughes 1976, Reynolds
1998). The members of this order have superficial
ascostromata with ovoid asci in fascicles and hyaline to
dark, one to multiseptate ascospores. The sooty molds
are highly pleomorphic and often highly pleoanamorphic. The order includes many anamorphic
species, all dematiaceous, including several conidiomatal, mycelial (often with dry-spored, blastic
phragmo- or dictyoconidia) or presumably spermatial
(usually phialidic) hyphomycete genera or pycnidial
synanamorphs (Hughes 1976).
Mycosphaerellaceae. The Mycosphaerellaceae is characterized by small pseudothecial ascomata that are
immersed in host tissue, single and superficial, or
imbedded in a pseudoparenchymatal stroma, papil-
r
FIG. 2. Dothideomycete phylogeny. 50% majority rule consensus tree of 45 000 trees obtained by Bayesian inference and
MCMCMC under GTR+I+C applied across seven partitions. Only orders and families with more than two members under the
current classification of Eriksson (2005) are shown in shadow. Bar indicates the nucleotide substitutions per site. Nodes of
interest are labeled alphabetically and support values are shown above and below. Bayesian PP 5 posterior probability, ML BP
5 maximum likelihood bootstrap, MP BP 5 maximum parsimony bootstrap, WP 5 weighted parsimony bootstrap. Gaps (–)
show a collapsed node and asterisks show the presence of a differently resolved node under the specific statistical sampling
method used.
1048
MYCOLOGIA
late, ostiolate, lacking interascal tissue. Asci vary from
ovoid to saccate to subcylindrical, usually stipitate, with
or without an apical chamber, lacking any other apical
mechanism. Ascospores are hyaline to slightly pigmented, 1-septate, but in some cases also 3-septate, and
sometimes are enclosed in a sheath. Mycosphaerella has
close to 30 anamorph genera associated with it, most
of which have cicatrized, sympodially proliferating
conidiogenous cells and single or acropetally catenate,
dry conidia. The two clades delineated within Mycosphaerella here also were recognized in a separate study
employing multiple genes to resolve relationships in
Mycosphaerella (Hunter et al 2006). Node I1 contains
the type of Mycosphaerella, M. punctiformis, and the
bulk of Mycosphaerella species, while the second clade
(above I4) appears to contain more extremotolerant
species (Crous et al unpubl data).
Mycosphaerella is distinguished from Davidiella
(Cladosporium anamorphs) by lacking irregular lumens or inclusions in its ascospores and not having
anamorphs with protruberant, thickened, darkened,
Cladosporium-like scars (Braun et al 2003, Aptroot
2006). As shown in this study Davidiella with its
Cladosporium anamorphs (type species Davidiella
tassiana, anamorph Cladosporium herbarum) clusters
in a well supported clade apart from Mycosphaerella
s.str. (Mycosphaerellaceae), and thus a new family is
proposed for clade I1.
Davidiellaceae Schoch, Spatafora, Crous et Shoemaker, fam. nov.
Ascomata Mycosphaerellae similia, sed lumen ascosporarum forma variabile et anamorphe Cladosporium.
Ascomata immersed to erumpent, small or medium-sized; separate or aggregated, uniloculate, apical
pore present, periphysate; wall of several layers of
brown, thickened, pseudoparenchymatal cells. Pseudoparaphyses lacking. Asci bitunicate, 8-spored, obovoid to ellipsoid or subcylindrical, fasciculate, with
or without apical chamber. Ascospores hyaline to pale
brown, smooth to somewhat roughened, mucous
sheath sometimes present, one-septate, thick-walled,
with irregular lumens. Anamorphs are species of
Cladosporium.
Typus. Davidiella tassiana (De Not.) Crous & U.
Braun, Mycol. Prog. 2:8. 2003.
The position of a single representative of the
Piedraiaceae, Piedraia hortae, is refined here as
associated with the Capnodiales and allies but not
the Myriangiales as reported earlier (Lindemuth et al
2001). This species was described with an ascus
containing only one thin wall (Shoemaker and Egger
1982). The specialized parasites in this family are
almost exclusively associated with human hair in
tropical regions. It is shown with low parsimony
bootstrap support (I3) with Trimmatostroma abietis,
a meristematic anamorph species isolated from conifer needles and rock surfaces. This species was
shown to be closely related to Mycosphaerella and its
allies in a recently published molecular phylogeny
(Selbman et al 2005).
Myriangiales. The Myriangiales are reported to be
related to the Dothideales (node M), although without
any significant bootstrap support for this placement.
They generally have ascostromata without ostioles in
monoascal locules. The species of the type genus,
Myriangium, has globose asci scattered at many levels
in an undifferentiated stromatic mass (Sivanesan
1984). The order includes saprobic, epiphytic or
biotrophic organisms. The anamorphs of this order,
when known, generally are acervular coelomycetes
with polyphialidic conidiogenous cells, such as the
Sphaceloma anamorphs of Elsinoë species (Kirk et al
2001).
Paraphysate Dothideomycetes.
We hereby propose a new subclass for the
pseudoparaphysate taxa supported by node D1.
Pleosporomycetidae Schoch, Spatafora, Crous et
Shoemaker, subclass nov.
Ascomata perithecialia vel hysterothecialia vel
cleistothecialia, immersa vel erumpentia. Hamathecii
pseudoparaphyses cellulares vel trabeculatae, maturae
nonnumquam deliquescentes. Asci bitunicati, plerumque basilares, nonnumquam lateraliter extendentes, cylindrici vel clavati vel oblongi vel saccati.
Ascosporae colore, forma septisque variabiles, plerumque heteropolares sed nonnumquam etiam symmetricae.
Ascomata perithecioid, hysterothecioid or cleistothecioid, conchate or dolabrate, immersed, erumpent or superficial; globose, sphaeroid, turbinate,
ovoid, obpyriform, conoid, doliiform, dimidiate.
Hamathecium of wide to narrow cellular or trabeculate pseudoparaphyses, deliquescing at maturity in
some. Asci bitunicate, usually basal, at times extending laterally, cylindric, clavate, oblong or saccate.
Ascospores variable in pigmentation, shape and
septation, usually with bipolar asymmetry, but some
symmetrical.
Type order. Pleosporales Luttrell ex M.E. Barr.
Represented order. Pleosporales Luttrell ex M.E. Barr.
Pleosporales. The Pleosporales is the largest order in
the Dothideomycetes. It contains several well known
plant pathogens such as Cochliobolus heterostrophus, the
causative agent for southern blight on corn, Leptosphaeria maculans, causing black leg on rape seed and
SCHOCH ET AL: DOTHIDEOMYCETES
Phaeosphaeria nodorum causing stagonospora blotch in
cereals. In this analysis a strain of Delitschia winteri is
placed above node D, supporting the rest of the
Pleosporales according to Eriksson’s broad concept
(2001). Delitschia shares features common to several
bitunicate species occurring on dung; they are darkly
pigmented, usually strongly constricted ascospores
with germ slits (Barr 2000). The family Delitschiaceae
was described by Barr (2000) for species previously
placed in the Sporormiaceae. The delineation is based
on an ostiole containing periphyses and asci with wide
outer ascus walls and an ocular chamber containing
refractive rods. This placement was confirmed with
nuc SSU rDNA sequence comparisons (Kruys 2005). A
combined nuc SSU analysis of Delitschia winteri
grouped it close to another species of the genus, D.
didyma (AF242264), confirming the identification of
the strain used (results not shown). Members of this
family are hypersaprotrophic on old dung and
exposed wood.
There was also strong support for the monophyly of
Pleosporales, with Lophium mytilinum branching at its
most basal node (D1). This species is found as
a saprobe on wood and on cones of conifers and is
listed incertae sedis as part of the Mytilinidiaceae
(Eriksson 2006). The family contains species with
characteristic conch shaped ascomata. Analyzing
additional taxa from the Mytilinidiaceae and related
groups also will be important to investigate ancestral
character states for the Pleosporales but they should
be placed as Pleosporomycetidae incertae sedis for
now.
The morphology of ascospores has played an
important role in delimiting families in the Pleosporales. However, as noted from some of the first
molecular based phylogenies of the Dothideomycetes,
several family relationships might be poorly supported (Lindemuth et al 2001). Perhaps the strains
chosen are not good exemplars for their families or
are derived from misidentified specimens. However it
seems unlikely that this can account for all the
relationships (FIG. 2) and a reassessment at this level
of classification seems urgent. Here we will discuss
only briefly a selection of highlighted families
(FIG. 2).
The most basal node inside the Pleosporales (D2)
supports two members of the Testudinaceae, provisionally included among Ascomycota incertae sedis
by Eriksson (2006). Members of this family are mainly
isolated from soil and produce reduced, cleistothecioid ascostromata. This clade unexpectedly contains
the ostiolate marine species, Verruculina enalia
(Didymosphaeriaceae) as also noted in an earlier
phylogenetic analysis (Kruys 2005). The next well
supported clade above node D3 supports the Spor-
PHYLOGENY
1049
ormiaceae. These fungi are found commonly on
dung but some occur on other substrates (e.g. wood,
soil and plant debris). A large number of species in
this group have germ slits. This morphological
variability was confirmed in a phylogenetic study
using DNA sequences from multiple ribosomal loci
(Kruys 2005).
The Lophiostomataceae and Melanommataceae
are inferred as paraphyletic in the next set of clades
(above D4 and D5), with one clade including two
species of Lophiostoma (Lophiostomataceae 1). This
clade also contains one species of Trematosphaeria
heterospora, which was classified as Lophiostoma
heterosporum (Barr 1992). The second clade (Lophiostomataceae 2) includes members of the Lophiostomataceae and Pleomassariaceae as well as Melanommataceae. Node D5 contains a diverse group of
species isolated from diseased and decaying plants
as well as soil (each currently classified under
a different family). This overlapped with relationships
reported before, using molecular-based phylogenies
(Liew et al 2000, 2002), but like many of the other
clades will require more intense sampling to address
family and genus descriptions.
The more terminal branches in the Pleosporales
(D6) include well studied families containing important plant pathogens, saprobes and animal pathogens
with numerous anamorphs. Didymella cucurbitacearum forms a clade with the anamorphs Ascochyta
pisi and Phoma herbarum (D8), parasites on agricultural crops. Leptosphaeria (Leptosphaeriaceae),
shown on a single branch, is a large genus with pale
to dark brown and septate ascospores. Members of
this family have flask-shaped pseudothecia with
narrow asci and a characteristic thin apex. Many
species are associated with coelomycetous anamorphs. Phoma anamorphs are particularly common
(Camara et al 2001, Verkley et al 2004). The Phaeosphaeriaceae (D9) are distinguished from the Leptosphaeriaceae by ascomal wall morphology and all
have pycnidial coelomycetes, mostly classified in
Stagonospora, characterized by holoblastic or sometimes annellidic conidiogenesis and the production
of phragmoconidia. Unnamed pycnidial microconidial anamorphs also are reported in some species
(Leuchtmann 1984). In a poorly supported clade
a trio of species without any clear phylogenetic
placement are noted. Two of these species are
anamorphs, Coniothyrium palmarum and Pyrenochaeta
nobilis, linked to the teleomorphs Leptosphaeria and
Herpotrichia.
The next well supported node (D10) contains the
Pleosporaceae, which have ascostromata that are
mainly flask-shaped pseudothecia embedded in the
substrate with 1-septate to muriform ascospores. In
1050
MYCOLOGIA
addition to species found in marine environments
and as parasites on animals a number of important
grass and cereal crop parasite genera, Cochliobolus,
Pyrenophora and Lewia, are included in this family.
The sexual states are normally well linked with single
anamorph genera. Important anamorph species include the well known genera Alternaria (with
Ulocladium paraphyletic within it), Stemphylium, the
so-called helminthosporia (Bipolaris, Curvularia,
Drechslera, Exserohilum) and a few other genera such
as Dendryphion and Dendryphiopsis.
Dothideomycetes incertae sedis.—A number of orders
could not be placed in any of the two subclasses defined
and will be discussed in more detail. Two orders,
Jahnulales and Patellariales, currently listed by Eriksson
(2006) are not included in this analysis but a separate
comparison using deposited sequences from nuc SSU
obtained from GenBank combined with our complete
taxa revealed them to be separate from the groups
referred to in this paper (data not shown).
Members of Hysteriales have been reported with
pseudoparaphyses in apothecioid ascomata with elongated openings (von Arx and Müller 1975, Barr 1987,
Luttrell 1974) and are often saprobes on wood or weak
parasites of woody plants. Four members of the
Hysteriales agreeing mainly with Luttrell’s original
definitions are included (FIG. 2) and it is clear that
these are not a monophyletic group, a proposition also
mentioned by Luttrell (1973). Farlowiella carmichaeliana could not be resolved with any certainty.
The phylogeny also supports a relationship between the dung fungus Phaeotrichum benjaminii and
Tyrannosorus pinicola (FIG. 2). Phaeotrichum is characterized by dark-brown, septate spores and cleistothecioid ascostromata. T. pinicola produces ostiolate ascostromata with characteristic long, sharp
spines and have been isolated from wood and plant
material. The multiple germ slits that were described
for T. pinicola may be linked to the terminal germ
pores characteristic of P. benjaminii.
Node E supports Kirschsteiniothelia aethiops with its
Dendryphiopsis atra anamorph. These two species also
appear unrelated to other species in the genus
(Shearer 1993) based on nuc SSU rDNA data and
the genus is reportedly heterogenous (Hawksworth
and Eriksson 2003). K. aethiops does not have close
associations with the Pleosporaceae and should be
placed in a separate family.
The Tubeufiaceae clade (above node G) contains
species with a variety of nutritional modes. They often
are reported as saprobes from terrestrial and freshwater environments, but some species are hyperparasites and others can parasitize insects. Teleomorphs
consist of brightly colored ascostromata, with long,
hyaline, multiseptate ascospores (Rossman 1987).
The best-known anamorphs of the Tubeufiaceae are
helicosporous hyphomycetes and well known genera
include Helicodendron, Helicomyces and Helicoon. Recent DNA sequence-based comparisons did not find
strong correlation between these anamorph forms
and phylogenetic groups. (Tsui et al 2006). Combining recent focused phylogenies into a large scale
dataset is required before placement of this group in
the current classification.
Botryosphaeriaceae. The position of the Botryosphaeriaceae (H) within the Dothideomycetes has
been enigmatic. The taxonomy of this group of plantassociated fungi has relied mostly on anamorph
descriptions; sequence data recently have linked
several anamorph genera to the genus Botryosphaeria
(Jacobs and Rehner 1998). Associated anamorphs
were divided into two groups, those with thin-walled,
hyaline conidia (Fusicoccum), and those with thickwalled, pigmented conidia (Diplodia) (Denman et al
2000). In a recent phylogenetic study employing LSU
sequence data to resolve relationships among members of the Botryosphaeriaceae, Crous et al (2006)
segregated Botryosphaeria into several genera, supported by morphological differences of their anamorphs. From the phylogenetic results obtained in
this study, it is clear that the Botryosphaeriaceae
deserves an order separate from the Pleosporales and
Dothideales, which is introduced below.
Botryosphaeriales Schoch, Crous & Shoemaker, ord.
nov.
Family. Botryosphaeriaceae Theiss. & P. Syd., Ann. Mycol.
16:16 (1918).
Type. Botryosphaeria Ces. & De Not., Comment Soc.
crittog. Ital. 1:211 (1863)..
Type species. B. dothidea (Moug. : Fr.) Ces. & De Not.,
Comment Soc. crittog. Ital. 1:212 (1863).
Ascomata unilocularia vel plurilocularia, pariete
multistratoso fusco inclusa, singularia vel aggregata,
raro in stromate submersa. Asci bitunicati, endotunica crassa, stipitati vel sessiles, clavati, camera apicali
distincta, pseudoparaphysibus hyalinis, septatis, ramosis vel simplicibus intermixti. Ascosporae hyalinae vel
pigmentatae, unicellulares vel septatae, ellipsoideae
vel ovoideae, nonnumquam appendicibus vel tunica
gelatinosis praeditae. Anamorphoses: conidiomata
pycnidialia, unilocularia vel multilocularia, saepe in
stromate submersa, cellulis conidiogenis phialidicis,
conidia hyalina vel pigmentata, tenui- vel crassitunicata proferentibus, quae nonnumquam appendicibus
vel tunica gelatinosis praedita sunt.
Ascomata uni- to multilocular with multilayered
dark brown walls, occurring singularly or in clusters,
frequently embedded in stromatic tissue. Asci bituni-
SCHOCH ET AL: DOTHIDEOMYCETES
cate, with a thick endotunica, stalked or sessile,
clavate, with a well developed apical chamber,
intermixed with hyaline, septate pseudoparaphyses,
branched or not. Ascospores hyaline to pigmented,
septate or not, ellipsoid to ovoid, with or without
mucoid appendages or sheath. Anamorphs have unito multilocular pycnidial conidiomata, frequently
embedded in stromatic tissue, with hyaline, phialidic
conidiogenous cells, and hyaline to pigmented, thinto thick-walled conidia, which sometimes have mucoid appendages or sheaths.
Conclusion.—This multigene phylogeny contributes
to the overall phylogenetic classification of the
Dothideomycetes. We emend a previously proposed
subclass, the Dothideomycetidae, and propose a new
one, the Pleosporomycetidae, based on the presence
or absence of pseudoparaphyses as defined by Barr
(1987) based on Luttrell (1955). The orders according to Eriksson (2006) are largely upheld with the
exception of the Hysteriales, but we also expand this
classification with an additional order, the Botryosphaeriales, and redefine the Capnodiales to include
the currently defined Mycosphaerellaceae and Piedraiaceae. A new family, the Davidiellaceae, is proposed to accommodate Davidiella species with
Cladosporium anamorphs. Several clades did not
correlate with familial relationships under Eriksson’s
classification (2006) and should be addressed in
subsequent analyses. Similarly a number of small clades
are incertae sedis and remain to be addressed in the
future. The strains used in this study, although
validated by morphological examinations in previous
publications (e.g. Berbee 1996) as well as by comparisons with sequences from GenBank, should continue
to be validated by more intensive taxon sampling in
a number of clades. The value of additive sampling in
this study, where two strains used in previous studies
could be shown to be misidentified, supports this.
One large gap in this analysis is the absence of
lichenized lineages. A single unidentified Trypethelium species was included, but numerous lichenized
ascostromatic bitunicate species (such as those in the
Pyrenulales) remain candidates for placement in the
Dothideomycetes. In fact a study by Del Prado et al
(2006) shows good support for a placement of the
lichenized Trypetheliaceae within the Dothideomycetes. In addition, numerous lineages remain unresolved in this class. For example the current
classification of Eriksson (2006) contains 23 families
placed in orders but more than 40 families remain
listed as Chaetothyriomycetes et Dothideomycetes
incertae sedis. It appears likely that, in the process
of combining the comprehensive body of work
already done on the biology, ontogeny and morphol-
PHYLOGENY
1051
ogy of these fungi within a molecular-based phylogenetic context, they will continue to surprise and
challenge us well into the future.
ACKNOWLEDGMENTS
We thank Walter Gams for assistance with the Latin
diagnosis and are grateful to Gary Samuels, Clement Tsui
and Gillian Turgeon for photographs provided. Computational assistance by Scott Givan and Chris Sullivan from the
Center for Gene Research and Biotechnology at Oregon
State University is appreciated. The efforts of Ben O’Rourke
at Oregon State University and Lisa Bukovnik at Duke
University were vital in generating sequence data. We thank
Richard Oliver for providing data from the Phaeosphaeria
nodorum genome project. The authors also acknowledge
the important contributions of Ewald Groenewald, Mahdi
Arzanlou and other personnel at CBS. Finally, we acknowledge financial support from the National Science Foundation (DEB-0228725, Assembling the Fungal Tree of Life and
DEB-0090301, Research Coordination Network: A Phylogeny for Kingdom Fungi).
LITERATURE CITED
Altekar G, Dwarkadas S, Huelsenbeck JP, Ronquist F. 2004.
Parallel Metropolis-coupled Markov chain Monte Carlo
for Bayesian phylogenetic inference. Bioinformatics 20:
407–415.
Aptroot A. 2006. Mycosphaerella and its anamorphs 2.
Conspectus of Mycosphaerella. CBS Biodiversity Series
5. Utrecht, The Netherlands,
Barr ME. 1987. Prodromus to class Loculoascomycetes.
Amherst, Massachusetts: M.E. Barr Bigelow. 168 p.
———. 1992. Notes on the Lophiostomataceae (Pleosporales). Mycotaxon 45:191–221.
———. 2000. Notes on coprophilous bitunicate ascomycetes. Mycotaxon 76:105–112.
Berbee ML. 1996. Loculoascomycete origins and evolution
of filamentous ascomycete morphology based on 18S
rRNA gene sequence data. Mol Biol Evol 13:462–470.
Braun U, Crous PW, Dugan F, Groenewald JZ, de Hoog GS.
2003. Mycol Prog 2:3–18.
Camara MPS, Palm ME, van Berkum P, Stewart EL. 2001.
Systematics of Paraphaeosphaeria, a molecular and
morphological approach. Mycol Res 105:41–50.
Crous PW, Slippers B, Wingfield MJ, Rheeder J, Marasas
WFO, Phillips AJL, Alves A, Burgess T, Barber P,
Groenewald JZ. 2006. Phylogenetic lineages in the
Botryosphaeriaceae. Stud Mycol 55:235–253.
del Prado RI, Schmitt I, Kautz S, Palice R, Lücking R,
Lumbsch HT. 2006. Morphological and molecular
evidence place the Tryphetheliaceae in the Dothideomycetes. Mycol Res 110:511–520.
Denman S, Crous PW, Taylor JE, Kang JC, Pascoe I,
Wingfield MJ. 2000. An overview of the taxonomic
history of Botryosphaeria and a re-evaluation of its
anamorphs based on morphology and ITS rDNA
phylogeny. Stud Mycol 45:129–140.
1052
MYCOLOGIA
Eriksson OE. 1981. The families of bitunicate ascomycetes.
Opera Botanic 60:1–220.
———, Baral H-O, Currah RS, Hansen K, Kurtzman CP,
Laessøe T, Rambold G, eds. 2001. Outline of Ascomycota. Myconet 7:1–88.
———, ed. 2006. Outline of Ascomycota. Myconet 12:1–82.
Guindon S, Gascuel O. 2003. A simple, fast, and accurate
algorithm to estimate large phylogenies by maximum
likelihood. Syst Biol 52:696–704.
Hawksworth DL, Eriksson O. 2003. Saccharicola, a new
genus for two Leptosphaeria species on sugar cane.
Mycologia 95:426–433.
Henssen A, Thor G. 1994. Developmental morphology of
the ‘‘Zwischengruppe’’ between Ascohymeniales and
Ascoloculares. In: Hawksworth DL, ed. Ascomycete
Systematics. Problems and Perspectives in the Nineties.
New York: Plenum Press. p 43–61.
Hughes SJ. 1976. Sooty molds. Mycologia 48:693–820.
Hunter GC, Wingfield BD, Crous PW, Wingfield MJ. 2006. A
multi-gene phylogeny for species of Mycosphaerella
occurring on Eucalyptus leaves. Stud Mycol 55:147–161.
Kirk P, Cannon P, David J, Stalpers J. 2001. Ainsworth and
Bisby’s Dictionary of the Fungi. 9th ed. Wallingford,
UK: CAB International.
Kruys Å. 2005. Phylogenetic relationships and species
richness of coprophilous ascomycetes [Doctoral dissertation]. Umeå, Sweden: Umeå University. 28 p.
Leuchtmann A. 1984. Über Phaeosphaeria Miyake und
andere bitunicate Ascomyceten mit mehrfach querseptierten Ascosporen. Sydowia 37:75–194.
Liew EC, Aptroot A, Hyde KD. 2002. An evaluation of the
monophyly of Massarina based on ribosomal DNA
sequences. Mycologia 94:803–813.
———, ———, ———. 2000. Phylogenetic significance of
the pseudoparaphyses in Loculoascomycete taxonomy.
Mol Phylogenet Evol 16:392–402.
Lindemuth R, Wirtz N, Lumbsch HT. 2001. Phylogenetic
analysis of nuclear and mitochondrial rDNA sequences
supports the view that loculoascomycetes (Ascomycota)
are not monophyletic. Mycol Res 105:1176–1181.
Liu YJ, Hall BD. 2004. Body plan evolution of ascomycetes,
as inferred from an RNA polymerase II phylogeny. Proc
Natl Acad Sci USA 101:4507–12.
Lumbsch HT, Lindemuth R. 2001. Major lineages of
Dothideomycetes (Ascomycota) inferred from SSU
and LSU rDNA sequences. Mycol Res 105:901–908.
———, Schmitt I, Lindemuth R, Miller A, Mangold A,
Fernandez F, Huhndorf S. 2005. Performance of four
ribosomal DNA regions to infer higher-level phylogenetic relationships of inoperculate euascomycetes
(Leotiomyceta). Mol Phylogenet Evol 34:512–24.
Luttrell ES. 1951. Taxonomy of Pyrenomycetes. U Missouri
Stud Sci 24.
———. 1955. The ascostromatic Ascomycetes. Mycologia
47:511–532.
———. 1973. Loculoascomycetes. In: Ainsworth GC, Sparrow FK, Sussman AS, eds. The Fungi: an advanced
treatise. IVA. London: Academic Press. p 59–90.
———. 1974. Parasitism of fungi on vascular plants.
Mycologia 65:1229–1418.
Lutzoni F, Kauff F, Cox CJ, McLaughlin D, Celio G,
Dentinger B, Padamsee M, Hibbett D, James TY,
Baloch E, et al. 2004. Assembling the Fungal Tree of
Life: progress, classification, and evolution of subcellular traits. Am J. Bot 91:1446–1480.
Miadlikowska J, Lutzoni F. 2004. Phylogenetic classification
of peltigeralean fungi (Peltigerales, Ascomycota) based
on ribosomal RNA small and large subunits. Am J Bot
91:449–464.
Nannfeldt JA. 1932. Studien über die Morphologie und
Systematik der nicht-lichenisierten inoperculaten Discomyceten. Nova Acta Regiae Societatis Scientiarum
Upsaliensis, Ser. IV 8(2):1–368.
Reeb V, Lutzoni F, Roux C. 2004. Contribution of RPB2 to
multilocus phylogenetic studies of the euascomycetes
(Pezizomycotina, Fungi) with special emphasis on the
lichen-forming Acarosporaceae and evolution of polyspory. Mol Phylogenet Evol 32:1036–1060.
Reynolds DR. 1998. Capnodiaceous sooty mold phylogeny.
Can J Bot 76:2125–2130.
Rossman AY. 1987. The Tubeufiaceae and similar Loculoascomycetes. Mycol Pap 157:1–71.
Shearer CA. 1993. A new species of Kirschsteiniothelia
(Pleosporales) with an unusual fissitunicate ascus.
Mycologia 85:963–969.
Shoemaker RA, Egger KN. 1982. Piedraia hortae. Fung Can
No. 211.
———, Holm L, Eriksson OE. 2003. Proposal to conserve
the name Dothidea with a conserved type (Fungi:
Dothideomycetes). Taxon 52:623–625.
Silva-Hanlin DMW, Hanlin RT. 2000. Small subunit ribosomal RNA gene phylogeny of several loculoascomycetes and its taxonomic implications. Mycol Res 103:
153–160.
Sivanesan A. 1984. The bitunicate Ascomycetes and their
anamorphs. In: Cramer J, ed. Vaduz, Liechtenstein: J.
Cramer.
Spatafora JW, Mitchell TG, Vilgalys R. 1995. Analysis of
genes coding for small-subunit rRNA sequences in
studying phylogenetics of dematiaceous fungal pathogens. J Clin Microbiol 33:1322–1326.
Tehler A. 1990. A new approach to the phylogeny of
Euascomycetes with a cladistic outline of Arthoniales
focusing on Roccellaceae. Can J Bot 68:2458–2492.
Tsui CKM, Sivichai S, Berbee M. 2006. Molecular systematics
of Helicoma, Helicomyces and Helicosporium and their 2
teleomorphs inferred from rDNA sequences. Mycologia
98:94–104.
Verkley GJM, da Silva M, Wicklow DT, Crous PW. 2004.
Paraconiothyrium, a new genus to accommodate the
mycoparasite Coniothyrium minitans, anamorphs of
Paraphaeosphaeria, and four new species. Stud Mycol
50:323–335.
von Arx J, Müller E. 1975. A re-evaluation of the bitunicate
ascomycetes with keys to families and genera. Stud
Mycol 9:1–159.
Winka K, Eriksson OE, Bång Å. 1998. Molecular evidence
for recognizing the Chaetothyriales. Mycologia 90:822–
830.
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