Mycol. Res. 108 (11): 1271–1282 (November 2004). f The British Mycological Society
1271
DOI: 10.1017/S0953756204001054 Printed in the United Kingdom.
Mycosphaerella punctiformis revisited : morphology, phylogeny,
and epitypification of the type species of the genus
Mycosphaerella (Dothideales, Ascomycota)
Gerard J. M. VERKLEY1, Pedro W. CROUS1, J. Z. (Ewald) GROENEWALD1, Uwe BRAUN2
and André APTROOT1
1
Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands.
F.B. Biologie, Institut für Geobotanik und Botanischer Garten, Martin-Luther-Universität, Neuwerk 21,
D-06099 Halle (Saale), Germany.
E-mail : verkley@cbs.knaw.nl
2
Received 21 January 2004; accepted 15 July 2004.
Mycosphaerella punctiformis, the type species of the genus Mycosphaerella, is epitypified by material collected on Quercus
robur in The Netherlands. The teleomorph is described in planta, and the Ramularia anamorph, for which the new name
R. endophylla is proposed, and the Asteromella spermatial state are characterized in vitro. Sequence data of the nuclear
ribosomal DNA are presented and analyzed together with other Mycosphaerella spp. with Ramularia and several other
anamorphs. Several strains originating from Quercus, Acer and Tilia showed diverging ITS sequences, indicating that
the M. punctiformis complex may comprise more than a single phylogenetic species, but this could not be confirmed by
the analysis of our dataset. An endophytic phase is established for the first time in the life-cycle of M. punctiformis, as the
species was repeatedly isolated from surface sterilized green healthy leaves of Quercus robur in summer at the epitype
locality.
INTRODUCTION
The genus Mycosphaerella is one of the largest genera
of ascomycetes, comprising many plant pathogens of
economically important crops, but also saprobic species.
Teleomorph morphology is relatively simple and uniform in Mycosphaerella, but the genus is unequalled in
the diversity of the associated anamorphs. Indeed, 27
anamorphic genera have been associated with Mycosphaerella (von Arx 1983, Sutton & Hennebert 1994),
23 of which were accepted by Crous et al. (2000).
Klebahn (1918) and Laibach (1922) suggested segregating groups of species from Mycosphaerella based
on their association with a particular anamorph, but
genera they proposed did not become widely used.
Recent molecular studies indicate that characters used
to define the anamorph genera, such as conidiomatal
structure, and conidial shape, size, and septation, are
not always phylogenetically informative, and that some
generic concepts for the anamorphs need to be revised
(Crous et al. 2000, Crous, Kang & Braun 2001, Verkley
et al. 2004). However, a group of species with Cladosporium anamorphs was recently segregated under the
name Davidiella (Braun et al. 2003) ; it is a close sister
group of other Mycosphaerella.
Mycosphaerella punctiformis, the type species of
Mycosphaerella, was originally described as Sphaeria
punctiformis from fallen leaves of Quercus robur.
Microscopical examination of the lectotype material of
M. punctiformis deposited in L, confirmed the identity.
However, the over 200 yr old herbarium specimen does
not provide an unambiguous application of the name,
because recent molecular work has shown that M.
punctiformis as currently circumscribed comprises
cryptic species that are morphologically indistinguishable. Several strains in the CBS collection that had been
morphologically identified as M. punctiformis from
Quercus, Acer and Tilia, were found to be heterogeneous in their sequences of the internal transcribed
spacer (ITS) region of the nuclear ribosomal RNA gene
array. As no ex-type strain is available, we tried to
obtain ribosomal DNA from the type material of M.
punctiformis, but failed. In accordance with Art. 9.7 of
the Code, we sought to settle the application of the name
by selecting an epitype for M. punctiformis. The main
purpose of this paper is to epitypify M. punctiformis
with material recently collected from the type host
Quercus robur in The Netherlands, and to give a full
phenotypic characterization of the teleomorph, and
(syn)anamorphs in culture. Because the anamorph will
Mycosphaerella punctiformis revisited
be the only sporulation observed in most ecological and
endophyte studies, we consider it useful to also formally name this conidial state. Fresh ascomata of
M. punctiformis were collected on dead fallen leaves
of the type host Quercus robur, checked for agreement
with the lectotype material, and ascospore isolates were
made. We also obtained ecological data from a biodiversity study of foliar ascomycetous endophytes of
Quercus in the epitype locality. We sequenced the ITS
region of rDNA of the available strains of M. punctiformis, and also included a number of additional taxa
in the sequence analyses to investigate the phylogenetic
relationships of M. punctiformis with other Mycosphaerella species with Ramularia and several other
anamorphs. Furthermore, partial small subunit (SSU)
sequences of the ex-epitype strain of M. punctiformis
were analysed with other data available in order to
obtain further support for the phylogenetic position
within the Mycosphaerella clade.
MATERIALS AND METHODS
Isolation from fruit bodies on decaying leaves
and endophytic mycelia from green leaves
Strains used in this study are listed in Table 1. Dead
fallen leaves with ascomata were collected in March
to May of 2002 and 2003 in the forested area ‘De
Stompert’ in The Netherlands, from three mature trees
of Quercus robur. Leaves were incubated in a moist
chamber for several hours in the laboratory at ca
20 xC. They were then cut into square pieces and
glued to the inside of Petri dish lids to allow ascospores
to be discharged on to 2% malt extract agar (MEA).
Germinating ascospores were examined after 24 h,
illustrated and transferred to MEA. Fresh green leaves
from the same trees were collected monthly between
May and November, put in plastic bags and transported to the lab. On the same day, leaves were sterilized in domestic bleach water (5 % chlorine) for 5 min,
followed by three rinses in sterile water. Small squares
of about 0.5 cm2 were placed onto MEA with 50 ppm
streptomycin, aureomycin and penicillin to inhibit
bacterial growth, placed on the laboratory bench in
diffuse daylight, and regularly checked for fungal
growth. Mycelia growing out of the margin were
transferred to 2% MEA and oatmeal agar (OA ;
Centraalbureau voor Schimmelcultures 2001) and preliminarily identified morphologically.
Phenotypic characterization
For microscopic examination, fruiting structures were
mounted in tap water. Line drawings were made with a
drawing tube, and photographic images with a Nikon
Coolpix 995 digital camera. For the description of
colony features and sporulating structures, isolates
were transferred onto OA and 3% MEA plates and
placed in an incubator at 15 x under n-UV (12 h
1272
rhythm). Colours are described according to Rayner
(1970).
DNA extraction and sequencing
Strains were transferred from agar cultures to 2 ml
liquid medium (2 % malt extract) and incubated on a
rotary shaker (300 rpm) for 3 wk at room temperature.
Liquid cultures were transferred to 2-ml tubes, centrifuged and washed twice with sterile water. DNA was
extracted using the FastDNAkit (Omnilabo 6050073,
BIO 101, CA) according to the manufacturer’s instructions. For ITS sequence analysis a part of the
ribosomal RNA gene cluster was amplified by PCR
using primer pairs V9G (de Hoog & Gerrits van den
Ende 1998) and LR5 (Vilgalys & Hester 1990). Part of
the 18S rRNA gene (SSU) was amplified using primers
NS1 and NS4 (White et al. 1990). PCR was performed
in 50 ml reaction volumes, each reaction containing
10–100 ng of genomic DNA, 25 pM of each primer,
40 mM dNTP, 1.0 unit Supertaq DNA polymerase and
5 ml 10r PCR buffer (SphaeroQ, Leiden). PCR was
performed in an Applied Biosystems (Foster City, CA)
thermocycler with the following program : 1 min at 95 x,
30 cycles (1 min 95 x, 1 min 55 x, 2 min 72 x) followed by
a final extension of 5 min at 72 x. PCR products were
cleaned with GFX columns (Amersham Pharmacia,
NJ) and analyzed on a 2 % agarose gel to estimate
concentrations. ITS1 and ITS4 (White et al. 1990) were
used as internal sequencing primers for the ITS region.
The SSU region was sequenced using the PCR primers.
Sequencing was performed with the BigDye terminator
chemistry (Part no. 403049, Applied Biosystems) following the manufacturer’s instructions. The sequencing
products were cleaned with G50 Superfine Sephadex
columns (Amersham Pharmacia 17-0041-01), and
separated and analyzed in an ABI Prism 3700 DNA
Analyzer (Applied Biosystems). Forward and reverse
sequences were matched using SeqMan (DNAstar,
Madison, WI).
Phylogenetic analyses
Pairwise and global alignment of consensus sequences
were performed in Bionumerics 3.0 (Applied Maths,
Kortrijk, Belgium), and manually adjusted where
necessary. Parsimony analysis was done using PAUP
v. 4.0b10 (Swofford 2003). The heuristic search was
performed with the following parameters : characters
unordered with equal weight, random taxon addition,
branch swapping with tree bisection-reconnection
(TBR) algorithm, branches collapsing if the maximum
branch length was zero. Maxtrees was set at 10 000.
Alignment gaps were treated as missing characters.
Parsimony bootstrap analyses were performed using
the full heuristic search option, random stepwise
addition, and 1000 replicates, with maxtrees set at 100.
Neighbour-joining analyses was performed using
PAUP, with GTR (Gamma=0.5, and rates for variable
G. J. M. Verkley and others
sites equal), and 1000 neighbour-joining bootstrap replications to test the stability of clades. BLAST searches
in GenBank revealed highest similarity to species of
Mycosphaerella. GenBank accession numbers, taxon
names and other information about the sequences from
GenBank used in this study are given in Table 1. GenBank accession numbers (marked with *) of sequences
generated in this study are also given in Table 1. A
strain of Davidiella tassiana (sub Mycosphaerella
tassiana) was defined as outgroup for the ITS dataset
and sequences of Botryosphaeria species were used as
outgroup for the SSU dataset. The alignments and trees
were lodged in TreeBASE (study accession S1126).
RESULTS
Phylogenetic analyses
The alignment of the ITS sequences comprised 513
characters, of which 168 (36 %) were parsimonyinformative. 23 of these characters were excluded from
the analysis because they were positioned in small insertions/deletions or regions with ambiguous position
homology. Furthermore, 322 uninformative characters
were also excluded, so that 145 characters were used in
the parsimony analysis. In the neighbour joining
analysis in total 213 characters were included, as constant characters were excluded, but autapomorphic
characters were included to obtain accurate branch
lengths in the phylogram. The heuristic search yielded
580 most parsimonious trees (MPT) of 535 steps
(C.I.=0.505, R.I.=0.878, R.C.I.=0.443, and homoplasy index=0.495). The strict consensus tree is shown
in Fig. 1. Several highly supported multi-taxon clades
were the same in the parsimony and neighbour joining
analyses (neighbour joining trees not shown). Among
these was a clade comprising all included strains
with Ramularia anamorphs (parsimony 99 %/neighbour
joining minimum 100 %), which in the parsimony
analysis formed a sister group to the clade with the
cereal pathogens Mycosphaerella graminicola and Septoria passerini (100/92). The support for the two clades
together was, however, lower (61/<50). Further highly
supported clades were the one with Cercospora spp.
(90/97), a clade with M. crystallina, M. heimii, M.
heimioides and M. colombiensis (99/95), and a clade
with M. africana, M. keniensis, M. aurantia, M. hedericola, Mycosphaerella sp. (from Coprosma sp.),
M. confusa, and Passalora fulva (91/81). The Ramularia
clade was rather unresolved in parsimony and neighbor joining analyses. In the parsimony analysis,
only a clade comprising four strains identified as
M. punctiformis from Quercus, Acer and Tilia was
well-supported (100/95). With their closest sister M.
phacae-frigidae, these strains also obtained good support in both analyses (91/77).
BLAST results of the SSU sequence of M. punctiformis (AY490775) supported the close association of
M. punctiformis with other Mycosphaerella species. The
1273
alignment of the SSU sequences included 1067 characters, of which 1006 were constant, 21 were parsimony
uninformative and 40 were parsimony informative. The
heuristic search yielded eleven most parsimonious trees
of 81 steps (C.I. 0.852, R.I. 0.919, R.C. index 0.783, and
H.I. 0.148). The strict consensus tree is shown in Fig. 2.
The topology of the eleven trees only differed in the
internal ordering of groups in the Mycosphaerella
clade. Two main clades are delimited in the SSU tree,
the first clade contains isolates of Mycosphaerella (98 %
bootstrap support) and the other isolates of Davidiella
(100 % bootstrap support). The sequence of M. punctiformis groups closest to the sequences of a Mycosphaerella sp. isolated from Acacia (AY251116) and a
sequence of Septoria tritici (AY251117). However,
this association does not have significant bootstrap
support.
Phenotypic characterization
(Figs 3–10)
A description of the teleomorph in planta: Leaf spots
not observed. Ascomata developing on fallen dead
leaves, predominantly hypophyllous, black, subepidermal, erumpent to superficial, globose, 70–110 mm diam ;
apical ostiole 5–10 mm wide ; wall consisting of 2–3
layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, cylindrical,
straight, 8-spored, 30–45r5–7(–9) mm. Ascospores
multiseriate, overlapping, hyaline, guttulate, thinwalled, straight, fusoid-ellipsoidal with obtuse ends,
widest just above the septum, medianly 1-septate, constricted at the septum, tapering towards both ends, but
more prominently towards the lower end, (6–)8–
9(–10)r(2–)3 mm (av. 9r3 mm). Germinating ascospores hyaline, distorting, forming germ tubes 4–6 mm
diam apically, parallel to the long axis from both
ascospore cells, and simultaneously also laterally,
from one or both ascospore cells, at an angle of 90 x or
less to the long axis (Germination pattern D; Crous
1998).
Free conidia possibly belonging to M. punctiformis
were occasionally observed in late summer on older leaf
lesions caused by pathogens such as Septoria quercicola
and Discula sp.
Colony description (diffuse daylight, 15 x) : Colonies
on OA reaching 28–31 mm diam in 27 d, spreading
(low), sometimes in the centre with some elevated mycelium, margin even or slightly lobed, glabrous, pale
Honey to Olivaceous Buff or Rosy Vinaceous to Rosy
Buff, colony surface glabrous or with appressed pure
white aerial hyphae or conidiophores ; in the centre
submerged and superficial mycelium Rosy Buff to
Salmon and concolourous on reverse, or becoming
Dark Violet to dark Purple due to the deposition of
violet pigment on the outer surface of vegetative
hyphae, surrounding medium then often becoming
Coral to red by diffusing pigments, and Coral to Flesh
on reverse. In a few isolates, the colony was dominated
by olivaceous colours (underneath a white covering of
GenBank accession no.
ITS
AY251078
Teleomorph
Anamorph
Origin
U42476
U42477
AY251096
AY251094
AY251092
AY251098
AY251097
Botryosphaeria rhodina
B. ribis
Davidiella tassiana
Davidiella state unknown
Davidiella state unknown
Davidiella state unknown
Davidiella state unknown
Lasiodiplodia theobromae
Fusicoccum sp.
Cladosporium herbarum
Cl. cladosporioides
Cl. colocasiae
Cl. sphaerospermum
Cl. uredinicola
Mycosphaerella africana
M. africana
M. aurantia
M. colombiensis
?M. confusa
M. crystallina
M. crystallina
M. fijiensis
M. fijiensis
M. musicola
M. musicola
M. laricina
M. fragariae
M. fragariae
M. fragariae
M. fragariae
M. fragariae
M. graminicola
M. graminicola
M. graminicola
M. graminicola
M. graminicola
M. graminicola
M. grossulariae
M. hedericola
M. heimii
M. heimii
M. heimii
M. heimii
M. heimioides
M. keniensis
M. latebrosa
M. latebrosa
M. latebrosa
M. marksii
Unknown
Unknown
Unknown
Pseudocercospora colombiensis
Ps. rubi
Ps. crystallina
Ps. crystallina
Ps. fijiensis
Ps. fijiensis
Ps. musae
Ps. musae
Pseudocercospora sp.
Ramularia grevilleana
R. grevilleana
R. grevilleana
R. grevilleana
R. grevilleana
Septoria tritici
S. tritici
S. tritici
S. tritici
S. tritici
S. tritici
S. ribis
Unknown
Pseudocercospora heimii
Ps. heimii
Ps. heimii
Ps. heimii
Ps. heimioides
Unknown
Septoria aceris
S. aceris
S. aceris
Unknown
No data available
No data available
ATCC 66670 (=‘STE-U 5101’) ; CCA-treated Douglas-fir pole, New York, USA
ATCC 66669 (=‘STE-U 5100’) ; Creosote-treated southern pine pole, New York, USA
STE-U 4323; Colocasia esculenta, Fiji Islands
CBS 188.54 (=‘STE-U 3686’, ATCC 11290)
ATCC 46649 (=‘STE-U 5390’) ; Fungicolous on Cronartium fusiforme f. sp. quercum
on Quercina nigra leaves, Alabama, USA
STE-U 794 (ex-type); Eucalyptus viminalis, South Africa
CBS 680.95 (=STE-U 796; ex type); Eucalyptus viminalis, South Africa
CBS 110500; Eucalyptus globulus, Australia
STE-U 1106; Eucalyptus, Colombia
CBS 256.35
STE-U 801 (ex type); Eucalyptus bicostata, South Africa
CBS 681.95, STE-U 802 (ex type); Eucalyptus bicostata, South Africa
ATCC 22116, PF7; Musa sp., Philippines
ATCC 36054, PFD9; Musa sp., Honduras
ATCC 22115; Musa sp., Philippines
PM11, ATCC 36143; Musa, Honduras
CBS 326.52; Larix decidua, Switzerland
CBS 259.36; Fragaria sp., The Netherlands
CBS 719.84; Fragaria sp., The Netherlands
CBS 298.34; Fragaria sp., The Netherlands
ATCC 24113; Fragaria sp., Illinois, USA
STE-U 656 ; Fragaria sp., South Africa
CBS 100330 (=IPO 6566.1); Triticum aestivum, The Netherlands
CBS 100335; Triticum aestivum, The Netherlands
CBS 392.59; Triticum aestivum
IPO 323; Triticum aestivum, The Netherlands
T1; Triticum aestivum, Minnesota, USA
STE-U 658 ; Triticum sp., South Africa
CBS 235.37; Ribes nigrum, The Netherlands
CBS 441.86; Hedera helix, France
CMW5705
CMW5707
No data available
CMW5713
STE-U 1312; Eucalyptus, Indonesia
STE-U 1084; Eucalyptus grandis, Kenya
CBS 183.97; Acer pseudoplatanus, The Netherlands
CBS 687.94; Acer pseudoplatanus, The Netherlands
CBS 652.85; Acer pseudoplatanus, The Netherlands
CBS 682.95 (=‘STE-U 842’) ; Eucalyptus grandis, South Africa
AY251117
AY251114
1274
AF173314
AY490773
AY150331
AF222838
AF362058
AF222839
AY490757
AY266152
AY266150
AF181706
AY288148
AY152590
AY152595
AY152597
AY152596
AF297235
AF173312
AY152601
AY152602
AY152603
AF181692
AF181693
AF362068
AY152581
AY490772
AF452508
AF452509
AF222841
AF452512
AF222842
AF173300
AY490768
AY152553
AY490769
AY152600
SSU
Mycosphaerella punctiformis revisited
Table 1. Fungal isolates included for ITS and SSU sequence analyses (in alphabetical order of the teleomorph names).
AY490775*
AY251115
AY251116
AY251110
AY251111
AY251104
AF279583
AY251108
Stenella parkii
Ramularia sp. ?
Septoria populicola
S. populicola
S. populicola
S. populicola
S. populicola
Ramularia sp.
Ramularia sp.
Ramularia sp.
Ramularia sp.
R. endophylla
R. endophylla
R. endophylla
R. endophylla
R. endophylla
R. endophylla
R. endophylla
Septoria pyricola
S. pyricola
Ramularia sp. ?
Phloeospora ulmi
Septoria sp. (in culture)
S. quercicola
Pseudocercospora stromatosa
Ramularia collo-cygni
Ramularia sp.
Ramulispora sorghi
R. sorghi
Cercospora apii
C. beticola
C. beticola
C. kikuchii
C. kikuchii
C. kikuchii
C. zebrina
Septoria apiicola
S. apiicola
S. apiicola
S. castaneicola
S. epambrosiae
S. hippocastani
S. lamiicola
S. lamiicola
S. passerinii
S. passerinii
Passalora dodonaeae
CBS 387.92 (=‘STE-U 353; ex type); Eucalyptus grandis, Brazil
CBS 234.55; Phaca frigida, Switzerland
CBS 100045; Populus trichocarpa, Washington, USA
CBS 100052; Populus trichocarpa, Washington, USA
CBS 100044; Populus trichocarpa, Washington, USA
CBS 100051; Populus trichocarpa, Washington, USA
CBS 100047; Populus trichocarpa, Washington, USA
CBS 515.69; Acer pseudoplatanus, The Netherlands
CBS 742.79; Tilia sp., Germany
CBS 943.97; Quercus sp., The Netherlands
CBS 184.97; Acer pseudoplatanus, The Netherlands
CBS 942.97; Quercus sp., Belgium
CBS 113871 (SS) ; Quercus robur, The Netherlands
CBS 113265 (SS; ex-epitype) ; Quercus robur, The Netherlands
CBS 113868; leaf endophyte Quercus robur, The Netherlands
CBS 113869; leaf endophyte Quercus robur, The Netherlands
CBS 113870; leaf endophyte Quercus robur, The Netherlands
KC1
CBS 222.31; Pyrus communis
CBS 640.72; Pyrus communis, The Netherlands
CBS 288.49; Angelica sylvestris
CBS 344.97; Ulmus glabra, Austria
CBS 113113; Coprosma sp., New Zealand
CBS 663.94; Quercus robur, The Netherlands
STE-U 1731; Protea sp., South Africa
STE-U 3837; Acacia sp., Venezuela
STE-U 2045; Hordeum sp., Germany
‘ascomycete 2’; Quercus robur, Germany
STE-U 905 ; Sorghum sp., South Africa
STE-U 906 ; Sorghum sp., South Africa
CA1, ATCC 12246
CBS 539.71; Beta vulgaris, Romania
MPPD12120, CB4; Beta vulgaris, Minnesota, USA
CBS 128.27 (ex type) ; Glycine max, Japan
CK 39; Glycine max, Illinois, USA
CK 35; Glycine max, Illinois, USA
STE-U 3955; Trifolium pratense, Canada
CBS 395.52 (=IMI 092627); Apium sp., The Netherlands
CBS 389.59; Apium graveolens, Italy
CBS 400.54 (=IMI 092628); Apium graveolens, The Netherlands
CBS 102377; Castanea sativa, The Netherlands
Ambrosia artemisiifolia
CBS 411.61; Aesculus hippocastanum, Germany
CBS 109113; Lamium album, Austria
CBS 102328; Lamium album, The Netherlands
ATCC 26516 ; Hordeum vulgare, Minnesota, USA
P78 ; Hordeum vulgare, Minnesota, USA
STE-U 1223; Dodonaea sp., South Africa
1275
AF173310
AJ417496
AY259131
AY259132
AY166268
AY152576
AY266165
AY152577
AY166260
AY266161
AY260078
AY152572
AY152573
AY152574
AY152588
AF279582
AY490770
AY152563
AY152564
AF181697
AF181699
M. parkii
M. phacae-frigidae
M. populicola
M. populicola
M. populicola
M. populicola
M. populicola
‘ M. punctiformis’
‘M. punctiformis’
‘M. punctiformis’
‘M. punctiformis’
M. punctiformis
M. punctiformis
M. punctiformis
M. punctiformis
M. punctiformis
M. punctiformis
M. punctiformis
M. pyri
M. pyri
M. rubella
M. ulmi
Mycosphaerella sp.
Mycosphaerella sp.
Mycosphaerella stromatosa
Mycosphaerella sp.
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
G. J. M. Verkley and others
AY152599
AY490758
AY152583
AY152584
AY152585
AY152586
AY152587
AY490759
AY490760
AY152593
AY152594
AY490761
AY490762
AY490763
AY490764
AY490765
AY490766
AF222848
AY152591
AY152592
AY490767
AY152575
AY490774
AY490771
AY251095
AY251113
AY251106
AY251107
AY152559
AY152561
1276
conidiophores) and greyish Sepia to Hazel or Olivaceous on reverse.
Colonies on MEA reaching 21–30 mm diam in 27 d,
restricted and up to 5 mm high in the centre, margin
weakly to distinctly lobed, glabrous or finely felty of pure
white aerial hyphea, Buff, pale Olivaceous or Rosy Buff,
colony surface Pale Vinaceous or Pale Violet, and then
often the surrounding medium becoming Coral to red
by diffusing pigments, or greyish, but largely covered by
pure white aerial hyphae or conidiophores ; reverse
Dark Purple to Blood Colour, or Fawn to Vinaceous
Buff with Dark Brick, Brick and Cinnamon areas.
TAXONOMY
Ramularia endophylla Verkley & U. Braun, sp. nov.
(Figs 11–16)
Conidiophora unicellulares (=cellulae conidiogenae), simplicia, subcylindrica vel cylindrica, (6–)10–30r2.5–4(–5) mm,
recta vel geniculata-sinuosa, hyalina, levia ; cicatrices conidiales leniter incrassatae et fuscae, circa 1 mm latae ; conidia
hyalina, levia vel sublevia, hila incrassata, fusca, refractiva,
0.5–1(–1.5) mm lata ; conidia primaria solitaria, ovoidea,
ellipsoidea vel subcylindrica, continua, apice rotundato, basin
versus leniter attenuata, 6–15r2–4 mm ; conidia secundaria
catenata, saepe ramicatenata, in OA praecipue ellipsoidea vel
cylindrica, in MEA ovoidea vel ellipsoidea-fusiformia, recta
vel curvata, 0–1-septata, in OA 7–29r3–4(–5) mm, in MEA
(4–)7–10(–15)r(3–)4–5 mm.
Typus: The Netherlands : Utrecht : Soesterberg, ‘ De
Stompert’, on dead leaf of Quercus robur (‘B3 ’), April 2003,
G. Verkley s.n. [ex-epitypus Mycosphaerella punctiformis]
(CBS 113265–holotypus; culture kept metabolically inactive,
in liquid nitrogen and lyophilized).
* GenBank accession no. of LSU sequence=AY490776.
CBS 119.46 (=‘STE-U 3688’) ; Lycopersicon esculentum, The Netherlands
CBS 145.37 (‘ STE-U 4303’) ; Oryza sativa, Arkansas, USA
CBS 102336; Knautia arvensis, The Netherlands
CBS 182.93; Succissa pratensis, France
CBS 354.29 (=‘STE-U 4301’)
CBS 355.58 (=‘STE-U 4302’) ; leaf of Rosa sp.
CBS 149.53 (=‘ATCC 11669); leaf of Citrus sinensis, Angola
STE-U 1869; Leucadendron sp., South Africa
Pas. fulva
Pas. janseana
Septoria scabiosicola
S. scabiosicola
Unknown
S. rosae
Pseudocercospora angolensis
Ps. protearum var. leucadendri
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Mycosphaerella state unknown
Sphaerulina polyspora
? Sphaerulina rhemiana
Unknown
Unknown
AY251069
AY251109
AY251103
Origin
Anamorph
Teleomorph
SSU
ITS
GenBank accession no.
Table 1. (Cont.)
Mycosphaerella punctiformis revisited
Conidiophores simple, subcylindrical or cylindrical,
(6–)10–30r2.5–4(–5) mm, straight or geniculatesinuous, hyaline, smooth-walled, arising from terminal
or intermediary hyphal cells at the colony surface, often
without a basal septum ; conidial scars somewhat
thickened and darkened, about 1 mm wide ; conidia
formed holoblastically, hyaline, walls smooth to
minutely roughened, hila conspicuous, thickened, darkened, refractive, 0.5–1(–1.5) mm wide ; primary conidia
solitary, ovoid, or ellipsoid to subcylindrical, aseptate,
rounded at the top and somewhat attenuated towards
the base, 6–15r2–4 mm; secondary conidia catenate,
often in branched, acropetal chains, on OA predominantly ellipsoid to cylindrical, on MEA ovoid to
ellipsoid-fusiform, straight to curved, 0–1-septate, ends
with a single hilum rounded to attenuated, branching
ends often with hila on short projections, on OA 7–29r
3–4(–5) mm, on MEA (4–)7–10(–15)r(3–)4–5 mm.
Asteromella spermatial state
Description in vitro : Spermogonia submerged or on the
agar surface, pycnidial, globose, mostly aggregated in
larger complexes containing several merging cavities
and one or several rather undifferentiated ostioles,
black to dark brown ; conidiomatal walls composed of
G. J. M. Verkley and others
1277
90
56
51
66
90
63
100
96
58
100
57
100
100
98
65
60
AY152573
AY152576
C. beticola
C.beticola
CB4
AY260078
C. zebrina
C.zebrina
AY260078
AY152587
M.populicola AY152587
AY152585
M.populicola AY152585
M. AY152583
populicola
AY152583
M.populicola
AY152584
M.populicola AY152584
AY152586
M.populicola AY152586
CBS
183.97
M.latebrosa
CBS183.97
latebrosa
AY152553
M.latebrosaM.CBS652.85
CBS
652.85
M.latebrosa AY152553
AY152575
M.ulmiMycosphaerella
AY152575ulmi
AY152581
M. grossulariae
M.grossulariae
AY152581
AY152591
M.pyri AY152591
M. pyri
AY152592
M.pyri AY152592
AY259131
Ramulisp.s orghii AY259131
Ramulispora sorghii
AY259132
Ramulisp.s orghii AY259132
AF181706
M.musicola
AF181706
M. musicola
PM
11
Ps.musae
PM11
PFD9
Paracercospora
fijiensis
var. difformis
Pa.fijiensis
v diff
PFD9
PF7
Pa. fijiensis var.
Pa.fijiensis
v fijiensis
fij PF7
AF222839
M.crystallinaM.AF222839
crystallina
CBS
685.91
M.crystallina
CBS685.91
AF222842
M. heimioides
M.heimioides
AF222842
AF452512
M.heimii AF222841
AF452508
M.heimii AF452512
M. heimii
AF452509
M.heimii AF452508
AF222841
M.heimii AF452509
AF222838
Pseudocercospora
colombiensis
Ps.colombiensis
AF222838
99
52
52
AY152573
S.apiicola
apiicola
AY152572
S.apiicolaSeptoria
AY152572
AY152574
S.apiicola AY152574
AF279582
S. epambrosiaeAF279582
S.epambrosiae
AY152563
S.lamiicola
AY152563
S. lamiicola
AY152564
S.lamiicola AY152564
AY152559
S.scabiosicola
AY152559
S. scabiosicola
AY152561
S.scabiosicola AY152561
CBS
411.61 S. hippocastani
S.hippocas
tani CBS411.61
AY152577
C.kikuchii Cercospora
AY152577
kikuchii
CK39
C.kikuchii CK39
CK35
C. kikuchii CK35
C.kikuchii
CB4
C. beticola AY152576
C.beticola
CA1
C. apiiCA1
C.apii
85
91
70
83
98
100
91
68
Ramularia - clade
99
74
59
61
71
100
97
CBS
441.86 M. hedericola
M.hedericola
CBS441.86
AF362058
M. confusa
M.sp. Coprosma
V1817
CBS
113113
Mycosphaerella
sp. Coprosma
M.confusa AF362058
AF173300
M.
keniensis
M.keniensis AF173300
AF173314
M. africana
M.africana
AF173314
CBS
680.95 M. africana
M.africana
CBS680.95
AY150331
M. aurantia
M.aurantia
AY150331
AY251069
Passalora
fulva
Pas.fulva AY251069
AY152590
M.
laricina
M.laricina AY152590
AY152588
S. castaneicola
S.castaneicola
AY152588
AY152599
M. parkii
M.parkii
AY152599
AY152600
M. marksii
M.marksii
AY152600
CBS
724.79 ‘AY152594
M. punctiformis’ ex Tilia
M.spec
CBS
515.69
‘
M.
punctiformis
’ ex Acer
M.punctiformis CBS724.79
AY152593
‘M. punctiformis
’ ex Quercus
M.punctiformis
CBS515.69
AY152594
punctiformis’ ex Acer
M.spec‘M.AY152593
CBS
234.55 M. phacae-frigidae
M.phacae-frigid
CBS234.55
CBS
942.97
M.punctiformis
CBS942.97
AF222848
Ramularia
sp.
KC1
Ramularia sp.KC1 AF222848
CBS
113870
M.punctiformis
CBS113870
CBS
113871
M.punctiformis
CBS113871
CBS
113869
M.punctiformis
CBS113869
CBS
113265 ex - epitypeCBS113265
M.punctiformis
CBS
113868
M.punctiformis CBS113868
AY152596
M.fragariae AF297235
AY152595
M.fragariae AY152597
AY152597
fragariae
M.fragariaeM.
AY152596
AF297235
M.fragariae AY152595
M. punctiformis
AF173312
M.fragariae AF173312
AF173310
Ra. collo-cygni AF173310
Ram.collo-cygni
CBS
288.49 M. rubella
M.rubella
CBS288.49
AJ417496
Ramularia sp. ‘ascomycete
2’
Ram.sp.ascom.2
AJ417496
AY152601
M.graminicola AY152601
AY152602
S.tritici AF362068
AF362068
M.graminicola
AY152602
M. graminicola
AF181692
M.graminicola AF181692
AF181693
M.graminicola AF181693
AY152603
M.graminicola AY152603
AF181697
S.passerinii
AF181697
S. passerinii
AF181699
S.passerinii AF181699
CBS
663.94
S.
quercicola
S.quercicola CBS663.94
AY251078
Davidiella tassiana
Dav.tassiana
AY251078
Fig. 1. Strict consensus tree of 580 most parsimonious trees of 535 steps obtained in a heuristic search of 168 parsimonyinformative characters of the ITS1-5.8SrDNA-ITS2 region calculated in PAUP. Numbers at the branches are
bootstrap values obtained from 1000 replications and rounded to the nearest integer, shown only for branches supported
by more than 50 %. Species are labelled by teleomorph name, if known (anamorph names are given in Table 1).
Mycosphaerella punctiformis revisited
56
AY251103 Passalora janseana
1278
3
AY251113 Septoria rosae
AY251104 Cercospora zebrina
AF279583 S. epambrosiae
AY251114 M. latebrosa
50
AY251115 Mycosphaerella sp.
63
AY251116 Mycosphaerella sp.
CBS 113265 M. punctiformis
AY251117 S. tritici
Mycosphaerella clade
AY251108 P. dodonaea
98
AY251109 P. fulva
85
AY251110 Ramulispora sorghi
AY251111 R. sorghi
76
100
AY251106 Pseudocercospora angolensis
AY251107 Ps. protearum var. leucadendri
AY251094 Cladosporium cladosporioides
AY251095 Sphaerulina polyspora
100
AY251092 C. colocasiae
AY251096 Davidiella tassiana
Davidiella clade
AY251098 C. sphaerospermum
4
AY251097 C. uredinicola
U42477 Botryosphaeria ribis
U42476 B. rhodina
1 change
Fig. 2. One of eleven most parsimonious trees obtained from a
heuristic search of the SSU sequence alignment. Bootstrap
support values from 1000 replicates are shown at the nodes
and the scale bar represents a single change. Branches that
were maintained in the Strict consensus tree are thickened and
the tree is rooted to Botryosphaeria ribis and Botryosphaeria
rhodina.
an outer layer of thick-walled, brown textura angularis,
and an inner layer of hyaline, irregular to isodiametric
cells ; spermatogenous cells phialidic, determinate, hyaline, discrete or integrated in simple, septate, more
rarely branched, hyaline spermatiophores with acropleurogenous openings ; spermatia ellipsoid to subcylindrical, with rounded ends, hyaline, smooth-walled,
aseptate, 3–4(–5)r1–1.5 mm, whitish in mass.
Sphaeria punctiformis Pers., Ann. Bot. (Usteri) 11 : 26.
1794 : Fr., Syst. Mycol. 2 : 525 (1823).
Sphaerella punctiformis (Pers. : Fr.) Rabenh., Herb.
Vivum Mycol., ed. nov., cent. 3, no. 264 (1856).
Mycosphaerella punctiformis (Pers. : Fr.) Starbäck,
Bih. Kongl. Svenska Vetensk.-Akad. Handl. 15(3,
2) : 9 (1889).
Typus: The Netherlands : On lower surface of dead leaves of
Quercus (Fagaceae), Persoon s.n. (L-Persoon – lectotypus hic
designatus); Utrecht : Soesterberg, ‘ De Stompert ’, G. Verkley
Figs 3–4. Mycosphaerella punctiformis, epitype (CBS herb.
7949). Fig. 3. Ascospores and asci in planta. Fig. 4. Germinating ascospores on MEA. Bars=10 mm.
G. J. M. Verkley and others
1279
5
6
7
8
9
10
11
13
12
14
Figs 5–14. Mycosphaerella punctiformis in vitro (diffuse daylight, 18 xC). Figs 5–7. Isolates on MEA, after 27 d.
Fig. 5. CBS 113870. Fig. 6. CBS 113868. Fig. 7. CBS 113869. Figs 8–10. Isolates on OA, after 27 d. Fig. 8. CBS 113870.
Fig. 9. CBS 113869. Fig. 10. CBS 113868. Figs 11–14. Conidia and conidiogenous cells on OA. Bars=10 mm.
Mycosphaerella punctiformis revisited
Fig. 16. Mycosphaerella punctiformis (CBS 113265 – ex-epitype).
Conidiogenous cells and conidia on MEA. Bar=10 mm.
1280
Fig. 15. Mycosphaerella punctiformis (CBS 113265 – ex-epitype). Conidiogenous
cells and conidia on OA. Bar=10 mm.
G. J. M. Verkley and others
s.n., on dead leaf of Quercus robur (‘ B3’), April 2003 (CBS
herb. Nr 7949 – epitypus hic designatus) ; living single ascospore (SS) culture CBS 113265 – (ex-epitype ; also with the
holotype of Ramularia endophylla).
The lectotype is the only material under this name in
the Persoon herbarium that was not classified in
another (often invalid) variety by himself. It is typical
for the species, with cylindrical asci, and ascospores
8–10r2–3 mm.
Endophytic isolates examined : The Netherlands : Utrecht:
Soesterberg, ‘ De Stompert ’, ex living leaf of Quercus robur,
‘ AugB3H8’, Aug. 2002 (CBS 113868) ; loc. cit., substr.,
‘ AugB2L12 ’, Aug. 2002 (CBS 113869), and ‘ AugB3H7’
(CBS 113870).
DISCUSSION
Previous work showed that ITS sequences are fairly
constant within most species of Mycosphaerella, and
that some species may not even be discriminated by ITS
sequences (Verkley et al. 2004). ITS sequence divergences among Mycosphaerella states which are identified
as M. punctiformis found on dead leaves of Quercus,
Tilia, and Acer, indicate that this morphospecies could
in fact represent a species complex. M. phacae-frigidae,
which grouped with four M. punctiformis strains, can
be distinguished morphologically from M. punctiformis
by the larger ascospores (11–13r3–3.5 mm in the
holotype of M. phacae-frigidae in ZT ; A. Aptroot, unpubl.). M. punctiformis, as we epitypify it here, has been
fully characterized phenotypically on the basis of isolates from Quercus. Future work including morphological analysis of strains from other hosts, and also
sequence analysis of additional genes, may provide evidence to delimit M. punctiformis s. str. from other cryptic
species. The host range of M. punctiformis in this restricted sense is therefore still unknown. The characters
of the teleomorph from which CBS 113265 was isolated
comply with the original material of M. punctiformis in
Persoon’s herbarium in L. The main aim of the work
presented here, is to link the name M. punctiformis to
this material, and in accordance with Art. 9 of the
Code, to epitypify M. punctiformis with herbarium
specimen CBS 7949 (teleomorph on leaves), and an exepitype strain CBS 113265. Other M. punctiformis
strains which originated from Tilia, Acer, and Quercus
differ in ITS sequence by more than 20 positions from
the epitype strain and other strains of M. punctiformis
s. str. However, the ITS data proved insufficient to resolve possible cryptic species within the M. punctiformis
complex. Therefore, all isolates studied here are for the
moment considered as M. punctiformis s. lat.
We repeatedly isolated endophytic Ramularia strains
from surface-sterilized, fresh, green leaves of Quercus
robur trees collected between June and September.
Because they were morphologically and genetically
identical to the epitype strain, we were able to prove
that M. punctiformis can asymptomatically colonize
1281
living Quercus leaves. Its presence becomes evident by
the spermogonia, which develop in large numbers when
oak leaves or parts hereof go into senescence naturally
or due to activities of fungi or other invaders. Although
R. endophylla conidia were occasionally seen near leaf
lesions, we were unable to confirm that conidial
sporulation of M. punctiformis does occur in planta or
on dead leaves in nature. This is in accordance with
Braun (1998), who listed the Ramularia anamorph of
M. punctiformis as an insufficiently known taxon,
formed in culture only. The life-cycle of M. punctiformis seems to be similar to that described in M. buna,
a fungus with a Pseudocercospora anamorph which
endophytically colonizes Fagus crenata foliage in Japan
(Kaneko & Kakishima 2001, Kaneko, Kakishima &
Tokumasu 2003).
On oaks in The Netherlands, M. punctiformis is
commonly accompanied by the weakly pathogenic
Septoria quercicola, which forms pycnidia within small
leaf spots. We recently also discovered its teleomorph
in small numbers on dead leaves, including those of the
epitype specimen. The teleomorph of S. quercicola
differs from M. punctiformis in the wider asci (35–50r
9–12 mm) and longer ascospores (13–20r3.5–5 mm, av.
17r4.5 mm), which are not constricted at the septum
and taper about equally towards both ends. Our ITS
sequence analyses indicate that this Mycosphaerella
species, which is probably different from all published
species on oaks (Gilman & Wadley 1952) and for which
an applicable name has not yet been found, is relatively
distant from taxa of the Ramularia clade, as well as
other taxa with Septoria anamorphs.
Host specificity in the M. punctiformis complex is still
insufficiently known. Brefeld & Tavel (1891) regarded
M. punctiformis as a plurivorous species. They noted
that it was less abundant on oak than M. maculiformis,
a species originally described from Corylus. According
to Brefeld & Tavel, M. maculiformis can be distinguished from M. punctiformis by the more densely
arranged ascomata, cylindrical asci and larger ascospores. However, they have been seen as synonymous
for a long time, and the type specimens of both
species were recently re-examined and found to contain
(at least) morphologically indistinguishable fungi.
Klebahn (1918) studied the ascomata of M. punctiformis on Tilia, Corylus, and Quercus and briefly
described and illustrated the Ramularia anamorphs in
culture. Klebahn noted that there were only minor
differences between the teleomorphs from the various
tree species, and that the isolates showed only some
differences in pigmentation but were otherwise indistinguishable. He tentatively classified these fungi as
host-specific forms of M. punctiformis. Von Arx (1949,
Müller & von Arx 1962) considered M. maculiformis as
a synonym of M. punctiformis, which he regarded as
plurivorous. Later authors followed this concept (Barr
1972, Sivanesan 1984), but as is shown here, the
situation is more complex and may involve more than
one species.
Mycosphaerella punctiformis revisited
All Mycosphaerella species with Ramularia anamorphs grouped in a single, monophyletic group which
obtained high bootstrap support particularly in the
parsimony analysis. This was also the case in earlier
molecular studies, in which fewer taxa had been
included (Crous et al. 2001, Goodwin, Dunkle &
Zismann 2001, Verkley et al. 2004). As in those studies,
M. graminicola and Septoria passerinii form the closest
sister group, but support for the joined clades remains
limited. The epitypification of the type species of Mycosphaerella will enable the unambiguous application
of the name M. punctiformis, and facilitate the naming
of possible future segregates from Mycosphaerella.
ACKNOWLEDGEMENTS
Irma van Kempen is kindly thanked for isolating and sequencing
the oak endophytes, and Mieke Starink-Willemse for sequencing
additional strains.
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Corresponding Editor: H. T. Lumbsch