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. REFERENCES Arx, J. A. von (1949) Beiträge zur Kenntnis der Gattung Mycosphaerella. Sydowia 3: 28–100. Arx, J. A. von (1983) Mycosphaerella and its anamorphs. Proceedings of the Koninklijke Nederlandse Akademie van Wetenschappen, ser. C 86: 15–54. Barr, M. E. (1972) Preliminary studies on the Dothideales in temperate North America. Contributions of the University of Michigan Herbarium 9: 523–638. Braun, U. (1998) A monograph of Cercosporella, Ramularia and allied genera (phytopathogenic Hyphomycetes). Vol. 2. IHW-Verlag, Eching. Braun, U., Crous, P. W., Dugan, F., Groenewald, J. Z. & de Hoog, G. S. 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