mycological research 111 (2007) 809–826 journal homepage: www.elsevier.com/locate/mycres Phylogeny and taxonomy of the oak powdery mildew Erysiphe alphitoides sensu lato Susumu TAKAMATSUa,*, Uwe BRAUNb, Saranya LIMKAISANGa, Sawwanee KOM-UNa, Yukio SATOc, James H. CUNNINGTONd a Department of Bioresources, Graduate School, Mie University, 1577 Kurima-Machiya, Tsu 514-8507, Japan Martin-Luther-University, Institute of Biology, Geobotany, Herbarium, Neuwerk 21, D-06099 Halle (Saale), Germany c Laboratory of Biology, Department of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu-shi, Toyama 939-0398, Japan d Department of Primary Industries – Knoxfield, Private Bag 15, Ferntree Gully Delivery Centre, Victoria 3156, Australia b article info abstract Article history: Phylogenetic analyses of Erysiphe alphitoides s. lat. using sequences of the rDNA ITS region Received 7 March 2007 and the D1/D2 domains of the 28S rDNA revealed a complex consisting of several geneti- Received in revised form cally and morphologically distinguished taxa, including the already described Erysiphe 8 May 2007 alphitoides s. str. and E. hypophylla. The ascomata (chasmothecia) of E. hypophylla are mor- Accepted 25 May 2007 phologically very similar to those of E. alphitoides, but the two species are easily distinguish- Published online 7 June 2007 able by their symptoms, as well as the shape and size of the conidia. The fungus on Quercus Corresponding Editor: phillyraeoides, distributed in warmer regions in southern Japan, is genetically clearly sepa- Hermann Voglmayr rated from E. alphitoides s. str., and morphologically characterized by having chasmothecia with appendages consistently shorter than the chasmothecial diameter. This fungus, Keywords: named Erysiphe quercicola in this paper, is also able to infect some other oak species, and Erysiphe sect. Microsphaera it is genetically identical with anamorphs on some tropical trees of other host genera. Col- Microsphaera alphitoides lections of E. alphitoides s. lat. on Quercus acutissima and Q. variabilis, both belonging to Quer- Quercus cus sect. Cerris, are genetically distinct from E. alphitoides s. str., E. hypophylla and Sequence analyses E. quercicola. They form two genetically and morphologically differentiated groups. The hy- rDNA ITS region pophyllous taxon on Q. acutissima and Q. variabilis, named Erysiphe hypogena in this paper, is 28S rDNA characterized by forming distinctive persistent hypophyllous mycelial patches, causing necrotic discolouration of the host tissue. The epiphyllous taxon on these hosts, for which the name E. epigena is proposed, differs in having epiphyllous mycelium, smaller chasmothecia with fewer appendages, and does not cause leaf discolouration. ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction The genus Quercus (Fagaceae), mainly distributed in Asia, Europe and North America, comprises in the current circumscription about 320 species (Quercus s. str.) and about 500 species in the traditional wider sense (Quercus s. lat.; Wagenitz 1981). Several species of powdery mildew are known to infect oaks. These include Erysiphe abbreviata (syn. Microsphaera abbreviata), E. alphitoides (syn. M. alphitoides), E. calocladophora (syn. M. calocladophora), E. extensa (syn. M. extensa), and * Corresponding author. E-mail address: takamatu@bio.mie-u.ac.jp 0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2007.05.013 810 E. hypophylla (syn. M. hypophylla) (Braun 1987; Braun & Takamatsu 2000; Braun et al. 2003). E. alphitoides s. lat. is common, widespread in Asia and Europe on numerous species of the genus Quercus, and has been introduced in various other parts of the world (e.g. Braun 1987, 1995; Chen et al. 1987; Heluta 1989; Bunkina 1991; Nomura 1997). In Europe, it is morphologically rather uniform (Braun 1995), whereas in Asia, above all in China, Japan and Korea, its chasmothecia are more variable (Homma 1937; Chen et al. 1987; Otani 1988; Nomura 1997; Shin 2000). Microsphaera hypophylla was described about 50 y ago, from the European part of Russia on Quercus robur, from where it spread rapidly westward (Blumer 1967; Braun 1987). Various authors compared the morphology of the chasmothecia of E. alphitoides and E. hypophylla and discussed differences (Kochman 1960; Roll-Hansen 1961; Cruchet 1962; Blumer 1967). However, the stability of the differences between E. alphitoides and E. hypophylla, above all with regard to the teleomorph, was disputed and the status of E. hypophylla as an independent species was called into question (Speer 1973; Záhorovská 1986, 1988; Heluta 1989; Braun & Takamatsu 2000). Differences in the conidial shape and size seem to be the only striking difference between the two species (Braun 1987). But the anamorph of E. hypophylla is rare, and if present, is often obscured by evanescent mycelium. Furthermore, the habit of E. alphitoides can be amphigenous, i.e. it can also form hypophyllous mycelium and ascomata. Another problem with the taxonomy of the E. alphitoides complex concerns records on various trees and shrubs of unrelated host plant families, viz. Aesculus hippocastanum in Europe (Germany and Ukraine) (see Marczenko 1976; Braun 1980), as well as several tropical fruits and trees, e.g. Anacardium occidentale, Bixa orellana, Citrus spp., Hevea brasiliensis, Mangifera indica, and Acacia spp. (Limkaisang et al. 2006). Hence, morphological re-examinations of the whole E. alphitoides complex, including E. hypophylla, and genetic analyses, using sequences of the rDNA ITS region and the D1/D2 domains of the 28S rDNA, were carried out to elucidate its phylogeny and taxonomic structure. S. Takamatsu et al. DNA extraction and amplification Sources of the powdery mildew specimens used for the molecular analyses and their DNA database accession numbers are listed in Table 1. Whole-cell DNA was isolated from chasmothecia or mycelia using the chelex method (Walsh et al. 1991; Hirata & Takamatsu 1996). The ITS region including 5.8S rDNA and the 50 end of 28S rDNA, including the variable domains D1 and D2, were separately amplified by two sequential PCRs using partial nested primer sets. PCR reactions were conducted with TaKaRa Taq DNA polymerase (TaKaRa, Tokyo) in a thermal cycler TP-400 (TaKaRa) under the following thermal cycling conditions: an initial denaturing step of 2 min at 95  C, thermocycling for 30 cycles where each cycle consisted of 30 s at 95  C, followed by 30 s at 52  C for annealing, and 30 s at 72  C for extension, and a final extension cycle of 7 min at 72  C. A negative control, which lacked template DNA, was included for each set of reactions. The PCR product was subjected to electrophoresis in 1.5 % agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH8.0), and the DNA product of each amplification was then excised from the ethidium bromide stained gel and purified using the JETSORB Kit (Genomed, Oeynhausen) as per the manufacturer’s protocol. Nucleotide sequences of the PCR products were obtained for both strands using direct sequencing in a DNA sequencer CEQ2000 XL (Beckman Coulter, Fullerton, CA). The sequence reactions were conducted using the CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter) as per the manufacturer’s instructions. For amplification of the ITS region, primers ITS5 (White et al. 1990) and P3 (Kusaba & Tsuge 1995) were used for the first amplification. One microlitre of the first reaction mixture was used for the second amplification with the partially nested primer sets ITS5 and ITS4 (White et al. 1990). The ITS5/ITS4 fragment was subjected to cycle-sequencing using primers ITS1, ITS4, T3, and T4 (Hirata & Takamatsu 1996). For amplification of 28S rDNA, primers PM3 (Takamatsu & Kano 2001) and TW14 (Mori et al. 2000), and NL1 (Mori et al. 2000) and TW14 were used for the first and second amplifications, respectively. Primers NL1, NL2, NL3 (Mori et al. 2000), and NLP2 were used for cycle-sequencing. Materials and methods Phylogenetic analysis Morphological studies The sequences were initially aligned using the Clustal X package (Thompson et al. 1997). The alignment was then visually refined with a word-processing program, using colour-coded nucleotides. The alignments were deposited in TreeBASE (http://www.treebase.org/) under accession number S1804. Phylogenetic trees were obtained from the data using MP method in PAUP 4.0 (Swofford 2001) and Bayesian analysis in MRBAYES 3.1.1 (Huelsenbeck & Ronquist 2001). MP analyses were done with the heuristic search option using the ‘tree bisection–reconstruction’ (TBR) algorithm with 100 random sequence additions to find the global optimum tree. All sites were treated as unordered and unweighted, with gaps treated as missing data. The strength of the internal branches of the resulting trees was tested with BS analyses using 1K replications with step-wise addition option set as simple (Felsenstein 1985). BS values higher than 50 % were given. Specimens of Erysiphe alphitoides s. lat. (including E. hypophylla) collected on various Quercus spp., mainly in Asia and Europe, were examined by standard light microscopy (Olympus, BX 50, Hamburg) and phase-contrast optical instruments and devices. To observe anamorphs from herbarium material, specimens were rehydrated before examination by boiling a small piece of infected leaf, with the mycelium downwards, in a drop of lactic acid on a slide as described by Shin & La (1993) and Shin (2000). After boiling, the mycelium was scraped off the leaf and mounted in lactic acid for LM. The specimens examined are deposited in MUMH (Herbarium, Faculty of Bioresources, Mie University, Tsu, Japan) and HAL (Martin Luther University, Institute of Botany, Geobotany, Herbarium, Halle, Germany). 811 Erysiphe alphitoides sensu lato Table 1 – Sources of Erysiphe alphitoides s. lat. material used for molecular analyses and DNA database accession numbers Group Fungal species Host species Location and year Voucher no.a Accession no.b A A A A A A A A A A A A A A A A A A A A A A Erysiphe alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides alphitoides Quercus alba dentata dentata crispula crispula crispula crispula crispula petraea robur robur robur robur robur robur robur robur robur serrata serrata sp. sp. Bariloche, Argentina; 2004 Hokkaido, Japan; 2003 Hokkaido, Japan; 2003 Shiga, Japan; 1996 Toyama, Japan; 1996 Toyama, Japan; 1997 Toyama, Japan; 2003 Mie, Japan; 2004 Geneva, Switzerland; 1998 Vilnius, Lithuania; 1999 Neuchâtel, Switzerland; 1999 Couterbug, UK; 1999 Kaunas, Lithuania; 1999 Geneva, Switzerland; 1995 San Martin, Argentina; 2004 Geneva, Switzerland; 1997 Nyon, Switzerland; 2000 Bariloche, Argentina; 2004 Ehime, Japan; 1998 Gifu, Japan; 2000 Victoria, Australia; 1994 Victoria, Australia; 1993 MUMH 3178 MUMH 2619 MUMH 2620 MUMH 242 MUMH 1950 MUMH 1953 MUMH 2358 MUMH 3272 MUMH 1448 MUMH 773 MUMH 631, HAL 1946 F MUMH 960 MUMH 756 VPRI 22226 MUMH 2471 MUMH 1442 MUMH 1443 MUMH 3169 MUMH 550 MUMH 1160 MUMH 3250, VPRI 20423 MUMH 3259, VPRI 18763 AB292703 AB292700 AB292701 AB237783 (AB237811)c,d AB292696 AB292697 AB292698 AB292706 AB257435c AB292710 AB292708 AB237784 (AB237812)c,d AB292709 AF298538c AB292699 AB257430c AB257431c AB292702 AB292707 AB292695 AB292704 AB292705 B B B B B B B B B B B quercicola quercicola quercicola quercicola quercicola quercicola quercicola quercicola quercicola quercicola quercicola crispula crispula crispula crispula phillyraeoides phillyraeoides robur serrata sp. sp. sp. Toyama, Japan; 1997 Toyama, Japan; 1997 Toyama, Japan; 1997 Toyama, Japan; 1997 Mie, Japan; 1995 Nara, Japan; 1999 SA, Australia; 1993 Shiga, Japan; 2005 Iran; 2004 Pechaboon, Thailand; 2004 Victoria, Australia; 1994 MUMH 1952, HAL 1972 F MUMH 1954, HAL 1971 F MUMH 1955, HAL 1967 F MUMH 1956, HAL 1968 F MUMH 124 MUMH 885, HAL 1969 F VPRI 19013 MUMH 3796, HAL 1974 F MUMH 3242, HAL 1973 F MUMH 3229, HAL 1975 F VPRI 20422 AB292688 AB292689 AB292690 AB292691 AB193590 (AB197135)c,d AB193591 (AB237813) c,d AB295454 AB292694 AB292693 AB292692 AB295455 C C C Erysiphe sp. Erysiphe sp. Erysiphe sp. cuspidata crispula crispula Shiga, Japan; 1996 Toyama, Japan; 2003 Nagano, Japan; 2004 MUMH 294 MUMH 2361 MUMH 3269 AB292712 AB292711 AB292713 D D D D hypophylla hypophylla hypophylla hypophylla cuspidata robur serrata serrata Toyama, Japan; 1997 Tochigi, Japan; 1981 Toyama, Japan; 2003 Nagano, Japan; 2003 MUMH 1957 VPRI 22120 MUMH 2373 MUMH 2405 AB292714 AF298544c AB292715 AB292716 E E E E E hypogena hypogena hypogena hypogena hypogena acutissima acutissima acutissima acutissima variabilis Nara, Japan; 1996 Niigata, Japan; 1997 Mie, Japan; 2005 Mie, Japan; 2004 Aichi, Japan; 2005 MUMH MUMH MUMH MUMH MUMH 900, HAL 1993 F 1962, HAL 1994 F 3795, HAL 1996 F 3211, HAL 1995 F 3794, HAL 1997 F AB292727 AB292723 AB292726 AB292724 AB292725 F F F epigena epigena epigena acutissima acutissima variabilis Mie, Japan; 2004 Mie, Japan; 2005 Mie, Japan; 2003 MUMH 3211, HAL 1995 F MUMH 3795 MUMH 2193, HAL 1998 F AB292721 AB292722 AB292720 G G G epigena epigena epigena variabilis variabilis variabilis Shiga, Japan; 1996 Aichi, Japan; 2000 Aichi, Japan; 2000 MUMH 148, HAL 2001 F MUMH 1065 MUMH 1958, HAL 2000 F AB292718 AB292717 AB292719 – Erysiphe sp. crispula Tokyo, Japan; 1997 MUMH 1951 AB292728 a Sources: HAL, Herbarium Martin Luther University, Germany; MUMH, Mie University, Mycological Herbarium, Japan; VPRI, Plant Disease Herbarium, Institute for Horticultural Development, Victoria, Australia. b DDBJ, EMBL, and GenBank database accession number of the nucleotide sequence data. c Sequence retrieved from DDBJ database. d Sequences of the ITS and 28S rDNA were separately deposited in a DNA database under the accession numbers. Parenthesis means the accession number of 28S rDNA sequence. 812 For Bayesian phylogenetic analyses, the best-fit evolutionary model was determined for each data set by comparing different evolutionary models via the Akaike information criterion (AIC) using PAUP and MrModeltest 2.2 (Nylander 2004). MRBAYES was launched with random starting trees for 1 M generations and the Markov chains were sampled every 100 generations, which resulted in 10K sampled trees. To ensure that the Markov chain did not become trapped in local optima, we used the MCMCMC algorithm, performing the estimation with four incrementally heated Markov chains. To establish whether the Markov chains had reached stationarity, we plotted the likelihood scores of sampled trees against generation time. Stationarity was deemed to have been reached when the likelihood of the sample points reached a stable equilibrium (Huelsenbeck & Ronquist 2001). Bayesian PP values higher than 0.9 were given. Results Phylogeny within Erysiphe alphitoides s. lat. ITS tree The 56 ITS sequences of Erysiphe alphitoides s. lat. (Table 1) were 555–557 bp in length. The maximum sequence divergence among these sequences was 3.1 % in p-distance. These sequences were aligned with the sequences of E. abbreviata (GenBank AB271785) and E. castaneigena (AF298545), which were used as outgroup taxa. The alignment data matrix consisted of 58 sequences and 560 characters, in which 47 (8.4 %) characters were variable and 30 (5.4 %) characters were informative for parsimony analysis. This parsimony analysis using PAUP generated 776 062 MP trees of 56 steps (CI ¼ 0.8929, RI ¼ 0.9701, RC ¼ 0.8662). Tree topologies were almost consistent among the trees, except for the relationships with the group A (Fig 1). All other major clades were commonly supported among the trees. One of the trees is thus shown in Fig 1 as a representative. The 56 sequences from E. alphitoides s. lat. were divided into seven distinct groups and one independent sequence (MUMH 1951). The sequence MUMH 1951 appeared only once among the 56 sequences. We thus ignore this sequence from the following discussion. All groups but group A were supported as monophyletic groups by strong or moderate BS and PP values. The Bayesian analysis resulted in an almost identical tree topology. Group A consisted of 27 (nearly) identical sequences and was paraphyletic to other groups. Groups A, B, C and D were composed of isolates from Quercus sect. Quercus, viz. Q. alba, Q. dentata, Q. crispula, Q. petraea, Q. robur, and Q. serrata. Two isolates from Q. phillyraeoides (sect. Cerris) belonged to group B. Groups E, F, and G consisted of isolates from Quercus sect. Cerris, viz. Q. acutissima and Q. variabilis. Isolates belonging to group A were collected in numerous parts of the world such as Japan, Australia, Argentina and European countries. Isolates of group B were collected from Japan, Australia, Thailand, and Iran, but not from European countries. The other groups, viz. C, D, E, F, and G, were composed of isolates from Japan. Both groups E and F were isolated from a single leaf of MUMH 3211 and MUMH 3795. S. Takamatsu et al. Phylogeny within Erysiphe alphitoides s. lat. 28 S tree The 33 28S rDNA sequences of Erysiphe alphitoides s. lat. (691–792 bp in length) were aligned with the sequences of E. abbreviata AB271785, which was used as outgroup taxon. The maximum sequence divergence among these sequences was 1.6 % in p-distance. The alignment data matrix consisted of 34 sequences and 792 characters, in which 24 (3.0 %) characters were variable and 19 (2.4 %) characters were parsimonyinformative. This parsimony analysis using PAUP generated only a single most parsimonious tree of 286 steps (CI ¼ 0.9286, RI ¼ 0.9831, RC ¼ 0.9128; Fig 2). The seven groups recognised in the ITS tree were also generated in the 28 S tree with moderate to strong BS and PP supports. The Bayesian analysis generated an almost identical tree topology. Phylogenetic relationships among the genus Erysiphe To investigate the phylogenetic relationships of Erysiphe alphitoides s. lat. within the genus Erysiphe, ITS sequences of E. alphitoides s. lat. were aligned with sequences of the sects. Erysiphe and Microsphaera of the genus Erysiphe retrieved from DDBJ database. The sequences of E. alphitoides s. lat. were selected to represent the respective groups. The alignment data matrix consisted of 91 sequences and 607 characters, in which 223 (36.7 %) characters were variable and 157 (25.9 %) characters were parsimony informative. E. glycines was used as outgroup taxon based on the report of Takamatsu et al. (1999). This parsimony analysis using PAUP generated 38,444 MP trees of 589 steps (CI ¼ 0.5365, RI ¼ 0.7944, RC ¼ 0.4262). One of the trees is shown in Fig 3. The Bayesian analysis generated a similar tree topology. The seven groups recognised in the ITS and 28S rDNA analyses also appeared in the tree of the genus Erysiphe, and grouped into a large clade although the BS and PP supports were weak. In addition to E. alphitoides s. lat., the following Erysiphe and Oidium species were included in group A with identical or one-base different sequences, viz. E. euonymi-japonici ex Euonymus japonicus (Celastraceae), O. mangiferae ex Mangifera indica (Anacardiaceae), E. hypophylla ex Paeonia lutea (Paeoniaceae), E. pseudolonicerae ex Cocculus trilobus (Menispermaceae), and E. wallrothii ex Vaccinium hirtum (Ericaceae). Similarly, the following Oidium species were included in group B, viz. O. citri ex Citrus spp. (Rutaceae), O. heveae ex Hevea brasiliensis (Euphorbiaceae), O. anacardii ex Anacardium occidentale (Anacardiaceae), O. mangiferae ex Mangifera indica, O. bixae ex Bixa orellana (Bixaceae), and Oidium sp. ex Acacia spp. (Mimosaceae). Conversely, the groups C, D, E, F, and G were composed of only E. alphitoides s. lat. E. castaneigena and E. abbreviata, both of which are parasitic to Fagaceae, and E. liriodendri were sister to E. alphitoides s. lat. although BS and PP supports were weak. We conducted similar phylogenetic analysis using 28S rDNA sequences. The 28S tree resulted in a phylogenetic relationship of E. alphitoides s. lat. similar to the ITS tree (Fig 3) within the genus Erysiphe. As only a few 28S rDNA sequences of the genus Erysiphe are available in the DNA database, we have not presented the tree here, but it is deposited in TreeBASE under the accession number indicated above. 813 Erysiphe alphitoides sensu lato MUMH 148 Q. variabilis JAPAN MUMH 1065 Q. variabilis JAPAN G 61 MUMH 1958 Q. variabilis JAPAN E. epigena 0.93 60 MUMH 2193 Q. variabilis JAPAN MUMH 3795 Q. acutissima JAPAN F 61 MUMH 3211 Q. acutissima JAPAN MUMH900 Q. acutissima JAPAN MUMH 1962 Q. acutissima JAPAN E 99 MUMH 3795 Q. acutissima JAPAN 1.00 MUMH 3794 Q. variabilis JAPAN E. hypogena MUMH 3211Q. acutissima JAPAN MUMH 1957 Q. cuspidata JAPAN 87 MUMH 2373 Q. serrata JAPAN D 0.99 MUMH 2405 Q. serrata JAPAN 86 E. hypophylla VP RI22120 Q. robur JAPAN 1.00 MUMH 294 Q. cuspidata JAPAN 65 MUMH 2361 Q. crispula JAPAN C Erysiphe sp. 0.91 MUMH 3269 Q. crispula JAPAN MUMH 1951 Q. crispula JAPAN VPRI 19013 Q. robur AUSTRALIA VPRI 20422 Quercus sp. AUSTRALIA MUMH 124 Q. phillyraeoides JAPAN 65 MUMH 885 Q. phillyraeoides JAPAN MUMH 1956 Q. crispula JAPAN MUMH 3796 Q. serrata JAPAN MUMH 1954 Q. crispula JAPAN 100 MUMH 1955 Q. crispula JAPAN 1.00 MUMH 3242 Quercus sp. IRAN MUMH 1952 Q. crispula JAPAN MUMH 3229 Quercus sp. THAILAND MUMH 2358 Q. crispula JAPAN MUMH 960 Q. robur UK MUMH 2619 Q. dentata JAPAN AJ309201 Q. robur FRANCE MUMH 1953 Q. crispula JAPAN MUMH 1443 Q. robur SWITZERLAND MUMH 756 Q. robur LITHUANIA MUMH 631 Q. robur SWITZERLAND AJ417498 Q. robur GERMANY MUMH 3178 Q. alba ARGENTINA MUMH 1442 Q. robur SWITZERLAND ITS MUMH 3169 Q. robur ARGENTINA A MUMH 2620 Q. dentata JAPAN 58 sequences MUMH 3272 Q. crispula JAPAN 560 characters E. alphitoides MUMH 242 Q. crispula JAPAN 56 steps AJ309200 Q. robur FRANCE AJ417497 Q. robur GERMANY CI = 0.8929 MUMH 773 Q. robur LITHUANIA RI = 0.9701 MUMH 1160 Q. serrata JAPAN RC = 0.8662 MUMH 3250 Quercus sp. AUSTRALIA MUMH 3259 Quercus sp. AUSTRALIA VPRI22226 Q. robur SWITZERLAND MUMH 1448 Q. petraea SWITZERLAND MUMH 1950 Q. crispula JAPAN MUMH 2471 Q. robur ARGENTINA MUMH 550 Q. serrata JAPAN AF298545 Erysiphecastaneigena KOREA AB271785 Erysipheabbreviata USA 90 0.99 55 B E. quercicola 0.5 substitutions Fig 1 – One of the 776 062 MP trees based on ITS sequences from 56 isolates of Erysiphe alphitoides s. lat. and two outgroup taxa. The tree was obtained by a heuristic search employing the random stepwise addition option of PAUP. Gaps were treated as missing data. Percentage BS support (1K replications; >50 %) and PP (>0.90) are shown on and under branches, respectively. A–G indicate groups found in E. alphitoides s. lat. Bold lines denote branches present in the strict consensus tree. Taxonomy Erysiphe alphitoides (Griffon & Maubl.) U. Braun & S. Takam., Schlechtendalia 4: 5 (2000), s. str. (emend.) (Figs 4A, 5–6) Anamorph: Oidium alphitoides Griffon & Maubl., Bull. Soc. Mycol. Fr. 26: 132 (1910). Oidium dubium Jacz., Trudy po Mikologii i Fitopatologii Ucenogo Komiteta: 231 (1910). Oidium quercinum var. gemmiparum Ferraris, Ann. Mycol. 7: 69 (1909). 814 S. Takamatsu et al. 28S rDNA 34 sequences 792 characters 28 steps CI = 0.9286 RI = 0.9831 RC = 0.9128 MUMH 3250 Quercus sp. AUSTRALIA MUMH 3259 Quercus sp. AUSTRALIA MUMH 242 Q. crispula JAPAN MUMH 1448 Q. petraea SWITZERLAND MUMH 773 Q. robur LITHUANIA MUMH 631 Q. robur SWITZERLAND 63 A MUMH 960 Q. robur UK E. alphitoides MUMH 2471 Q. robur ARGENTINA MUMH 1442 Q. robur SWITZERLAND MUMH 1443 Q. robur SWITZERLAND MUMH 3169 Q. robur ARGENTINA MUMH 550 Q. serrata JAPAN 63 MUMH 1160 Q. serrata JAPAN 62 MUMH 124 Q. phillyraeoides JAPAN 0.98 MUMH 885 Q. phillyraeoides JAPAN 99 1.00 85 MUMH 1956 Q. crispula JAPAN B MUMH 3796 Q. serrata JAPAN E. quercicola MUMH 3242 Quercus sp. IRAN MUMH 3229 Quercus sp. THAILAND 66 MUMH 294 Q. cuspidata JAPAN 0.95 MUMH 2361 Q. crispula JAPAN C 96 MUMH 2373 Q. serrata JAPAN 1.00 MUMH 2405 Q. serrata JAPAN Erysiphe sp. D E. hypophylla MUMH 3794 Q. variabilis JAPAN E 63 MUMH 900 Q. acutissima JAPAN MUMH 1962 Q. acutissima JAPAN E. hypogena MUMH 3795 Q. acutissima G JAPAN MUMH 148 Q. variabilis JAPAN 96 58 1.00 0.93 MUMH 1065 Q. variabilis JAPAN MUMH 1958 Q. variabilis JAPAN MUMH 2193 Q. variabilis JAPAN 86 MUMH 3795 Q. acutissima JAPAN 1.00 MUMH 3211 Q. acutissima JAPAN G E. epigena F AB271785 Erysiphe abbreviata USA 0.5 substitutions Fig 2 – A single MP tree based on the sequences of domains D1 and D2 of the 28S rDNA from 33 isolates of Erysiphe alphitoides s. lat. and one outgroup taxon. The tree was obtained by a heuristic search employing the random stepwise addition option of PAUP. Gaps were treated as missing data. Percentage BS support (1K replications; >50 %) and PP (>0.90) are shown on and under branches, respectively. A–G indicate groups found in Erysiphe alphitoides s. lat. Basionym: Microsphaera alphitoides Griffon & Maubl., Bull. Soc. Mycol. Fr. 28: 100 (1912). Microsphaera querci Sawada, Bull. Gov. Forest Exp. Sta. Meguro, Tokyo 50: 122 (1951). Typus: Switzerland: Neuchâtel, on Quercus robur, 4 Sep. 1999, S. Takamatsu (MUMH 631 – neotypus hic designatus; HAL 1946 F – isoneotypus). rDNA sequence ex-type: AB292708. Mycelium amphigenous, mainly epiphyllous, in white patches or effuse, persistent on the upper leaf surface. Hyphae branched, septate, 3–7 mm wide, hyaline, thin-walled, smooth or almost so. Appressoria lobed, solitary or in opposite pairs, 6–10 mm diam. Anamorph abundant. Conidiophores arising terminally from the mother cell, mostly central, erect, straight, rarely curved or flexuous, up to 80 mm long, on the lower leaf surface often longer, foot-cells cylindrical, 15–40  6–10 mm, Synonyms: Microsphaera dentatae Liou, Contr. Lab. Bot. Natl. Acad. Peiping 1: 19 (1931). Microsphaera alni var. dentatae (Liou) F.L. Tai, Bull. Chinese Bot. Soc. 1: 22 (1935). Oidium citri AB237793 Oidium citri AB237791 Oidium heveae AB 193587 Oidium heveae AB 193606 ITS Oidium ana cardii AB 237786 Oidium mangiferae AB 237796 91 sequences Oidiumbixae AB 237788 Oidium bixae AB 237787 607 characters B Oidium sp. ex Acacia AB 237809 589 steps Oidium sp. ex Acacia AB 237804 100 MUMH 1956 Q. crispula CI = 0.5365 MUMH 3796 Q. serrata 1.0 MUMH 3242 Quercus sp. RI = 0.7944 MUMH 124 Q. phillyraeoides Oidium mangiferae AB 237802 RC = 0.4262 Erysiphe euonymi-japonici AB 250228 Erysiphe euonymi-japonici AB250229 Oidium mangiferae AB 237795 Oidium mangiferae AB 237798 Erysiphe hypophylla ex Paeonia AB 257432 Erysiphe hypophylla ex Paeonia AB 257433 MUMH 550 Q. serrata A MUMH 2471 Q. robur MUMH 3250 Quercus sp. MUMH 1443 Q. robur MUMH 242 Q. crispula Erysiphe pseudolonicerae AB015915 Erysiphe wallrothii AB015930 87 MUMH 148 Q. variabilis MUMH 1065 Q. variabilis G 1.0 MUMH 1958 Q. variabilis MUMH 2193 Q. variabilis MUMH 3795 Q. acutissima F MUMH 3211 Q. acutissima MUMH 900 Q. acutissima MUMH 1962 Q. acutissima 98 MUMH 3795 Q. acutissima E MUMH 3794 Q. variabilis 1.0 MUMH 3211 Q. acutissima MUMH 1957 Q. cuspidata 86 MUMH 2373 Q. serrata D MUMH 2405 Q. serrata 87 1.0 VPRI22120 Q. robur MUMH 294 Q. cuspidata 1.0 MUMH 2361 Q. crispula C MUMH 3269 Q. crispula Erysiphe castaneigena AF298545 Erysiphe abbreviata AB271785 Erysiphe liriodendri AF011302 87 Erysiphe pisi AF011306 81 Erysiphe pisi AF073348 85 1.0 Erysiphe howeana AF011301 Oidium sp. ex Glycine AB078800 1.0 Erysiphe baeumleri AB015933 89 98 Erysiphe baeumleri AB015919 1.0 Erysiphe trifolii AB015913 1.0 Erysiphe cruciferarum AF031283 Erysiphe polygoni AF011308 94 Erysiphe polygoni AF011307 97 1.0 Erysiphe betae AF011290 Oidium sp. exConvolvulus AF154328 1.0 Erysiphe heraclei AB000942 92 Erysiphe friesii AB000939 1.0 Erysiphe convolvuli AF011298 100 Erysiphe convolvuli AF154327 1.0 Erysiphel espedezae AB015921 100 Erysiphel espedezae AB015923 1.0 Erysiphe syringae AB015920 Erysiphe magnifica AF011312 Erysiphe aquilegiae AB015929 Erysiphe aquilegiae AF154322 100 Erysiphe aquilegiae AB000944 1.0 Erysiphe macleayae AB016048 Erysiphe blasti AB015918 Erysiphe paeoniae AB257436 100 100 Erysiphe paeoniae AB257437 1.0 Erysiphe paeoniae AB257438 1.0 Erysiphe staphyleae AB015922 Erysiphe katumotoi AB015917 Erysiphe pulchra AB015935 Erysiphe helwingiae AB015916 76 Erysiphe vanbruntiana AB015925 0.99 Erysiphe japonica AB000941 100 Erysiphe japonica AB015924 1.0 Erysiphe viburni AF298541 85 Erysiphe weigelae AB015931 100 0.99 Erysiphe weigelae AB015932 1.0 Erysiphe huayinensis AB015914 Erysiphe juglandis AB015928 100 Erysiphe glycines AB015927 Erysiphe glycines AB015934 1.0 5 substitutions Fig 3 – One of the 38 444 MP trees based on ITS sequences from 91 sequences of Erysiphe spp. and E. alphitoides s. lat. The tree was obtained by a heuristic search employing the random stepwise addition option of PAUP. Gaps were treated as missing data. Percentage BS support (1K replications; >70 %) and PP (>0.95) are shown on and under branches, respectively. Two specimens of E. glycines were used as outgroup. Sequences from E. alphitoides s. lat. are shown in boldface. A–G indicate groups found in E. alphitoides s. lat. Bold lines denote branches present in the strict consensus tree. 816 S. Takamatsu et al. Fig 4 – Symptoms of species of the Erysiphe alphitoides complex. (A) E. alphitoides (from HAL 1946 F). (B) E. quercicola (from HAL 1969 F). (C) E. hypogena (from HAL 1979 F, left and HAL 1982 F, right). (D) E. epigena (from HAL 1977 F). Bars [ 1 cm. followed by 1–3 shorter cells. Conidia solitary, primary conidia obovoid-ellipsoid, apex rounded, base subtruncate, secondary conidia doliiform when mature, ends truncate or subtruncate, immature ones sometimes ellipsoid-cylindrical, 25–40(–45)  13–25 mm, length:breadth ratio 1.3–2.3, mostly below 2, germ tubes terminal to subterminal, short to moderately long, usually terminating with a lobed appressorium. Chasmothecia amphigenous, mainly epiphyllous, scattered to gregarious, 75–140 mm diam, rarely larger. Peridial cells angular-irregular in outline, 10–30 mm diam. Appendages more or less equatorial, 5–25, straight to somewhat curved, 0.75–2 times as long as the chasmothecial diam (70–225 mm), 6–10 mm wide below, wall almost smooth to verruculose, 0–1-septate, colourless or only pigmented at the very base, thick-walled throughout or thick-walled below and thinner upwards, apically (3–)4–6 times regularly and mostly densely branched, branched part 35–80  25–70 mm, rarely with somewhat elongated primary branches, tips of the ultimate branchlets uncinate to circinate when mature. Asci 4–15, broadly ellipsoid-ovoid (-subglobose), saccate, subsessile to short-stalked, 40–70(–80)  30–55 mm, oculus not very evident, 10–18 mm diam, (6–)8-spored. Ascospores broadly ellipsoid-ovoid, colourless, 13–26  7–15 mm. Collections examined: See Table 1. Host range and distribution: on a wide range of oak species, including Quercus dentata, Q. crispula, Q. petraea, Q. robur, Q. serrata, Fagaceae (occasionally attacking Aesculus hippocastanum, Hippocastanaceae; Mangifera indica, Anacardiaceae; Paeonia lutea, Paeoniaceae), Asia (Armenia, Azerbaijan, China, Georgia, India, 817 Erysiphe alphitoides sensu lato Fig 5 – Erysiphe alphitoides, anamorph. (A) Appressoria. (B) Conidiophores. (C) Primary conidia. (D) Secondary conidia (partly with germ tubes). Bars [ 10 mm. U. Braun del. Iran, Iraq, Israel, Japan, Kazakhstan, Korea, Lebanon, Nepal, Russia, Sri Lanka, Turkey, Turkmenistan), North Africa (Ethiopia, Morocco), all Europe, introduced in South Africa, Australia, New Zealand, North and South America (Amano 1986; Braun 1987; Sharma & Khare 1995; Crous et al. 2000). Comments: Type material of this species could not be traced and is very probably not preserved (Braun 1987). Hence, we propose to neotypify this species with a collection that has been included in the present molecular examinations, so as to fix the application of the name E. alphitoides. The exact host range of this species is not yet known. Records of this species from southern Asia (e.g. India, Nepal, Sri Lanka) are uncertain, i.e. they could also belong to the tropical-subtropical Erysiphe quercicola sp. nov. Numerous other oak species recorded as hosts of E. alphitoides (s. lat.) need to be re-examined. Furthermore, the whole taxonomic situation is complicated as particular host species may be infected by several powdery mildew species in the E. alphitoides complex, e.g. Q. crispula, Q. robur and Q. serrata are all hosts of E. alphitoides s. str., E. hypophylla (clade D), and E. quercicola sp. nov., and mixed infections are not uncommon, as we found while sequencing specimens in this study. E. alphitoides s. str. is able to attack North American oaks, e.g. Q. alba (in Argentina, see Table 1) and Q. rubra (in Europe, see Braun 1995). Most hosts of E. alphitoides s. str. pertain to Quercus sect. Quercus. In Europe, E. alphitoides s. str. may infect several other species of sect. Cerris, e.g., Q. castaneifolia, Q. ceris and Q. macrolepis (Braun 1995). Erysiphe hypophylla (Nevod.) U. Braun & Cunningt., Schlechtendalia 10: 92 (2003) (Figs 6 and 7) Anamorph: Oidium (Pseudoidium) sp. Basionym: Microsphaera hypophylla Nevod., Griby SSSR 1: 4 (1952). Synonym: Microsphaera silvatica Vlasov, Trudy Inst. Lesa 16: 160 (1954). Typus: Russia: Moscow, on Quercus robur, 3 Oct. 1950, G. S. Nevodovsky, (LE–holotypus). Mycelium hypophyllous, in thin patches or effuse, white, but soon evanescent. Hyphae straight to flexuous, somewhat geniculate-sinuous, branched at more or less right angles and often near septa, hyphal cells (25–)45–50(–60)  3–6 mm. Appressoria lobed, 5–10 mm diam. Anamorph sparsely developed or lacking, conidiophores erect, 40–110 mm long, 6–10 mm wide, foot-cells cylindrical, 20–60 mm long, followed by 1–2 shorter cells or cells of about the same length. Conidia formed singly, cylindrical, subcylindrical or 818 S. Takamatsu et al. equatorial, straight to curved, (5–)10–25, (0.5–)0.75–1.5(–2) times as long as the chasmothecial diam (75–200 mm), 6–11 mm wide below, wall almost smooth to verruculose, colourless, thick-walled throughout or thick-walled below and thinner upwards, aseptate or with a single basal septum, usually colourless, occasionally pigmented at the very base, apically (3–)4–6(–8) times regularly and mostly densely branched, branched part 50–80  40–60 mm, primary branches sometimes elongated, rarely deeply cleft, tips of the ultimate branchlets uncinate to circinate when mature. Asci 4–12, broadly ellipsoid-ovoid (-subglobose), saccate, subsessile to short-stalked, (40–)50–70(–80)  (25–)30–50(–55) mm, oculus (thin-walled apical portion of ascus) not very evident, 10– 20 mm diam, (6–)8-spored. Ascospores broadly ellipsoid-ovoid, colourless, 13–25(–30)  9–15 mm. Collections examined: See Table 1. Host range and distribution: on various oak species, including Quercus cuspidata, Q. petraea, Q. robur, Q. serrata (Fagaceae); Asia (Central Asia, China, Japan), almost all Europe, introduced in New Zealand. Fig 6 – Erysiphe alphitoides, teleomorph (very similar and barely distinguishable: E. hypophylla). (A) Chasmothecium. (B) Asci. Bars [ 25 mm (chasmothecium), 10 mm (asci). U. Braun del. ellipsoid-cylindrical, 25–45(–60)  10–18.5 mm, length:breadth ratio 1.9–3.5. Chasmothecia strictly hypophyllous, scattered to subgregarious, (80–)100–155 mm diam. Peridial cells angularirregular in outline, 10–30 mm diam. Appendages more or less Comments: The differentiation between Erysiphe hypophylla and E. alphitoides s. str. may be complicated, especially when the latter species is amphigenous. Furthermore, mixed infections of oaks by E. alphitoides s. str. and E. hypophylla are not uncommon (e.g. in the collection on Q. cuspidata, Japan, MUMH 1957). Various authors discussed differences in the ascomata between the two species (Kochman 1960; Roll-Hansen 1961; Cruchet 1962; Blumer 1967), which could not, however, be confirmed by detailed re-examinations comparing the variability of all teleomorphic characters. Thus, the conidial shape remains the only clear difference between the two species. Due to uncertainties about the status of Microsphaera hypophylla as a true species distinct from M. alphitoides, Braun & Takamatsu (2000) hesitated to transfer it to Erysiphe. It was supposed that the differences in the conidial shape could be Fig 7 – Erysiphe hypophylla, anamorph. (A) Appressoria. (B) Conidiophores. (C) Primary conidia. (D) Secondary conidia. Bars [ 10 mm. U. Braun del. 819 Erysiphe alphitoides sensu lato influenced by the mycelium growing on the upper or lower leaf surface. Detailed observations made during the course of the present examinations revealed consistent differences in the length of the conidiophores on the upper and lower leaf surfaces in E. alphitoides s. str. (shorter when epiphyllous, longer when hypophyllous). But, the conidial shape and size was unchanged, i.e. conidia of E. alphitoides s. str. persisted to be broadly doliiform when formed on the lower leaf surface. The same phenomenon has also been observed in E. quercicola sp. nov. Thus, the conidial shape in connection with a hypophyllous growth is sufficient to differentiate E. hypophylla from E. alphitoides s. str. and all other allied species, in spite of the limited applicability of these features due to the evanescent, often sparse, or even lack of, conidial formation. Doubts about the status of E. hypophylla were also caused by a lack of genuine collections of this species in Germany for more than 30 y, where oak powdery mildew has been continuously observed by one of the authors. In that time, all collections of ‘E. hypophylla’ turned out to be hypophyllous E. alphitoides s. str.. This was confirmed by the present molecular examinations showing that recent collections from Switzerland determined as ‘E. hypophylla’ represented hypophyllous E. alphitoides s. str. In the middle of the former century, an epidemic spread of E. hypophylla in Europe took place, but now this species seems to be extinct or very rare in this continent, possibly having been replaced by E. alphitoides s. str. Erysiphe quercicola S. Takam. & U. Braun, sp. nov. (Figs 4B and 8) MycoBank no.: MB 510529 Etym.: ‘quercicola’ refers to the host genus Quercus. Differt a E. brevioribus. alphitoides chasmotheciis cum appendicibus Typus: Japan: Nara, Ikoma Mt., on Quercus phillyraeoides (Fagaceae), 27 Nov. 1999, S. Takamatsu (MUMH 885 – holotypus; HAL 1969 F – isotypus). rDNA sequence ex-type: AB193591 (ITS) and AB237813 (28S). Anamorph: Oidium anacardii Noack, Bol. Inst. Estado São Paulo 9(2): 77 (1898). Synonyms: O. citri (J.M. Yen) U. Braun, Zentralbl. Microbiol. 137: 323 (1982). O. heveae Steinmann, De ziekten en plagen van Hevea brasiliensis in Nederlandsch-Indie: 91, Buitenzorg (1925). O. mangiferae Berthet, Bol. Agric. (São Paulo) 15: 818 (1914). Fig 8 – Erysiphe quercicola. (A) Appressoria. (B) Conidiophores. (C) Primary conidia. (D) Secondary conidia. (E) Chasmothecium. (F) Appendage. (G) Asci. Bars [ 25 mm (chasmothecium), 10 mm (asci). U. Braun del. 820 Mycelium amphigenous, effuse or forming patches, persistent, often causing brownish discolouration of the hosts leaves. Hyphae branched, 2–6 mm wide, hyaline, smooth or almost so, thin-walled. Appressoria solitary or in opposite pairs, 3–8 mm diam. Conidiophores erect, up to 90 mm long, shorter on the upper leaf surface, often longer below, foot-cells cylindrical, 20–40  7–11 mm, straight or curved at the base, followed by 1–2 cells, shorter, equal in length or occasionally somewhat longer. Conidia formed singly, primary conidia obovoid-ellipsoid, apex rounded, base subtruncate, secondary conidia doliiform when mature, ends truncate or subtruncate, immature ones sometimes ellipsoid-cylindrical, 25–40(–45)  12–22 mm, length/width ratio 1.5–2.3, mostly 2. Chasmothecia amphigenous, mainly epiphyllous, scattered to gregarious, (80–) 95–150 mm diam. Peridial cells angular-irregular in outline, 10– 30 mm diam. Appendages 5–30, more or less equatorial, straight to somewhat curved, 0.5–1(–1.25) times as long as the chasmothecial diam, 60–130(–150) mm, 6–10 mm wide at the base, thick-walled throughout or becoming gradually thinner towards the apex, almost smooth to verruculose-rugose, aseptate, hyaline or only pigmented at the very base, apex 4–6 times regularly and densely branched, occasionally deeply cleft, branched part 35–70(–80) mm broad and 20–50 mm high, ultimate tips recurved when mature. Asci 6–15, broadly ellipsopid-ovoid, saccate, occasionally subglobose or broadly subcylindrical, subsessile to short-stalked, 50–70  25–45 mm, oculus not very evident, (8–)10–18(–20) mm diam, (6–)8-spored. Ascospores broadly ellipsoid-ovoid (-subglobose), hyaline, 14–20  8–15 mm. Additional collections examined: Japan: Kanagawa Prefecture, Kawasaki-shi, Ikuta Green Park, on Quercus phillyraeoides, 23 Nov. 1983, S. Tanda (HAL 76 F, TUAMH 2114); Toyama, Yao, Shiraki Mt, on Q. crispula, 2 Feb. 1997, Y. Sato (MUMH 1954–1956, HAL 1971, 1967, 1968 F; Yao, Niho, on Q. crispula, 2 Feb. 1997, Y. Sato (MUMH 1952, HAL 1972 F); Shiga, Mt Kiyotaki, on Q. serrata, 2 Oct. 2005, S. Takamatsu & K. Matsuno (MUMH 3796, HAL 1974 F). – Iran: on Quercus sp., 11 Oct. 2004, S. A. Khodaparast (MUMH 3242, HAL 1973 F). – Thailand: Petchaboon, Numnao National Park, on Quercus sp., 24 Nov. 2004, C. Nakashima (MUMH 3229, HAL 1975 F). Host range and distribution: on Quercus crispula, Q. phillyraeoidea, Q. robur, Q. serrata, Quercus spp. (Fagaceae), Asia (Iran, Japan, Thailand), Australia; on Hevea brasiliensis (para rubber tree), Anacardium occidentale (cashew), Bixa orellana, Citrus spp., Mangifera indica (mango), and Acacia spp., widespread in tropical and subtropical Asia, Africa and South America. Comments: In all phylogenetic analyses, Erysiphe alphitoides s. lat. on Quercus phillyraeoides constituted separate clades supported by high BS values (99–100 %). Thus, this fungus, morphologically well characterized by having uniformly short chasmothecial appendages, warrants recognition as a separate species, described here as E. quercicola. This species is closely allied to E. alphitoides. In the absence of sequence data or ascomata, it is very difficult to differentiate E. quercicola from E. alphitoides as the anamorphs of the two species are not clearly differentiated. The appendages in E. alphitoides are usually 1–1.5 times as long as the chasmothecial diameter, but ascomata with relatively short appendages, about as long as the diameter or somewhat shorter, are not uncommon. As far as is known, E. quercicola with Q. phillyraeoides as principal host, S. Takamatsu et al. seems to be a species of subtropical to tropical distribution, with a wide host range, comparable with E. alphitoides. Collections of E. alphitoides s. lat. on Hevea brasiliensis, Anacardium occidentale, Bixa orellana, Citrus spp., and Mangifera indica have been recorded for a long time under various Oidium names (Braun 1987). Boesewinkel (1980) carried out inoculation experiments and demonstrated that Oidium mangiferae was synonymous with E. alphitoides s. lat. Molecular analyses showed that all records on these exotic hosts apart from two isolates of O. mangiferae, pertain to E. alphitoides s. lat. on Q. phillyraeoides, i.e. E. quercicola (Limkaisang et al. 2006). We did not find E. quercicola in temperate northern Asia or Europe. Erysiphe quercicola occurs on several species of oaks that are also infected by E. alphitoides, such as Q. crispula, Q. robur and Q. serrata. Erysiphe hypogena S. Takam. & U. Braun, sp. nov. (Figs 4C and 9) MycoBank no.: MB 510530 Etym.: ‘hypogena’ refers to the hypophyllous mycelium. Anamorph: Oidium (Pseudoidium) sp. Differt a E. alphitoides et E. quercicola myceliis hypophyllis, coloniis magnis, albidis, persistentibus, maculis decoloribus efferentibus; a E. hypophylla myceliis persistentibus et conidiis doliiformibus; et a E. epigena myceliis hypophyllis et chasmotheciis majoribus, saepe 100–175 mm diam, cum appendicibus numerosis, saepe 15–35, ad basim saepe septatis et brunneis. Typus: Japan: Niigata, Mt. Yahiko, on Quercus acutissima (Fagaceae), 25 Oct 1997, Y. Sato (MUMH 1962 – holotypus; HAL 1994 F – isotypus). rDNA sequence ex-type: AB292723. Mycelium strictly hypophyllous, forming large white patches, subcircular in outline to irregular, persistent, causing necrotic discolouration of the host tissue. Hyphae sparingly branched, 3–8 mm wide, septate, hyaline, smooth. Appressoria solitary or in opposite pairs, lobed, 8–10 mm diam. Conidiophores 70–100 mm long, foot-cells cylindrical, usually straight, 30–50  6–12 mm. Conidia solitary, doliiform, 25–38  14– 18 mm. Chasmothecia hypophyllous, scattered to subgregarious, (75–)100–175 mm diam. Peridial cells angular-irregular in outline, 10–30 mm diam. Appendages more or less equatorial, (10–)15–35, straight to somewhat curved, 0.5–2 times as long as the chasmothecial diam (75–200 mm), 6–10 mm wide, thickwalled throughout or wall becoming thinner towards the apex, almost smooth to verrucose, aseptate and hyaline or often 1–2-septate at the base, septa somewhat distant from the point of attachment and pigmented, brown portion up to 60 mm long, apex 4–6 times regularly and densely branched, primary branches sometimes elongated, up to 30 mm long, occasionally deeply cleft, branched part 35–70 mm broad and 25–50 mm high, ultimate tips recurved when mature. Asci 6–13, broadly ellipsoid-ovoid, saccate, subsessile to shortstalked, 50–75  30–45 mm, (6–)8-spored. Ascospores broadly ellipsoid-ovoid, colourless, 13–20  10–14 mm. Additional collections examined: Japan: Nara, Haibara, on Quercus acutissima, 29 Oct. 1996, S. Takamatsu (MUMH 900; HAL 1993 F); Muroji, on Q. acutissima, 10 Nov. 2002, S. Takamatsu (MUMH 1965, HAL 1982 F); Hairaba, on Q. acutissima, 10 Nov. 2002, S. Takamatsu (MUMH 1967, HAL 1981 F); Mie, Matsusaka, Mt Tsubone, on Q. acutissima, 12 Nov. 2005, S. Takamatsu (MUMH Erysiphe alphitoides sensu lato 821 Fig 9 – Erysiphe hypogena. (A) Appressoria. (B) Conidiophores. (C) Primary conidia. (D) Secondary conidia. (E) Chasmothecium. (F) Appendage. (G) Asci. Bars [ 25 mm (chasmothecium), 10 mm (asci). U. Braun del. 3795, HAL 1996 F), p.p. (hypophyllous); Mt Hossaka, on Q. acutissima, 4 Nov. 2003, S. Takamatsu (MUMH 2776, HAL 1980 F); Nagano, Shinshu University, on Q. acutissima, 25 Sep. 2005, S. Takamatsu (MUMH 3860, HAL 1979 F); Shinshu University, on Q. acutissima, 25 Sep. 2005, S. Takamatsu (MUMH 3878, HAL 1978 F); Aichi, Higashi Botanical Garden, on Q. variabilis, 14 Nov. 2005, S. Takamatsu & R. Divarangkoon (MUMH 3794, HAL 1997 F); Higashi Botanical Garden, on Q. variabilis, 31 Oct. 2005, S. Takamatsu & R. Divarangkoon (MUMH 1065), p.p. (hypophyllous); Shiga, Mt Ibuki, on Q. variabilis, 2 Nov. 2003, S. Takamatsu (MUMH 2309, HAL 1983 F); Mt Ibuki, on Q. variabilis, 6 Nov. 2004, S. Takamatsu (MUMH 3627, 3629, 2634, 3644; HAL 1986– 1989 F); Nagoya, Higashiyama Botanical Garden, on Q. variabilis, 21 Nov. 2005, S. Takamatsu & R. Divarangkoon (MUMH 4185, 4188; HAL 1984, 1985 F). Host range and distribution: on Quercus acutissima and Q. variabilis, Asia, Japan (and possibly China). Comments: As far as is known, Erysiphe hypogena is confined to Quercus acutissima and Q. variabilis, two species of Quercus sect. Cerris in Asia. In molecular sequence analyses, all collections examined formed a well-supported clade, clearly set apart from all other clades. Furthermore, this species is morphologically easily distinguishable from all allied species on oaks by forming persistent, hypophyllous, white patches, which cause necrotic discolouration of the attacked host tissue. This species is possibly also known from China, but Chinese collections of E. alphitoides and E. hypophylla on Q. acutissima and Q. variabilis (see Chen et al. 1987) have not yet been re-examined. Erysiphe epigena S. Takam. & U. Braun, sp. nov. (Figs 4D and 10) MycoBank no.: MB 510531 Etym.: ‘epigena’ refers to the epiphyllous mycelium, Anamorph: Oidium (Pseudoidium) sp. Differt a E. hypogena myceliis epiphyllis, sine maculis decoloribus, chasmotheciis minoribus, 65–115(125) mm diam, appendicibus minoribus, 5–18(25), ad basim saepe non septatis, hyalinis. Typus: Japan: Aichi, Nagoya, Higashiyama Botanical Garden, on Quercus variabilis (Fagaceae), 15 Nov. 1996, S. Takamatsu (MUMH 148 – holotypus; HAL 2001 F – isotypus). rDNA sequence ex-type: AB292718. Mycelium epiphyllous, white, forming patches or effuse, persistent, without causing any necrotic discolouration; hyphae sparingly branched, 2–8 mm wide, septate, hyaline, smooth. Appressoria solitary or in opposite pairs, lobed, 4–10 mm diam. Conidiophores 40–80 mm long, foot-cells cylindrical, usually 822 S. Takamatsu et al. Fig 10 – Erysiphe epigena. (A) Appressoria. (B) Conidiophores. (C) Primary conidia. (D) Secondary conidia. (E) Chasmothecium. (F) Asci. Bars [ 25 mm (chasmothecium), 10 mm (asci). U. Braun del. straight, 20–50  7–10 mm, followed by 1–2 shorter cells. Conidia solitary, primary conidia broadly obovoid, secondary conidia doliiform, (20–)25–35  (12–)15–20 mm, germ tubes terminal or subterminal, short to moderately long, subclavate or apex lobed. Chasmothecia epiphyllous, scattered to subgregarious, 65–115(–125) mm diam. Peridial cells angular-irregular in outline, 10–35 mm diam. Appendages more or less equatorial, 5–18(–25), straight to somewhat curved, 0.5–1.5 times as long as the chasmothecial diam (75–150 mm), 6–9 mm wide, thick-walled throughout or wall becoming thinner towards the apex, almost smooth to verrucose, aseptate and hyaline, occasionally with a single basal septum, colourless or occasionally brown at the very base, apex (3–)4–5(–6) times regularly and densely branched, primary branches sometimes elongated, up to 30 mm long, occasionally deeply cleft, branched part 30–50 mm broad and 20–40 mm high, ultimate tips recurved when mature. Asci 5–8, broadly ellipsoid-ovoid, saccate, subsessile to shortstalked, 40–50  30–40 mm, oculus not very conspicuous, 10– 18 mm diam, (6–)8-spored. Ascospores broadly ellipsoid-ovoid, colourless, 13–24  7–12 mm. Additional collections examined: Japan: Tokyo, Koganei city, Koganei Park, on Q. acutissima, 17 Oct. 1983, T. Kawai (TUAMH 2288, HAL 66 F); Mie, Matsusaka, Mt. Shirai, on Q. acutissima, 24 Oct. 2004, S. Takamatsu (MUMH 3211, HAL 1995 F); Nara, Tenri, on Q. acutissima, 22 Oct. 2000, S. Takamatsu (MUMH 1072, HAL F); Aichi, Nagoya, Higashiyama Botanical Garden, on Q. variabilis, 14 Nov. 1996, S. Takamatsu (MUMH 1958, HAL 2000 F); Nagoya, Nagoya University, on Q. variabilis, 17 Dec. 2003, S. Takamatsu (MUMH 2702, HAL 1976 F); Mie, Tsu, Mie University, on Q. variabilis, 7 Aug. 2003, S. Ito (MUMH 2193, HAL 1998 F). Host range and distribution: on Quercus acutissima and Q. variabilis, Asia, Japan (and possibly China). Comments: Erysiphe epigena is easily distinguishable from E. hypogena by forming epiphyllous mycelium without any discolouration of the host tissue. Furthermore, the ascomata are significantly smaller and possess a smaller number of appendages. But the discrimination between E. epigena and E. alphitoides, as well as E. quercicola is difficult. Other than the different host ranges, the characters of the anamorphs and teleomorphs of these are barely distinct, except for smaller ascomata with relatively few appendages in E. epigena. This species is possibly also known from China, but Chinese collections of E. alphitoides and E. hypophylla on 823 Erysiphe alphitoides sensu lato Q. acutissima and Q. variabilis (see Chen et al. 1987) have not yet been re-examined. Discussion The phylogenetic analyses clearly revealed the genetic heterogeneity of the Erysiphe alphitoides complex, which is separated into seven well-supported clades. The morphology of the whole complex around E. alphitoides s. lat. and E. hypophylla was carefully re-examined, based on numerous specimens, mainly from Asia and Europe, on various oak species. Morphological characters of the collections of each clade were compared with each other and with specimens from the other clades so as to detect morphological similarities and differences, respectively. E. alphitoides was described from Europe on Quercus robur (Griffon & Maublanc 1912). Sequences of all European collections on Q. petraea and Q. robur form a homogeneous cluster (Figs 2 and 3: group A) together with samples from Asia (Japan), Australia and South America (Argentina) on Q. alba, Q. crispula, Q. dentata and Q. serrata. This assemblage of the group A represents E. alphitoides s. str. The examination of the specimens concerned showed that they are also morphologically uniform by having amphigenous mycelium, persistent on the upper leaf surface, doliiform conidia and chasmothecia agreeing with the original concept of this species. E. alphitoides s. str. is common and widespread on a wide range of oak species. It has also been introduced in North America together with Q. robur, used as an ornamental tree or hedge (Braun 1987). Furthermore, E. alphitoides s. str. is undoubtedly able to attack some unrelated hosts, e.g., Aesculus hippocastanum (Marczenko 1976, as Microsphaera penicillata f. aesculi; Braun 1980). Braun (1980) observed horse chestnut trees attacked by E. alphitoides, which grew in direct contact with strongly infected oaks in Germany. The morphological features of the collections on A. hippocastanum perfectly coincided with those on oaks, but inoculation experiments or molecular sequence analyses confirming the identity of the samples concerned are not yet available. All collections of E. alphitoides s. lat. on Q. phillyraeoides from warmer regions in southern Japan (Figs 2–3: group B) are morphologically characterised by having uniformly short appendages (0.5–1  as long as the chasmothecial diam). Otherwise, the morphology of the powdery mildew on this host corresponds well with E. alphitoides s. str. (colonies amphigenous, persistent on the upper leaf surface, conidia doliiform, other chasmothecial characters very similar). The host range of this fungus is almost as wide as in E. alphitoides s. str., covering several oak species (Q. crispula, Q. phillyraeoides, Q. robur, and Q. serrata) as well as some unrelated hosts (Anacardium occidentale, Bixa orellana, Citrus spp., Hevea brasiliensis, Mangifera indica and Acacia spp.; see Limkaisang et al. 2006). Although morphologically only slightly different from E. alphitoides s. str. (group A), group B forms genetically distinct, well-supported (BS ¼ 100 %, PP ¼ 1.0) clades in the rDNA ITS, as well as the 28S rDNA trees, and has to be considered a new separate species. We, therefore, propose the name E. quercicola for this group. Key to the species of the Erysiphe alphitoides complex (E. alphitoides s. lat.) 1 Mycelium and chasmothecia strictly hypophyllous, mycelium evanescent; conidia more or less cylindrical, 25–45(–60)  10–18.5 mm; on various Quercus spp., Asia and Europe, introduced in New Zealand ........................................ .hypophylla Mycelium and chasmothecia strictly epiphyllous or amphigenous, when strictly hypophyllous mycelium persistent and causing discolouration of the host tissue; conidia doliiform to broadly ellipsoid-ovoid, (20–)25–40  (12–)14–20(–25) mm.2 2(1) Mycelium and ascomata strictly hypophyllous, forming large, white, persistent patches, causing necrotic discolouration of the host tissue, finally brown; chasmothecia usually 100–175 mm diam, with numerous appendages, usually 15–35, often with 1–2 septa and brown at the base, pigmented portion up to 60 mm long; confined to Quercus acutissima and Q. variabilis, Japan and (?) China ............................................................................................................................................. .hypogena Mycelium and chasmothecia strictly epiphyllous or amphigenous, mycelium persistent, without any necrotic discolouration; on other hosts or on Quercus acutissima and Q. variabilis, but then chasmothecia smaller, 65–115(–125) mm diam, with relatively few appendages, 5–18(–25), usually aseptate and colourless at the base or at most brown at the very base (E. alphitoides s. lat.) ...................................................................................................................................................................... .3 3(2) Chasmothecia rather small, 65–115(–125) mm diam, with relatively few appendages, 5–18(–25); confined to Quercus acutissima and Q. variabilis, Japan and (?)China ........................................................................................................ epigena Chasmothecia larger, up to 150 mm diam, appendages up to 30; on other hosts ..................................................................... .4 4(3) Mycelial patches often causing necrotic brownish discolouration of the green host leaves; chasmothecia with consistently short appendages, 0.5–1(–1.25) times as long as the chasmothecial diam; on Quercus phillyraeoides, also on additional oak species and several subtropical and tropical fruits and trees (Hevea brasiliensis, Anacardium occidentale, Bixa orellana, Citrus spp., Mangifera indica, and Acacia spp.), but on these hosts usually only forming the anamorph; common in subtropical and tropical areas ................................................................................................................................................ quercicola Green host leaves usually not discoloured; appendages 0.75–2 times as long as the chasmothecial diam, mostly 1–1.5 times; on a wide range of oak species, except Q. phillyraeoides, but also on Aesculus hippocastanum, Mangifera indica, Paeonia lutea; common in temperate areas .................................................................................................................................. ..alphitoides 824 Group D, comprising Q. cuspidata, Q. robur and Q. serrata as hosts, represents E. hypophylla, the collections concerned all showing similar morphology (mycelium strictly hypophyllous, evanescent, conidia more or less cylindrical). The identity of group C, embracing Japanese specimens on Q. cuspidata and Q. crispula, is not yet clear. Mycelium and ascomata are, as far as developed, hypophyllous, but the anamorph could not be traced. In the ITS tree, group C is close to group D (E. hypophylla), whereas in the 28S tree group C is closer to group A (E. alphitoides s. str.). As the anamorphs were absent from the specimens examined, a final conclusion on the identity and taxonomy of group C is not yet possible. The teleomorphs of E. alphitoides s. str. and E. hypophylla are morphologically too similar to be distinguished. The host species (oaks) of E. alphitoides s. str. (A), E. hypophylla (D), and E. quercicola (B), as well as group C, mainly belong in Quercus sect. Quercus. E. alphitoides s. lat. collections on Q. acutissima and Q. variabilis (Quercus sect. Cerris) constitute distinct groups (E, F, G) in the ITS, as well as 28S rDNA trees, clearly set apart from E. alphitoides s. str. and all other clusters. Group E represents a hypophyllous taxon, genetically and morphologically distinguishable from groups G and F, which is an epiphyllous fungus. The development of the two taxa on hosts of Quercus sect. Cerris seems to be comparable with the development of E. alphitoides s. str. and E. hypophylla on hosts of Quercus sect. Quercus. The hypophyllous taxon (group E) is characterised by having consistently hypophyllous mycelium, forming persistent white patches, and causing necrotic discolouration of the host tissue. The ascomata are rather large (100–160 mm diam) and possess numerous appendages (15–35), often with 1–2 basal septa, and frequently brown at the base (pigmented portion up to 60 mm long). These features easily distinguish this taxon from the epiphyllous one, as well as E. alphitoides s. str., E. hypophylla and E. quercicola. The epiphyllous fungus on Q. acutissima and Q. variabilis does not cause necrotic lesions of the host tissue and the ascomata are smaller (65–125 mm diam), with fewer appendages (5–18), usually aseptate and without well-developed, long, brown pigmentation at the base of the appendages. Thus, the two taxa on Japanese species of Quercus sect. Cerris need to be considered as separate new species, which undoubtedly co-evolved with oaks of this section. We, therefore, propose the name E. hypogena for the hypophyllous species and E. epigena for the epiphyllous one. Manos et al. (1999) carried out a molecular phylogenetic study of a wide range of oaks, in which separate parsimony analyses of each data set showed that individual gene trees were congruent and often complementary in supporting clades that generally corresponded to previously recognized taxonomic groups. These analyses also supported the recognition of a strictly Eurasian section Cerris clearly set apart from section Quercus. In addition to E. alphitoides s. lat. sequences, sequences from several other species including Oidium spp. from tropical and subtropical fruit trees belonged to the groups of E. alphitoides s. str. and E. quercicola sp. nov., as previously reported by Limkaisang et al. (2006). Erysiphe wallrothii on Vaccinium hirtum (GenBank AB015930) has an ITS sequence identical to E. alphitoides s. str. To exclude the possibility that the sequence of E. wallrothii was created by any kind of experimental error, independent DNA extraction, PCR, and sequencing were S. Takamatsu et al. performed using another specimen of the species (MUMH283), but the new data obtained confirmed the result. We then conducted morphological observation of E. wallrothii. The result indicated that the morphology of E. wallrothii specimen was consistent with the description of the species (Braun 1987), and differed from E. alphitoides by having (3–)5– 6(–7)-spored asci. Thus, E. wallrothii should be retained as a separate species, although it has an ITS sequence identical to E. alphitoides s. str. Erysiphe pseudolonicerae and E. euonymijaponici have ITS sequences only one base different from E. alphitoides s. str. The ultimate tips of appendages of E. pseudolonicerae are usually straight, not curved and differ from the appendage of E. alphitoides s. str., in which the ultimate tips are usually curved. Thus, E. pseudolonicerae should be also retained as a separate species. Boesewinkel (1979, 1981) reported that E. euonymi-japonici is infectious to some Citrus species, as well as its original host Euonymus japonicus. In the present study, E. euonymi-japonici belonged to the E. alphitoides s. str. group and O. citri from Citrus spp. belonged to the E. quercicola group, although they are commonly included in the E. alphitoides s. lat. As only two isolates of each O. citri and E. euonymi-japonici were used in this study, additional isolates should be analysed to clarify their identity. Sequences of powdery mildew from Paeonia lutea AB257432 and AB257433 were deposited in the DNA database as E. hypophylla (Bolay 2005; Takamatsu et al. 2006a). However, this fungus apparently belongs to the E. alphitoides s. str. group in its ITS and 28S rDNA sequences. Oidium spp. from tropical and subtropical trees, viz. O. citri, O. heveae, O. mangiferae, O. bixae, and Oidium sp. on Acacia spp., belonged to E. quercicola sp. nov. within the E. alphitoides complex. The morphological similarity of the powdery mildews on these tropical and subtropical trees to E. alphitoides s. lat. has been reported by several authors (Thankamma 1968; Boesewinkel 1980, 1981; Braun 1987) including some inoculation tests. The identification of tropical powdery mildews has been difficult because fruit bodies (chasmothecia), on which classification is based, are rarely produced or unknown. The present molecular analyses, as well as the morphological similarity, strongly suggest that all Oidium spp. on these tropical and subtropical trees are anamorphs of E. quercicola sp. nov., except for two of the four O. mangiferae isolates that belonged to E. alphitoides s. str. Boesewinkel (1980) reported that O. mangiferae is an anamorph of E. alphitoides s. lat. based on morphological observation and inoculation test. The present analyses support his arguments, even though we now know that isolates fall into two different types within this complex. Boesewinkel (1980) argued that E. alphitoides of oak was introduced into Europe by mango trees infected with O. mangiferae. The present molecular phylogenetic analysis does not reject his argument. However, in the phylogenetic tree of the genus Erysiphe (Fig 3), the seven E. alphitoides s. lat. groups form a large clade, although BS and PP supports are weak. In this context, an ancestral host at the root of the large clade should be oak based on parsimony criteria. The fungi within E. alphitoides s. str. and E. quercicola sp. nov. may have expanded their host ranges onto a wide range of trees including mango. It is interesting that E. alphitoides s. str. seems to have expanded its host range mainly onto trees of temperate regions, whereas E. quercicola sp. nov. expanded to trees of Erysiphe alphitoides sensu lato tropical and subtropical regions. This is consistent with the geographical distributions of the re-defined species. It is also noteworthy that these host expansions seem to have occurred only in these particular species and not in the other groups of E. alphitoides s. lat. A similar phenomenon was found in the erysiphaceous genus Golovinomyces (Matsuda & Takamatsu 2003; Takamatsu et al. 2006b). In this genus, several basal groups comprised only isolates from asteraceous plants. Isolates from a wide range of other plant families grouped in a large clade together with isolates from the tribe Lactuceae of the Asteraceae. This suggests that a host expansion had occurred from the tribe Lactuceae to other plant families, but not from other asteraceous tribes. Consequently, it seems that host expansions only occurred in a few particular erysiphaceous groups, but not in the Erysiphales in general. It would be interesting to investigate whether similar evolutionary events have occurred in other groups of the Erysiphales or other plant parasitic fungi. Acknowledgements We thank the corresponding editor and anonymous reviewers for suggestions and editorial comments. 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