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Persoonia 24, 2010: 38–48 www.persoonia.org RESEARCH ARTICLE doi:10.3767/003158510X494596

Molecular phylogenetic analyses reveal a close evolutionary relationship between Podosphaera (: Erysiphaceae) and its rosaceous hosts

S. Takamatsu1, S. Niinomi1, M. Harada1, M. Havrylenko 2

Key words Abstract Podosphaera is a of the powdery mildew fungi belonging to the tribe Cystotheceae of the Erysipha­ ceae. Among the host of Podosphaera, 86 % of hosts of the section Podosphaera and 57 % hosts of the 28S rDNA subsection Sphaerotheca belong to the . In order to reconstruct the phylogeny of Podosphaera and to evolution determine evolutionary relationships between Podosphaera and its host plants, we used 152 ITS sequences and ITS 69 28S rDNA sequences of Podosphaera for phylogenetic analyses. As a result, Podosphaera was divided into two molecular clock large clades: clade 1, consisting of the section Podosphaera on (P. tridactyla s.l.) and subsection Magnicel­ phylogeny lulatae; and clade 2, composed of the remaining member of section Podosphaera and subsection Sphaerotheca. powdery mildew fungi Because section Podosphaera takes a basal position in both clades, section Podosphaera may be ancestral in Rosaceae the genus Podosphaera, and the subsections Sphaerotheca and Magnicellulatae may have evolved from section Podosphaera independently. Podosphaera isolates from the respective subfamilies of Rosaceae each formed different groups in the , suggesting a close evolutionary relationship between Podosphaera spp. and their rosaceous hosts. However, topology comparison and molecular clock calibration did not support the possibility of co-speciation between Podosphaera and Rosaceae. Molecular phylogeny did not support delimitation of P. aphanis, P. clandestina, P. ferruginea, P. spiraeae and P. tridactyla in their current circumscriptions, which suggests the need for revision of these species.

Article info Received: 21 October 2009; Accepted: 10 January 2010; Published: 25 February 2010.

INTRODUCTION Erysiphaceae were trees (Mori et al. 2000a). Multiple host shifts from trees to herbs may have then occurred during the The Erysiphaceae is a group of obligate biotrophic fungi that Tertiary (Takamatsu 2004). 4 cause powdery mildew disease on about 1 × 10 angiosperm The powdery mildews belonging to the tribe Cystotheceae species (Amano 1986), and it consists of 16 genera and ap- have both herbaceous and woody plants as hosts and consist proximately 650 species (Braun & Takamatsu 2000, Braun of three genera, Cystotheca, Podosphaera and Sawadaea, of et al. 2002, Takamatsu et al. 2005a, b, Liberato et al. 2006). which the host ranges of the genera Cystotheca and Sawadaea The host range of this fungal group is strictly confined to an- are restricted to a narrow range of host families, i.e. all hosts giosperms and the fungi have never been reported to infect of Cystotheca belong to the Fagaceae, and Sawadaea mostly ferns or gymnosperms (Amano 1986). Molecular phylogenetic occurs on Aceraceae. All hosts of these two genera are trees. analyses demonstrated that the Erysiphaceae form a distinct The genus Podosphaera consists of two sections, Podosphaera monophyletic group (Mori et al. 2000b, Lutzoni et al. 2004, and Sphaerotheca. The section Podosphaera (formerly the Takamatsu 2004, Wang et al. 2006). Thus, the family Erysipha­ genus Podosphaera) parasitizes woody plants, and about 90 % ceae is derived from a single ancestral taxon that may have of its hosts belong to the Rosaceae. The section Sphaerotheca acquired parasitism just once. Molecular clock calibration sug- (formerly the genus Sphaerotheca) is further divided into the gested that the fungi originated in the late Cretaceous (Mori subsections Sphaerotheca and Magnicellulatae, each of which et al. 2000b, Takamatsu & Matsuda 2004), which is consistent forms a separate monophyletic clade derived from different an- with the hypothesis of Heluta (1992) and the fact that their host cestors (Takamatsu et al. 2000). More than 50 % of the hosts of range is restricted to angiosperms. the subsection Sphaerotheca are woody or herbaceous plants The Erysiphaceae are divided into five tribes and two basal belonging to the Rosaceae. On the other hand, all hosts of the genera. Both tree-parasitic and herb-parasitic fungi are included subsection Magnicellulatae are herbaceous plants scattered in three of the five tribes: Cystotheceae, Erysipheae and Phyl­ among 40 families that do not include the Rosaceae. lactinieae. Tree-parasitic fungi usually take basal positions in Thus, although the genus Podosphaera has a close affinity to these tribes and herb-parasitic fungi have derived positions. Rosaceae, subsection Magnicellulatae is unique in having no These observations, as well as the fact that the most basal rosaceous plant as host. This may be supported by the fact that genera of the Erysiphaceae, i.e. Parauncinula and Caespito­ Magnicellulatae has its own conidial germination type, i.e. the theca, infect trees, suggest that the early host plants of the Magnicellulatae type, which differs from the Fibroidium type of other taxa of Podosphaera (Cook & Braun 2009). 1 Graduate School of Bioresources, Mie University, 1577 Kurima-Machiya, Phylogenetic relationships within the tribe Cystotheceae were Tsu 514-8507, Japan; corresponding author e-mail: [email protected]. previously reported by Takamatsu et al. (2000). They reported 2 Department of , Centro Regional Universitario Bariloche, Universidad that host shifts from trees to herbs occurred at least twice in Nacional del Comahue, San Carlos de Bariloche, Rio Negro, . this tribe and that the subtribes Sphaerotheca and Magnicel­

© 2010 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. S. Takamatsu et al.: Relationship between Podosphaera and its rosaceous hosts 39

Table 1 Sources of Podosphaera material sequenced in this study and DNA database accession numbers.

Host Fungal species Location and year Designation1 Database ID no.2

Asteraceae Calendula officinalis fusca Bariloche, Argentina; 2001 MUMH 1933 AB525914 Calendula officinalis fusca Bariloche, Argentina; 2004 MUMH 2432 AB525915 officinale fusca Bariloche, Argentina; 2004 MUMH 2440 AB525916 Cannabaceae Humulus lupulus macularis Nagano, Japan; 2003 MUMH 2926 AB525917 Caricaceae Carica papaya caricae-papayae Chiang Rai, Thailand; 2002 MUMH 1853 AB525918 rubra negeri Lago Curruhue, Argentina; 2001 MUMH 1478 AB525919 Escallonia rubra negeri Bariloche, Argentina; 2001 MUMH 1479 AB525920 Escallonia virgata negeri Cerro Tronador, Argentina; 2004 MUMH 2515 AB525921 Geranium thunbergii fugax Nara, Japan; 1997 MUMH 343 AB525922 magellanica gunnerae Tierra del Fuego, Argentina; 1999 BCRU 03874 AB525923 (MUMH 1480) Gunnera magellanica gunnerae Tierra del Fuego, Argentina; 1999 BCRU 03890 AB525924 (MUMH 1481) Linaceae Linum usitatissimum lini Switzerland; 1998 MUMH 1392 AB525925 Onagraceae Epilobium ciliatum epilobii Chall Huaco, Argentina; 2001 MUMH 1873 AB525926 Rosaceae laevis clandestina Halle (Saale), Germany; 2009 MUMH 4968 AB525927 Aria alnifolia curvispora Toyama, Japan; 2001 MUMH 3266 AB525928 Cerasus incana salatai Tbilisi, Georgia; 2001 MUMH 2595 AB525929 monogyna clandestina Bariloche, Argentina; 2001 MUMH 2429 AB525930 Crataegus oxyacantha clandestina Lago Lacar, Argentina; 2001 MUMH 1869 AB525931 Crataegus sp. clandestina Bariloche, Argentina; 2001 MUMH 1868 AB525932 Fragaria chiloensis aphanis Lago Gutierrez, Argentina; 2001 MUMH 1871 AB525933 serratifolia sp. Aichi, Japan; 1997 MUMH 407 AB525934 aff. crenatoserrata Oidium sp. 3 Bariloche, Argentina; 2004 MUMH 2450 AB525935 Oidium sp. 3 Bariloche, Argentina; 2001 MUMH 1870 AB525936 Rosa rubiginosa pannosa Lago Gutierrez, Argentina; 2001 MUMH 1476 AB525937 Rosa rubiginosa pannosa Lago Lacar, Argentina; 2001 MUMH 1872 AB525938 Rosa maltiflora pannosa Yamanashi, Japan; 2000 MUMH 819 AB525939 cantoniensis spiraeae Villa La Angostura, Argentina; 2004 MUMH 2490 AB525940 Spiraea japonica clandestina Buenos Aires, Argentina; 2004 MUMH 2535 AB525941 Stachyurus praecox sp. Mie, Japan; 1999 MUMH 830 AB525942 Stephanandra incisa stephanandrae Mie, Japan; 1999 MUMH 831 AB525943 Verbenaceae Diostea juncea Oidium sp. 3 Cerro Tronador, Argentina; 2004 MUMH 2498 AB525944 Diostea juncea Oidium sp. 3 Bariloche, Argentina; 2009 MUMH 4937 AB525945 Diostea juncea Oidium sp. 3 Bariloche, Argentina; 2009 MUMH 4938 AB525946 Violaceae Viola maculata O. maculatae3 Bariloche, Argentina; 2002 BCRU 04343 AB525947 (MUMH 3050) Viola maculata O. maculatae3 Bariloche, Argentina; 2002 BCRU 04345 AB525948 (MUMH 3051)

1 BCRU: Institutional Herbarium of Centro Regional Universitario Bariloche, San Carlos de Bariloche, Argentina; MUMH: Mie University, Mycological Herbarium, Japan. 2 DDBJ, EMBL, and GenBank database accession number of nucleotide sequence data. 3 Oidium. lulatae evolved independently from separate ancestral taxa MATERIALS AND METHODS belonging to tree-parasitic Podosphaera species. However, they used only 26 ITS sequences in the analysis, which was DNA extraction and amplification inadequate to determine the phylogenetic relationships within The collection and location of host plants and the accession the genus Podosphaera. In this study, we conducted compre- numbers for the nucleotide sequence databases (DDBJ, EMBL hensive phylogenetic analyses by combining newly determined and GenBank) are provided in Table 1. Whole-cell DNA was sequences with sequences reported in Takamatsu et al. (2000) isolated from chasmothecia or mycelia via the chelex method and retrieved from DNA databases. (Walsh et al. 1991, Hirata & Takamatsu 1996). The ITS region The aims of this study were including the 5.8S rDNA, and the 5’ end of the 28S rDNA includ- 1. to reconstruct the phylogeny of the genus Podosphaera; ing the variable domains D1 and D2 were amplified separately 2. to discuss the evolution of Podosphaera with special refer- by two sequential PCR reactions using partially nested primer ence to host relationships; and sets. The PCR reactions were conducted using TaKaRa Taq 3. to consider species delimitation of Podosphaera from the DNA polymerase (TaKaRa, Tokyo, Japan) in a TP-400 thermal perspective of molecular phylogeny. cycler (TaKaRa) under the following thermal cycling conditions: We included sequences of the subtribe Magnicellulatae in the an initial denaturation step of 2 min at 95 °C, 30 cycles of 30 s present analyses, but we did not address them in the discussion at 95 °C, followed by 30 s at 52 °C for annealing, and 30 s at because this fungal group is too large and distinct to be ana- 72 °C for extension, and a final extension step of 7 min at 72 °C. lyzed in this paper. Therefore, the phylogeny of Magnicellulatae A negative control that lacked template DNA was included in has been discussed elsewhere (Ito & Takamatsu 2010). each set of reactions. The PCR products were subjected to 40 Persoonia – Volume 24, 2010 MP 4 Sphaerotheca Magnicellulatae Podosphaera Sphaerotheca Podosphaera (Bold) supported by 70% or (Bold) supported MPvalues higher bootstrap Nodes supported by 95% or Nodes supported of section section subsection subsection Cannabaceae Can = Caricaceae Car = Cercidiphyllaceae Cer = Escalloniaceae Esc = Fag = Fagaceae Ger = Geraniaceae Gun = Gunneraceae = Linaceae Lin Mor = Ona = Onagraceae subfamily R/A = Rosaceae subfamily R/M = Rosaceae Rosoideae Rosaceae subfamily R/R = subfamily R/S = Rosaceae Scr = Scrophlariaceae Stachyuraceae Sta = Ver = Verbenaceae Vio = Violaceae Argentina ARG = Australia AUSTR = GER = Germany KOR = JPN = Japan NZ = New Zealand SWI = Switzerland THAI = Thailand States of America USA = United P: Section or subsection P: Section HostHF: families of collection Place tree (T) = (S) = (H) = herb * Bpp values higher r r r r Fag Fag Fag Fag Fag Scr Gun Gun Scr Mor R/A R/A R/A R/A R/A R/A R/A R/A R/A R/A R/A R/A R/A R/A R/A R/R R/R R/R R/R R/R R/R R/R R/R Sta Ge R/S Car Ve Ve Ve Esc Esc Esc R/S R/S R/S R/S R/S R/R R/R R/R Can Lin R/A R/S R/S R/S R/M R/A R/S Ona Vio R/M R/M R/M R/M R/M Cer R/M R/M R/M R/M R/M HF P )

e AB026144 JPN (H) AB026144 JPN AF011288 USA (T) used as outgroup taxa. The tree is a phylogram of one of the 1 x 10 AF011289 USA (T)

Maloidea group AB026147 JPN (T) AB000933 USA (T) AB027231 JPN (T) P. tridactyla s. lat. P. tridactyla MUMH407 JPN (T) sp. AF073354 KOR (T) Amygdaloideae group ( 2 AF073353 AUSTR (T) MUMH3266 (T) JPN Cystotheca californica Quercus agrifolia Quercus sibiricum

Spiraeoideae group Rosoideae group ex Pourthiae vilosa ex ex ex Quercus agrifolia Molus domestica and ex ex AB000932 (T) JPN sp. ex sp. Photinia serratifolia Aria alnifolia . sibirica Molus domestica C. lanestris C. lanestris C. lanestris C. lanestris ex P ex sp. ex sp. AF011318 (T) USA C. lanestris . leucotricha odosphaera MUMH1480 ARG (H) AB046986 USA (H) P P MUMH1481 ARG (H) glauca Quercus AB027232 JPN (H) AY833658 SWI (T) AY833658 SWI Magnicellulatae . curvispora . curvispora Podosphaera AY833657 AUSTR (T) AY833656 AUSTR (T) P ex 1 . leucotricha odosphaera MUMH1872 ARG (S) P P MUMH831 JPN (S) AB026134 JPN (H) AB026136 JPN (H) MUMH1869 ARG (T) MUMH2450 ARG (T) MUMH830 JPN (S) MUMH3050 ARG (H) MUMH1476 ARG (S) AB026141 JPN (H) AB022385 (H) JPN MUMH2429 ARG (T) MUMH819 JPN (S) * * MUMH1853 THAI (T) MUMH2515 ARG (T) MUMH2595 GEOR (T) MUMH2926 JPN (H) MUMH1478 ARG (T) Prunus armeniaca MUMH4938 (S) ARG MUMH4937 (S) ARG AF073355 AUSTR (H) MUMH2498 (S) ARG sp. AF154321 AUSTR (T) MUMH2535 ARG (S) Veronica spicata AF011316 USA (T) C. wrightii sp. AF011322 USA (S) MUMH1871 ARG (H) MUMH1392 SWI (H) MUMH1479 ARG (T) ex AB026150 JPN (S) subsect. 84 sequences GQ856690 NZ (T) ex MUMH1870 ARG (T) Prunus cerasifera AB026143 JPN (S) AB026153 (S) JPN MUMH1873 (H) ARG Gunnera magellanica Rosa sp. AB022348 JPN (S) sp. MUMH1868 ARG (T) AF011317 USA (T) Prunus cerasifera Prunus armeniaca Prunus MUMH2490 (S) ARG AB026149 JPN (S) Gunnera magellanica ex MUMH4968 GER (T) ex Rosa rubiginosa ex AB026137 JPN (S) ananossa Sanguisorba officinalis Viola maculata AB026152 JPN (H) ex ex ex x ex Rosa Rosa rubiginosa Rosa maltiflora AB026140 JPN (T) ex Carica papaya AB026138 JPN (T) Agrimonia pilosa AF154325 KOR (S) Prunus incana Prunus ex ex . fuliginea . tridactyla Stephanandra incisa Prunus avium Geranium nepalense Diostea juncea Diostea Stachyurus praecox Stachyurus juncea Diostea Diostea juncea Diostea ex ex ex Fragaria grandiflora P P Humulus lupulus ex ex * ex ex Escallonia virgata Escallonia rubra Spiraea japonica Crataegus Crataegus oxyacantha Crataegus monogyna Filipendula purpurea ex ex ex ex ex . gunnerae . tridactyla . pannosa ex ex ex banksii Streblus Spiraea japonica Escallonia rubra P P P ex ex ex ex ex Fragaria chiloensis Fragaria AY833655 SWI (T) AY833655 SWI . tridactyla . tridactyla . tridactyla sp. ex . pannosa sp. ex sp. ex sp. ex . gunnerae Linum usitatissimum P P P P ex ex AY833654 SWI (T) P Epilobium ciliatum Spiraea thunbergii ex ex ex . salatai . salatai . pannosa . pannosa . pannosa . fugax * * . aphanis . ferruginea Spiraea japonica Pyracantha aff. crenatoserrata Pyracantha Spiraea cantoniensis sp. AY833659 KOR (T) Pyracantha crenulata Pyracantha AY833651 AUSTR (T) AY833653 AUSTR (T) P P P P P ex dioicus P P sp. ex . negeri * . negeri Prunus laurocerasus ex . aphanis ex Spiraea nipponica laevisAmelanchier Spiraea cantoniensis Prunus apetrata P ex Oidium P Oidium Oidium ex P Oidium ex * . spiraeae . macularis * AB000943 JPN (T) . negeri Spiraea salicifolia . lini . clandestina ex ex ex P P ex Oidium maculatae Oidium maculatae P P P sp. ex sp. ex Prunus * . clandestina . clandestina . clandestina . clandestina AY833652 KOR (T) . aphanis . aphanis * . stephanandrae . caricae-papayae ex Oidium P P P P P P P P sp. ex sp. ex * * . clandestina . clandestina . epilobii . piraeae AB000945 JPN (T) P P P Cercidiphyllum japonicum . spiraeae Prunus lusitanica . spiraeae * P Oidium Oidium P Prunus persica Prunus persica . spiraeae . ferruginea ex ex P P Prunus laurocerasus . spiraeae . clandestina . clandestina . clandestina * * ex ex P P P P Prunus japonica . tridactyla . tridactyla odosphaera ex P P * ex ex * . tridactyla Prunus grayana P . cercidiphylli . tridactyla . tridactyla * * ex ex P P P . tridactyla P * . tridactyla . tridactyla P P * . longiseta P * agnicellulatae * M clade 2 clade 1 * 1 change ITS /ITS tree MP 157 sequences 499 characters 496 steps = 0.5464 CI = 0.9299 RI RC = 0.5081 odosphaera Phylogenetic analysis of the nucleotide sequences of the ITS region including 5.8S for rDNA 157 sequences from P Fig. 1 Gaps were treated as missing data. Horizontal branch lengths are proportional to the number of nucleotide trees with 496 steps, which was obtained by a heuristic search employing the random stepwise addition option of PAUP. substitutions that were inferred to have occurred along a particular branch of the tree. S. Takamatsu et al.: Relationship between Podosphaera and its rosaceous hosts 41 electrophoresis in a 1.5 % agarose gel in TAE buffer, excised RESULTS from the ethidium bromide-stained gel, and purified using the JETSORB Kit (Genomed, Oeynhausen, Germany) according to Phylogeny inferred from ITS sequences the manufacturer’s protocol. The nucleotide sequences of the A total of 157 ITS sequences, including 33 sequences newly PCR products were obtained for both strands using direct se- determined in this study, were used for the current analysis. quencing in a CEQ2000XL DNA sequencer (Beckman Coulter, Cystotheca spp. were used as outgroup taxa based on Mori Fullerton, CA, USA). The sequence reactions were conducted et al. (2000a). The dataset consisted of 499 characters, of using the CEQ Dye Terminator Cycle Sequencing Kit (Beckman which 179 characters were variable and 140 characters were Coulter) according to the manufacturer’s instructions. informative for parsimony analysis. A total of 1 × 104 MP trees For amplification of the ITS region, primers ITS5 (White et with 496 steps (CI = 0.5464; RI = 0.9299; RC = 0.5081) were al. 1990) and P3 (Kusaba & Tsuge 1995) were used for the constructed by the MP analysis. One of the 1 × 104 MP trees, first amplification. One microlitre of the first reaction mixture excluding subtribe Magnicellulatae (shown in Appendix 2), is was used for the second amplification, along with the partially shown in Fig. 1. Parsimony ratchet analysis generated trees nested primer sets ITS5 and ITS4 (White et al. 1990). The with the same tree length and similar tree topologies. Therefore, ITS5/ITS4 fragment was subjected to cycle sequencing using we concluded that the tree shown in Fig. 1 is not the result of a the primers ITS1, ITS4, T3 and T4 (Hirata & Takamatsu 1996). local optimum. MrModeltest selected the GTR+I+Γ model as the For amplification of the 28S rDNA, primers PM3 (Takamatsu best for this dataset. Using this evolution model, MrBayes was 7 5 & Kano 2001) and TW14 (Mori et al. 2000a), and NL1 (Mori run for 1 × 10 generations, resulting in approximately 1 × 10 et al. 2000a) and TW14 were used for the first and second sampling trees. The first 59 390 trees were discarded (burn-in) amplifications, respectively. Primers NL1, NL2, NL3 and NLP2 because ASDSF dropped below 0.01. The remaining 40 611 (Mori et al. 2000a) were used for cycle-sequencing. trees were summarised in a majority-rule consensus tree, yielding the probability of each clade being monophyletic. The Phylogenetic analysis tree topology generated by the Bayesian analysis was almost identical to the MP tree, and thus the tree is not shown. The sequences were initially aligned using the Clustal X pack- age (Thompson et al. 1997). The alignment was then visually The 152 ITS sequences of Podosphaera species were divided refined with a word processing program using colour-coded into two large clades, clade 1 and clade 2. Clade 1 appeared nucleotides. The alignments were deposited in TreeBASE in the MP strict consensus tree, although statistical support of (http://www.treebase.org/) under the accession number S2604. this clade was low in both BS and Bpp analyses. Clade 1 con- Phylogenetic trees were obtained from the data using the sisted of 15 sequences of Podosphaera tridactyla s.l. (section maximum parsimony (MP) method in PAUP v4.0 (Swofford Podosphaera) on Prunus spp. (Rosaceae), 89 sequences of the 2001) and a Bayesian analysis in MrBayes v3.1.1 (Ronquist isolates belonging to the subsection Magnicellulatae of section & Huelsenbeck 2003). MP analyses were performed with the Sphaerotheca, and one sequence of Oidium sp. on Streblus heuristic search option using the ‘tree-bisection-reconstruction’ banksii. The basal nodes of clade 1 were occupied by the se- (TBR) algorithm with the stepwise addition option set as simple quences of Podosphaera tridactyla s.l. and the sequences of subsection Magnicellulatae formed a distinct clade (BS = 61 %; and maximum tree number as 1 × 104. All sites were treated as unordered and unweighted, with gaps treated as missing data. Bpp = 1.0) at a derived position. Clade 2 (BS = 95 %; Bpp = 1.0) The strength of the internal branches of the resulting trees was consisted of the sequences of the section Podosphaera para­ tested with bootstrap (BS) analyses (Felsenstein 1985) using sitizing the subfamilies Maloideae ( subfamily) and Spi­ raeoideae of the Rosaceae and all sequences of the subsection 1 000 replications with the stepwise addition option set as Sphaerotheca of section Sphaerotheca. The sequences of the simple and maximum tree number as 10 to save analysis time. section Podosphaera occupied a basal position of clade 2. BS values of 70 % or higher were shown. We also constructed The sequences of the subsection Sphaerotheca of section Sphae- MP trees with the parsimony ratchet method (Nixon 1999) in rotheca were placed at a derived position but did not form a PAUP and PAUPRat v1 (Sikes & Lewis 2001) to confirm that distinct monophyletic group. the MP tree generated by the MP analysis is not the result of a local optimum. Phylogeny inferred from the 28S rDNA region For Bayesian phylogenetic analyses, the best-fit evolutionary A total of 71 28S rDNA sequences, including 23 sequences model was determined for each dataset by comparing different newly determined in this study, were used for the current evolutionary models via the Akaike information criterion (AIC) analysis. Cystotheca spp. were used as outgroup taxa based using PAUP and MrModeltest v2.2 (Nylander 2004). MrBayes on Mori et al. (2000a). The dataset consisted of 680 characters, was launched with random starting trees and Markov chains of which 78 characters were variable and 47 characters were were sampled every 100 generations. To ensure that the Markov informative for parsimony analysis. A total of 1 × 104 MP trees chain did not become trapped in local optima, we used the with 128 steps (CI = 0.7031; RI = 0.9128; RC = 0.6418) were MCMCMC algorithm and performed the estimation with four constructed by the MP analysis. One of the 1 × 104 MP trees, incrementally heated Markov chains. The average standard excluding subtribe Magnicellulatae (shown in Appendix 1), is deviation of split frequencies (ASDSF) was observed to verify shown in Fig. 2. Parsimony ratchet analysis generated trees that the values dropped below 0.01. Support for individual nodes with the same tree length and similar tree topologies. Therefore, was tested by Bayesian posterior probabilities (Bpp) obtained we concluded that the tree shown in Fig. 2 is not the result of from a 50 % majority rule consensus. Bpp values of 0.95 or a local optimum. MrModeltest selected the GTR+I+Γ model as higher are shown. the best for this dataset. Using this evolution model, MrBayes was run for 5 × 106 generations, resulting in 50 001 sampling Host plants trees. The first 14 650 trees were discarded (burn-in) because The host plant data were extracted from the database ‘Host ASDSF dropped below 0.01. The remaining 35 351 trees were plants of the powdery mildew fungi v1.0’ (available from the summarised in a majority-rule consensus tree, yielding the corresponding author upon request), which was based on the probability of each clade being monophyletic. The tree topology table ‘Host plants of powdery mildew fungi and their distribution produced by the Bayesian analysis was almost identical to the by country’ (Amano 1986). MP tree and thus the tree is not shown. The tree topology of 42 Persoonia – Volume 24, 2010 - MP trees MP 4 Sphaerotheca Magnicellulatae Podosphaera Sphaerotheca (Bold) supported by 70% or higher(Bold) supported by bootstrapvaluesMP Podosphaera Nodes supported by 95% or higheror 95% by supported Nodes of of section section subsection subsection = Escalloniaceae Esc Fagaceae Fag= Geraniaceae = Ger = Gunneraceae Gun Linaceae= Lin Onagraceae = Ona Rosaceaesubfamily R/A = Amygdaloideae Rosaceaesubfamily R/M = Maloideae = Rosaceae R/R Rosoideae subfamily Rosaceaesubfamily R/S = Spiraeoideae = Verbenaceae Ver Violaceae= Vio = Argentina ARG Germany= GER Japan = JPN = Switzerland SWI United of America = USA States P: Section or subsectionor P: Section HF: Host familiesHost HF: of collectionPlace tree(T) = = shrub (S) herb = (H) * Bpp values HF Vio Vio Ger Lin Esc Esc Fag Fag R/R R/R R/R R/S R/S R/R R/A R/A Ver Ver Ver Gun R/M R/M R/M R/M R/M Ona R/M P ) used as outgroup taxa. The tree is a phylogram used of as one outgroup of taxa. the 1 x 10

Rosoideae Spiraeoideae group

Maloideae group P. tridactyla s. lat. P. tridactyla

Amygdaloideae group ( AB022353 USA (T) AB022353 AB022355 JPN (T) Cystotheca MUMH1481 ARG (H) and Quercus glauca Podosphaera Quercus agrifoliaQuercus ex AB022393 JPN (T) ex ex MUMH343 JPN MUMH343 (H) MUMH1869 ARG (T) MUMH1869 ARG AB022423 JPN (T) MUMH1871 ARG (H) MUMH1871 MUMH2429 ARG (T) MUMH2429 AB103367 IRAN (T) MUMH3051 ARG (H) MUMH3051 Gunnera magellanica Gunnera MUMH4968 GER (T) MUMH1870 ARG (T) MUMH2490 ARG (S) MUMH1873 ARG (H) MUMH1873 MUMH3050 ARG (H) MUMH3050 AB022384 JPN (H) ex ex MUMH1392 SWI (H) MUMH1476 ARG (S) MUMH1476 ARG MUMH2535 ARG (S) MUMH1479(T) ARG sp. MUMH1868 ARG (T) MUMH1478(T) ARG MUMH2498 ARG (S) MUMH4937 ARG (S) MUMH4938 ARG (S) Prunus japonica Cystotheca wrightii Cystotheca Prunus grayana ex Geranim thunbergii Cystotheca lanestris Cystotheca . gunnerae sp. AB022347 JPN sp. (S) Magnicellulatae Viola maculata ex Fragaria chiloensis P Crataegus oxyacantha Crataegus Cydonia oblonga Cydonia ex Viola maculata ex ex Crataegus monogyna Crataegus Crataegus ex Rosa Amelanchier ex ex ex Epilobium ciliatum ex Spiraea cantoniensis Rosa rubiginosa Pyracantha crenulata ex ex ex ex . fugax Spiraea japonica Spiraea . tridactyla ex ex Filipendula purpurea Diostea juncea Diostea juncea Escallonia rubra Diostea juncea Linum usitatissimum subsect. 44 sequences P ex P ex Escallonia rubra . longiseta P ex ex ex ex ex . aphanis ex ex sp. ex sp. ex P  sp. ex sp. ex sp. ex sp. ex sp. ex O. maculatae . pannosa . lini . epilobii  . clandestina . clandestina P O. maculatae P P P P . pannosa . spiraeae   . negeri . clandestina . clandestina . clandestina Oidium P P . negeri P P P  P . spiraeae P Oidium Oidium Oidium P   . clandestina P    agnicellulatae clade 1 M  clade 2 1 change odosphaera P Phylogenetic analysis of the divergent domains D1 and D2 sequences of the 28S rDNA for 71 sequences from Phylogenetic analysis of the divergent domains D1 and D2 sequences of the 28S rDNA 28S rDNA/ MP tree 71 sequences 680 characters 128 steps = 0.7031 CI = 0.9128 RI RC = 0.6418 Fig. 2 with 128 Gaps steps, were which treated was as obtained missing by data. a Horizontal heuristic branch search lengths employing are proportional the to random the stepwise number addition of option nucleotide of sub PAUP. stitutions that were inferred to have occurred along a particular branch of the tree. S. Takamatsu et al.: Relationship between Podosphaera and its rosaceous hosts 43

0.11 0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 Distance

Magnicellulatae

clade 1

Podosphaera

clade 2 Fig. 3 Estimated dates of divergence of major clades of Podosphaera based on the nucleotide Cystotheca sequences of the rDNA ITS region and nucleotide substitution rate of Erysiphaceae (2.52 × 10-9 sub- Tertiary Geological stitutions per site per year) reported by Takamatsu Eocene Oligocene Miocene Pliocene Epoch & Matsuda (2004). Ma, million years ago. 40 30 20 10 0 Time (Ma) the 28S rDNA sequences was almost identical to that of the Eocene, and the split of clade 1 and clade 2 occurred about ITS tree, but the statistical support for the major clades was 20 Ma in the Miocene (Fig. 3, 4). lower than that of the ITS tree. DISCUSSION Timing of evolutionary events Because a likelihood ratio test (LRT) significantly rejected the Host relationships molecular clock of the ITS dataset of 157 sequences, five se- Host plants of Podosphaera quences with extremely long or short terminal branches were The number of host plant species of Podosphaera, arranged removed from the dataset. The molecular clock hypothesis of by plant families, is shown in Table 2. Podosphaera is divided the reduced dataset consisting of 152 ITS sequences was not rejected by the LRT and thus was used to construct a UPGMA into two sections, Podosphaera (formerly genus Podosphaera) tree. The LRT did not reject the molecular clock hypothesis of and Sphaerotheca (formerly genus Sphaerotheca). The latter the 28S rDNA dataset consisting of 71 sequences. Thus, the section is further divided into two subsections, Sphaerotheca dataset was used to construct a UPGMA tree. The Kimura two- and Magnicellulatae, respectively. The subsection Magnicel­ parameter model (Kimura 1980) was used to calculate genetic lulatae forms a distinct monophyletic group that diverged from an ancestral by a host shift from Prunus (Rosaceae) to distances. The molecular clocks of the ITS (2.52 × 10-9 substitu- tions per site per year, ssy) and the D1/D2 domain of the 28S herbaceous plants (Takamatsu et al. 2000). Magnicellulatae is unique in its host range and morphological characteristics rDNA region (6.5 × 10-10 ssy) of the Erysiphales (Takamatsu & Matsuda 2004) were used to calculate evolutionary timing. compared with other Podosphaera species, suggesting that The molecular clocks suggested that Podosphaera split from this subsection evolved independently from other Podosphaera Cystotheca about 40 million years ago (Ma) at the end of the taxa. Thus, we discuss the evolution of this fungal group else- where (Hirata et al. 2000, Ito & Takamatsu 2010). There are 250 host species of the section Podosphaera spanning 10 orders 0.03 0.02 0.01 0 Distance and 13 families, of which 216 host species (86.4 %) belong to the Rosaceae. The subsection Sphaerotheca of section Sphaerotheca has 806 host species covering 15 orders and 28 families, of which 456 (56.6 %) belong to the Rosaceae. Of the non-Rosaceae hosts, 70 belong to the , 67 to the Geraniaceae, 65 to the Onagraceae and 54 to the Hydrangeaceae. Thus, the Rosaceae has the highest number Magnicellulatae of host species for both section Podosphaera and subsec- clade 1 tion Sphaerotheca. On the other hand, Magnicellulatae has 1 110 host species spanning 40 families. About half (45 %) Podosphaera of the hosts belong to the and none belong to the

clade 2 Rosaceae. Cystotheca Powdery mildews of Rosaceae Tertiary Geological Eocene Oligocene Miocene Pliocene Epoch The number of host species of powdery mildew fungi reported 40 30 20 10 0 Time (Ma) on Rosaceae is shown in Table 3 and is organised by plant subfamilies and fungal genera. Five powdery mildew genera Fig. 4 Estimated dates of divergence of major clades of Podosphaera based on the divergent domains D1 and D2 sequences of the 28S rDNA and the have been reported to occur on the Rosaceae. Of these, the nucleotide substitution rate of Erysiphaceae (6.5 × 10-10 substitutions per site genus Podosphaera has the highest number of host species in per year) reported by Takamatsu & Matsuda (2004). Ma, million years ago. the Rosaceae (672). Podosphaera occurs on all four subfamilies 44 Persoonia – Volume 24, 2010 of Rosaceae, and the highest number of host species is in the Relationships between host plants and subfamily Rosoideae (418 species). Phyllactinia is the genus phylogeny of Podosphaera having the next highest number of host species in Rosaceae Phylogenetic trees of Podosphaera excluding Magnicellulatae (110), and finally Erysiphe has 68 host species. Although all are shown in Fig. 1 and 2. Podosphaera taxa are divided into three sections of Erysiphe occur on Rosaceae, the number of four groups according to the subfamilies of Rosaceae: the host species differs depending on the plant subfamilies and Amygdaloideae group, the Maloideae group, the Spiraeoideae fungal sections. Although Golovinomyces and Leveillula also group and the Rosoideae group. The Amygdaloideae group occur on Rosaceae, the number of host species is less than (= P. tridactyla s.l.) is an assemblage that splits from the other 10 in both genera. Therefore, Rosaceae is the most important three groups first and forms a large group (clade 1) with the plant family as host for Podosphaera and vice versa. fungi belonging to Magnicellulatae. The first node of clade 1 was shared by the isolates from Amygdaloideae, which sug- Table 2 Number of host plant species of the genus Podosphaera. gests that the Amygdaloideae group is ancestral in clade 1 Number of host species and the Magnicellulatae diverged from an ancestor that was parasitic to the Amygdaloideae. This group is further divided into two small groups with large genetic divergence. Subgroup

Sphaerotheca 1 (BS = 95 %; Bpp = 0.95) split from other clade 1 groups Podosphaera Sphaerotheca first and consists of isolates from the subgenera Amygdalus, Plant order & family Sect. Sect. Total Laurocerasus and Padus of Prunus. Subgroup 2 (BS = 58 %; subsect. EUROSIDS I Bpp = 0.84), sister to Magnicellulatae, consists of isolates from Ulmaceae 1 2 3 the subgenera Cerasus and Prunus. Thus, the phylogeny of Moraceae 0 4 4 the Amygdaloideae group is closely related to the subgeneric- Urticaceae 0 2 2 Rosaceae 216 456 672 level of Prunus, which suggests the possibility of Elaeagnaceae 0 3 3 co-speciation between Prunus and the Amygdaloideae group. Betulaceae 1 0 1 However, in the phylogeny of the plant genus, subgenus Amyg­ Celastrales Celastraceae 0 3 3 dalus groups with Prunus, and the remaining three subgenera Salicaceae 3 0 3 Cerasus, Laurocerasus and Padus form another group (Bortiri Linaceae 0 1 1 Euphorbiaceae 0 70 70 et al. 2001, Lee & Wen 2001), which is not consistent with the grouping of powdery mildew fungi on Prunus. Because we used EUROSIDS only 15 sequences in the present analysis, more sequence Sapindales Anacardiaceae 0 8 8 data will be required to clarify the phylogenetic relationships 0 18 18 Onagraceae 0 65 65 within P. tridactyla s.l. Punicaceae 0 1 1 Powdery mildews on the subfamilies Maloideae, Spiraeoideae Geraniaceae 0 67 67 and Rosoideae formed a large clade (clade 2: BS = 95 %; Saxifragales Saxifragaceae 0 54 54 Hamamelidaceae 4 0 4 Bpp = 1.0) distinct from the Amygdaloideae group. Because the Cercidiphyllaceae 2 0 2 first split of clade 2 occurred within the Maloideae group, taxa belonging to the Maloideae group may be the most ancestral EUASTERIDS I & II in clade 2. The Maloideae group was further divided into two Solanales Convolvulaceae 0 1 1 groups that are parasitic to the tribes and Cratageae, Oleaceae 2 2 4 Verbenaceae 0 2 2 respectively. The former group split at the base of clade 2 and Gentianales Hydrophyllaceae 0 6 6 was sister to the other groups. The fungi parasitic to the tribe Asclepiadaceae 1 0 1 Cratageae formed a clade with the Spiraeoideae and Rosoideae Apiales Apiaceae 1 1 2 groups and occupied a basal position in the clade. Podosphaera Dipsacales Caprifoliaceae 4 1 5 cercidiphylli on Cercidiphyllum japonicum (Cercidiphyllaceae) was included in the Maloideae group in the phylogeny. Ebenaceae 2 0 2 The Spiraeoideae group formed a clade together with the Rosoi­ Ericaceae 12 11 23 Polemoniaceae 0 21 21 deae group (BS < 50 %; Bpp = 0.98). Although the two groups Cornales Cornaceae 1 0 1 did not form independent clades, they were generally situated Caryophyllales Polygonaceae 0 1 1 at different places in the tree (Fig. 1). Podosphaera epilobii on Phytolaccaceae 0 1 1 Epilobium (Onagraceae) and Oidium maculatae on Viola (Viola­ Tamaricaceae 0 1 1 ceae) were included in the Spiraeoideae group. Podosphaera Plumbaginaceae 0 1 1 Ranunculales Ranunculaceae 0 2 2 clandestina on Prunus (Amygdaloideae, Rosaceae) was also Papaveraceae 0 1 1 included in this group. Podosphaera lini on Linum (Linaceae), P. macularis on Humulus (Cannabaceae), P. caricae-papayae on Total 250 806 1056 Papaya (Caricaceae), P. negeri on Escallonia (Escalloniaceae),

Table 3 Number of host species of the powdery mildew fungi in Rosaceae.

Subfamily of Podosphaera Phyllactinia Erysiphe Golovinomyces Leveillula Rosaceae Erysiphe Microsphaera Uncinula Total Amygdaloideae 96 21 0 1 13 14 2 1 Maloideae 108 76 3 6 3 12 0 0 Rosoideae 418 11 32 1 6 39 6 1 Spiraeoideae 46 2 1 2 0 3 1 1 Unknown 4 0 0 0 0 0 0 0

Total 672 110 36 10 22 68 9 3 S. Takamatsu et al.: Relationship between Podosphaera and its rosaceous hosts 45 and P. fugax on Geranium (Geraniaceae) were included in the Taxonomic implications Rosoideae group. A fungus on Diostea juncea (Verbenaceae) Sections and subsections was identified as ‘Sphaerotheca verbenae’ (= Podosphaera xan­ The genus Podosphaera is divided into two sections, the sec- thii) by Havrylenko (1997). However, ‘Sphaerotheca verbenae’ tion Podosphaera, with appendages dichotomously branched belongs to subsection Magnicellulatae of section Sphaerotheca. at the apex, and the section Sphaerotheca, with hypha-like In this study, this fungus has a DNA sequence identical to that simple appendages. Section Sphaerotheca is further divided of P. negeri in both ITS and 28S rDNA regions and belongs to into two subsections, Sphaerotheca and Magnicellulatae, by the Rosoideae group. We thus treated this fungus as Oidium the size of the peridium cells of the chasmothecia. These sec- sp. in this study. tions and subsections were introduced by Braun & Takamatsu The present study revealed that more than 50 % of the hosts (2000) as morphological, i.e., non-monophyletic groups. The of Podosphaera (excluding Magnicellulatae) belong to the Ro­ present analysis revealed that the two subsections do not share saceae, and phylogenetic relationships of Podosphaera have a common ancestor. Therefore, the section Sphaerotheca is close affinity with the taxonomy of the Rosaceae. These results polyphyletic, consisting of two different groups derived from strongly suggest a close evolutionary relationship between different ancestral taxa. Section Podosphaera is a paraphyletic Podosphaera and Rosaceae. The Rosaceae may have been group situated at the basal part of clade 1 and clade 2. The the first host family for Podosphaera, and host shifts from the present analysis indicates that the section Podosphaera is an- Rosaceae to other plant families may have occurred sponta- cestral in the genus Podosphaera, and the subsections Sphaero­ neously during the evolution of Podosphaera. The Rosaceae theca and Magnicellulatae were derived from the Maloideae belongs to the Rosales of EUROSIDS I (APG II 2003). Of the group and Amygdaloideae group, respectively. Therefore, the host families analysed in this study, all plant families, except- genus Podosphaera is a natural unit supported by molecular ing Rosaceae, Cannabaceae and Moraceae, do not belong to phylogeny, but the sections Podosphaera and Sphaerotheca the Rosales. These results suggest that Podosphaera tend to are artificial, morphological units that are not supported by expand their hosts to closely related plant species when ex- phylogeny, as already stated by Braun & Takamatsu (2000). panding within a single plant family. Conversely, inter-family level host shifts seem to occur independently of host phylogeny. Species supported by phylogeny Similar phenomena have been also found in host expansions of The genus Gunnera consists of herbaceous plants distributed Golovinomyces and Phyllactinia of the Erysiphaceae (Matsuda in the Southern Hemisphere and tropical regions. Podosphaera & Takamatsu 2003, Takamatsu et al. 2008). gunnerae was first described as a powdery mildew of Gun­ nera by Havrylenko & Braun (1998). This species has been Is there any co-speciation between reported only in Argentinian Patagonia. Both ITS and 28S Podosphaera and Rosaceae? rDNA sequences clearly indicated that P. gunnerae forms an The above results suggest a close evolutionary relationship independent clade. This clade was situated at the basal part of between Podosphaera and Rosaceae, but did co-speciation Magnicellulatae and was sister to P. fuliginea. This suggests that occur between Podosphaera and Rosaceae? At least two P. gunnerae split from the other fungi in the early stage of the items should be evaluated to determine whether co-speciation evolution of Magnicellulatae. This species is the only powdery between organisms has occurred. One is similar or identical mildew species described for the genus Gunnera. phylogeny between the organisms concerned, and another is Podosphaera negeri was first described as Sphaerotheca spi­ timing of divergence of the organisms. Of the four subfamilies ralis in 1907 (Braun 1987) and revised as P. negeri by Braun et of the Rosaceae, the subfamilies Amygdaloideae, Maloideae al. (2006). This species has a unique characteristic with coiled and Rosoideae were supported as monophyletic groups by appendages, infects Escallonia (Escalloniaceae) and has been the rbcL sequence phylogeny, but Spiraeoideae was shown reported only in Argentinian Patagonia. Both ITS and 28S rDNA to be polyphyletic (Morgan et al. 1994, Potter et al. 2002). sequences clearly indicated that P. negeri forms an independ- Podosphaera isolates were mostly divided into different groups ent clade. Oidium sp. found on Diostea juncea has a sequence according to the subfamilies of Rosaceae, suggesting that identical to that of P. negeri in both ITS and 28S rDNA regions. the phylogeny of Podosphaera is mostly consistent with the Podosphaera fugax found on Geranium was sister to P. negeri, subfamily-level taxonomy of Rosaceae. Phylogenetic analyses but this was supported neither by BS nor by Bpp values. using rbcL and matK sequences showed that the first split is Oidium maculatae was first described on Viola maculata (Havry­ shared by the Rosoideae, suggesting that Rosoideae is the lenko & Takamatsu 2005). Both ITS and 28S rDNA sequences most ancestral of the Rosaceae (Morgan et al. 1994, Potter showed that this species forms a unique clade. et al. 2002). In Podosphaera phylogeny, the first split occurs between isolates from the Amygdaloideae and Maloideae, and Species not supported by phylogeny the isolates from the Rosoideae are placed at a derived part of the trees (Fig. 1, 2). Thus, the phylogeny of Podosphaera Podosphaera species parasitic to Prunus have been mostly does not conform to that of the Rosaceae. Evolutionary timing classified as P. tridactyla. Cunnington et al. (2005) reported of Podosphaera divergence, as calculated by the molecular that this species has large genetic variation and is divided into clocks of ITS and 28S rDNA regions, suggested that the split several groups, which was confirmed by the present analysis. of Cystotheca and Podosphaera occurred in the late Eocene These results indicate that P. tridactyla is a species complex (c. 40 Ma) and that the split of clade 1 and clade 2 occurred composed of several biological species. Prunus s.l. has been in the mid-Miocene (c. 20 Ma). Fossil records suggest that the divided into 5 to 6 subgenera, which were sometimes treated as traditional subfamilies Amygdaloideae and Maloideae are found separate genera (Bortiri et al. 2001, Lee & Wen 2001). Groups in the Eocene (DeVore & Pigg 2007). Therefore, divergence of P. tridactyla are mostly consistent with the delimitation of the of Podosphaera may have occurred later than the divergence subgenera. Podosphaera tridactyla may have specialised to the of the Rosaceae. In conclusion, there is no evidence that co- respective host groups along with the genetic divergence of Pru­ speciation occurred between Podosphaera and Rosaceae. nus. Some segregated species like P. longiseta (Sawada 1951) and P. salatai (Heluta et al. 2005) have been proposed to lie within P. tridactyla s.l., which is supported by molecular analysis. Comprehensive revision of P. tridactyla s.l. is required. 46 Persoonia – Volume 24, 2010

Podosphaera clandestina is a well known species described Braun U. 1987. A monograph of the Erysiphales (powdery mildews). Beihefte in 1851 and parasitizes 14 genera of the Rosaceae containing zur Nova Hedwigia 89: 1–700. Crataegus, Prunus and Spiraea as hosts (Braun 1987). Nine Braun U, Cook RTA, Inman AJ, Shin HD. 2002. The taxonomy of the powdery mildew fungi. In: Bélanger R, Bushnell WR, Dik AJ, Carver TLW (eds), ITS sequences of P. clandestina from Amelanchier, Crataegus, The powdery mildews: a comprehensive treatise: 13–55. APS Press, St. Prunus and Spiraea used in this study revealed that these se- Paul, USA. quences do not form a single clade. In particular, the sequences Braun U, Delhey R, Dianese JC, Hosagoudar VB. 2006. Miscellaneous notes of the isolates from Crataegus formed a distinct clade distantly on biotrophic micromycetes. Schlechtendalia 14: 85–97. related to other P. clandestina on Amelanchier, Spiraea and Braun U, Takamatsu S. 2000. Phylogeny of Erysiphe, Microsphaera, Uncinula Prunus. 28S rDNA sequence of P. clandestina on Cydonia (Erysipheae) and Cystotheca, Podosphaera, Sphaerotheca (Cystotheceae) was identical to that of isolates from Crataegus. Isolates from inferred from rDNA ITS sequences – some taxonomic consequences. Amelanchier, Prunus and Spiraea were closely related to each Schlechtendalia 4: 1–33. Cook RTA, Braun U. 2009. Conidial germination patterns in powdery mildews. other, but there were some nucleotide substitutions among Mycological Research 113: 616–636. them. A taxonomic re-evaluation of this species is necessary. Cunnington JH, Lawrie AC, Pascoe IG. 2005. Genetic variation within Podo- The fungus on Pyracantha was reported as P. clandestina by sphaera tridactyla reveals a paraphyletic species complex with biological Amano (1986) and Delhey et al. (2003). However, as far as specialization towards specific Prunus subgenera. 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P. xanthii ex Trichosanthes kirilowii AB462786 JPN 28S rDNA/ MP tree P. fusca ex Arctium lappa AB462767 JPN 71 sequences Oidium sp. ex Saintpaulia sp. VPRI20756 AUSTR 680 characters P. xanthii ex Cucurbita maxima AB462766 JPN 128 steps CI = 0.7031 P. phaseoli ex Vigna angularis AB462771 JPN RI = 0.9128 Podosphaera sp. ex Lycopus lucidus AB462778 JPN RC = 0.6418 P. fusca ex Helianthus annuus AB462781 JPN P. fusca ex Zinnia elegans AB462782 JPN Podosphaera sp. ex Glycine max AB462783 JPN 1 change Podosphaera sp. ex juncea AB462785 JPN P. xanthii ex Zehneria japonica AB462792 JPN P. balsaminae ex balsamina AB462788 JPN * P. xanthii ex Cucumis sativus AB462791 JPN Podosphaera sp. ex Solanum melongena AB462769 JPN P. euphorbiae-hirtae ex australis AB462770 JPN Podosphaera sp. ex Dunbaria villosa AB462775 JPN Podosphaera sp. ex Vigna unguiculata AB462784 JPN P. fusca ex Rudbeckia sp. AB462798 JPN Podosphaera sp. ex Boehmeria nivea AB462768 JPN P. fusca ex indica AB462773 JPN P. fusca ex Lactuca raddeana AB462776 JPN Podosphaera sp. ex Verbena x hybrida AB462780 JPN * Podosphaera sp. ex Cayratia japonica AB462765 JPN P. fusca ex Cucurbita sp. AB103368 IRAN P. fusca ex Xanthium strumarium AB103369 IRAN * P. fusca ex Calendula officinalis AB462762 JPN P. fusca ex Calendula officinalis MUMH1933 ARG Podosphaera sp. ex Peristrophe japonica AB462795 JPN P. fusca ex Gerbera hybrida AB462763 JPN P. balsaminae ex Impatiens noli-tangere AB462789 JPN P. fusca ex Helianthus annuus AB462774 JPN P. fusca ex Cosmos bipinnatus AB462797 JPN P. fusca ex Bidens frondosa AB462790 JPN P. balsaminae ex Impatiens textori AB462787 JPN Appendix 1 Phylogeny of subsection Magnicel­ P. intermedia ex trichotomum AB462777 JPN lulatae of the genus Podosphaera inferred from P. fusca ex iinumae AB462760 JPN the nucleotide sequences of the D1/D2 domains P. fusca ex Aster microcephalus AB462779 JPN of the 28S rDNA. This is a part of the phylogenetic tree of Podosphaera, but was not shown in Fig. 2. P. fusca ex Aster tataricus AB462793 JPN P. elsholtziae ex Ajuga reptans AB462794 JPN A bold line means that the node was supported * P. fusca Taraxacum officinale by an MP bootstrap value of 70 % or higher. An ex MUMH2440 ARG asterisk means that the node was supported by P. fusca ex Conyza canadensis AB462772 JPN a Bayesian posterior probability value of 0.95 or P. fusca ex Taraxacum albidum AB462764 JPN higher. Taxon name shown by bold type is the P. fusca ex Taraxacum officinale AB462796 JPN sequence determined in this study. P. sibirica ex Veronicastrum sibiricum AB462761 JPN 48 Persoonia – Volume 24, 2010

P. balsaminae ex Impatiens balsamina AB462800 JPN P. balsaminae ex Impatiens balsamina AB462799 JPN P. balsaminae ex Impatiens balsamina AB462803 JPN ITS / MP tree P. xanthii ex Gynostemma pentaphylla D84378 JPN P. xanthii ex Zehneria japonica D84387 JPN 157 sequences P. xanthii ex Verbena x hybrida AB046985 USA P. fusca ex Abelmoschus ficulneus AB040293 JPN 499 characters P. phaseoli ex Vigna angularis AB040297 JPN 496 steps Podosphaera sp. ex Crotalaria juncea AB040304 JPN Podosphaera sp. ex Glycine max AB040305 JPN CI = 0.5464 P. fusca ex Helianthus annuus AB040312 JPN * P. xanthii ex Cucurbita maxima AB040315 JPN RI = 0.9299 P. xanthii ex Trichosanthes kirilowii AB040316 JPN P. xanthii ex Cucumis sativus AB026146 JPN RC = 0.5081 P. xanthii ex Cucumis sativus AB040324 JPN P. xanthii ex Cucumis sativus AB040323 JPN Podosphaera sp. ex Saintpaulia ionantha AB040338 JPN Podosphaera sp. ex Saintpaulia sp. AB040339 AUSTR Podosphaera sp. ex Lycopus lucidus AB040343 JPN P. fusca ex Zinnia elegans AB040354 JPN P. fusca ex Zinnia elegans AB040355 JPN P. euphorbiae-hirtae ex Acalypha australis AB040306 JPN Podosphaera sp. ex mutabilis AB040308 JPN Podosphaera sp. ex Solanum melongena AB040333 JPN Podosphaera sp. ex Dunbaria villosa AB040334 JPN Podosphaera sp. ex Vigna unguiculata AB040340 JPN P. fusca ex Verononia sp. AB040348 JPN P. fusca ex Rudbeckia hirta AB040296 JPN P. fusca ex japonicus AB040307 JPN P. fusca ex Rudbeckia sp. AB040337 JPN P. fusca ex Coreopsis lanceolata AB046990 JPN P. fusca ex Cosmos bipinnatus AB040300 JPN * P. fusca ex Cosmos bipinnatus AB040302 JPN P. fusca ex Cosmos bipinnatus AB040303 JPN 1 change P. fusca ex Bidens frondosa AB040295 JPN P. fusca ex Carthamus tinctorius AB040298 JPN P. fusca ex Helianthus annuus AB040311 JPN P. fusca ex Helianthus x multiflorus AB040319 JPN P. fusca ex Cacalia hastata AB040314 JPN P. xanthii ex Euryops chrysanthemoides DQ205330 AUSTR P. fusca ex Coreopsis lanceolata EF442023 ITA P. balsaminae ex Impatiens textorii AB462801 JPN P. balsaminae ex Impatiens textorii AB462802 JPN P. fusca ex Euryops pectinatus AB046989 USA P. fusca ex Calendula officinalis AB040317 JPN P. balsaminae ex Impatiens textori AB040344 JPN P. fusca ex Farfugium japonicum AB040346 JPN P. fusca ex Syneilesis palmata AB040349 JPN Podosphaera sp. ex Peristrophe japonica AB026135 JPN P. fusca ex Calendula officinalis MUMH1933 ARG P. fusca ex Calendula officinalis MUMH2432 ARG P. balsaminae ex Impatiens noli-tangere AB462805 JPN P. balsaminae ex Impatiens noli-tangere AB462806 JPN * P. balsaminae ex Impatiens noli-tangere AB462807 JPN P. fusca ex Gerbera hybrida AB040309 JPN P. balsaminae ex Impatiens noli-tangere AB040318 JPN P. xanthii ex Physalis angulata EF050036 TAIWAN * P. fusca ex Physalis sp. AB040336 AUSTR Oidium sp. ex Verbena bonariensis AB462804 JPN Podosphaera sp. ex Cayratia japonica AB026151 JPN Podosphaera sp. ex Boehmeria nivea AB026139 JPN P. fusca ex Lactuca indica AB040294 JPN P. fusca ex Arctium lappa AB040310 JPN P. pseudofusca ex villosa AB040320 JPN P. fusca ex Tussilago farfara AB040345 JPN Podosphaera sp. ex Verbena x hybrida AB040347 JPN P. fusca ex Youngia denticulata AB040351 JPN P. fusca ex Lactuca raddeana AB040352 JPN P. xanthii ex Helianthus annuus EF010913 TAIWAN P. fusca ex Aster microcephalus AB040335 JPN P. fusca ex Aster tataricus AB040341 JPN * P. fusca ex Carpesium abrotanoides AB040350 JPN P. fusca ex Aster iinumae AB040353 JPN P. fusca ex Taraxacum officinale AB026148 JPN * P. fusca ex Taraxacum albidum AB040342 JPN P. fusca ex Matricaria matricarioides AB046988 USA P. fusca ex Taraxacum officinale AB046987 USA P. fusca ex Hypochaeris brasiliensis AY739113 BRA * P. intermedia ex Clerodendrum trichotomum AB026145 JPN P. elsholtziae ex Ajuga reptans AB026142 JPN P. fusca ex Conyza canadensis AB040313 JPN P. fusca ex nemorosum AB040332 LIT P. fusca ex Taraxacum officinale MUMH2440 ARG P. fusca ex Leontodon autumnalis AB040331 SWI

Appendix 2 Phylogeny of subsection Magnicellulatae of the genus Podosphaera inferred from nucleotide sequences of rDNA ITS region. This is a part of the phylogenetic tree of Podosphaera, but was not shown in Fig. 1. A bold line means that the node was supported by an MP bootstrap value of 70 % or higher. An asterisk means that the node was supported by a Bayesian posterior probability value of 0.95 or higher. Taxon name shown by bold type is the sequence determined in this study.