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Mycologia, 102(6), 2010, pp. 1369–1382. DOI: 10.3852/10-034 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897

ITS rDNA phylogeny of Iranian strains of Cytospora and associated teleomorphs Khalil-Berdi Fotouhifar Ghorban-Ali Hedjaroude

longevity of ornamental and forest shrubs and trees (Christensen 1940, Biggs 1989, Adams et al. 2006). In addition to causing disease some Cytospora species produce secondary metabolites that have antimicrobial and chemotherapeutic effects (Gurusiddaiah and Ronald 1981, Jayasuriya et al. 2003, Kokubun et al. 2003, Singh et al. 2007). Approximately 550 species of Cytospora have been described (Grove 1935; Gutner 1935; Gvritishvili 1982; Saccardo 1882–1931; Adams et al. 2002, 2005, 2006), but most of these species names are considered synonyms (Adams et al. 2006). Cytospora spp. are the anamorphs of the ascomycete genera Leucostoma (Nitschke) Ho¨hn., Valsa Fr., Valsella Fuckel and Valseutypella Ho¨hn. of family Valsaceae, Diaporthales, Ascomycota. Detailed historical overviews on the taxonomy of Cytospora and associated teleomorphs are given by Togashi (1930), Kobayashi (1970), Spielman (1980, 1985) and Adams et al. (2005). Von Ho¨hnel (1914, 1917, 1918, 1923) recognized different types of locule arrangements in Cytospora species and proposed several genera for the locule types. Recent monographs published by Spielman (1983, 1985) and Gvritishvili (1982) treated the anamorphic types recognized by von Ho¨hnel as subgenera or sections. Adams et al. (2005) abandoned the use of infrageneric ranks and proposed descriptive terms for locule arrangement types instead. Teleomorphic genera associated with Cytospora species are delimited by morphological characters that include number of ascospores per ascus, presence or absence of conceptacle and the dark parenchymatous fungal stroma around the perithecia. However there has been longstanding disagreement about the reliability of these characters for delimitation of the genera. Some specialists have supported distinctness of these genera (De´fago 1944; Urban 1957, 1958; Barr 1978, 1990), whereas others consider them as variations of species in genus Valsa (Petrak 1919, 1969; Hubbes 1960; Vasilyeva 1988, 1994; Adams et al. 2005, 2006). Vasilyeva (1988, 1994) mentioned that in the Diatrypaceae (Xylariales, Ascomycota) the presence or absence of the conceptacle is considered significant only at the species level and proposed the same treatment for members of the Valsaceae. Also Petrak (1919, 1969) and Mu¨ller and von Arx (1973) did not accept the idea that the polysporous nature of genus Valsella was sufficient to separate its species from those of genus Leucostoma. Gvritishvili (1982) viewed the conceptacle as a

Department of Plant Pathology, Faculty of Agricultural Science and Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj 31587-77871, Iran

Adrian Leuchtmann1 Plant Ecological Genetics, Institute of Integrative Biology (IBZ), ETH Zu ¨ rich, CH-8092 Zu ¨ rich, Switzerland

Abstract: Cytospora spp. and associated teleomorphic species (Ascomycota, Diaporthales, Valsaceae) are among the most common and widespread canker- and dieback-causing fungi on trees, shrubs and herbaceous plants worldwide. From specimens collected all over Iran a total of 114 isolates were morphologically identified, representing 20 Cytospora, one Leucostoma and five Valsa species from 38 plant species. Nine of the identified taxa were new records for Iran, and many new hosts were identified. The phylogenetic relationships of the Iranian strains, along with sequences of 13 reference strains from GenBank, were inferred from ITS1-5.8S-ITS2 nuclear rDNA sequences. Parsimony analysis established five distinct major clades and 12 subclades, which represented accepted species and genera. Some of these subclades corresponded to morphologically based taxonomic concepts of single Cytospora species, while others contained more than one morphospecies. Teleomorphic states were present in six subclades, and most clustered with the corresponding anamorphs. This suggests that morphological and phylogenetic species concepts overlap and that in most cases they are meaningful for correct species identification. Key words: Cytospora, ITS, Leucostoma, phylogeny, sequence analysis, Valsa INTRODUCTION

Species of Cytospora Ehrenb. and associated teleomorphs cause canker and dieback diseases of hardwood and coniferous trees and rarely in herbaceous plants, resulting in considerable economic losses worldwide. Diseases not only affect the cultivation of fruit trees but also seriously limit the productivity and Submitted 12 Feb 2010; accepted for publication 31 Mar 2010. 1 Corresponding author. E-mail: [email protected]

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MYCOLOGIA

character of subgeneric significance, and Vasilyeva (1994) placed the type species of Valseutypella into Valsa without subgeneric or section rank. Castlebury et al. (2002) and Rossman et al. (2007) reviewed the phylogeny of order Diaporthales including Valsaceae and suggested that neither the conceptacle nor polysporous asci unite taxa into related teleomorphic species. Adams et al. (2005, 2006) treated all species of these four teleomorphic genera within genus Valsa based on both morphological studies and the results of the DNA sequence analyses. Accounts of Iranian Cytospora and related teleomorphic species were made first by Fragoso (1918) describing C. silenes Gonz. Frag. on Silene boryi Boissier. Petrak and Esfandiari (1941), Esfandiari (1946, 1948), Petrak (1949), Steyaert (1953) and Scharif and Ershad (1966) also reported on many other Cytospora species occurring on native and nonnative plants in Iran. Ershad (1995) listed 17 species of Cytospora in the second edition of Fungi of Iran, including C. teheranica Esf. on Ligustrum vulgare L. that was described as a new species by Esfandiari (1951). In addition Ashkan and Hedjaroude (1981) described a new variety C. cincta Sacc. var. flavocirris Ashkan on Armeniaca vulgaris Lam. from Iran based on the color of the conidial tendrils. Despite the common occurrence of anamorphic states in Iran, teleomorphs of these species were found rarely in this area. In recent times only three teleomorphic species were reported from Iran (Ashkan 1994, Ashkan and Hedjaroude 1981); these are Leucostoma persoonii (Nitschke) Ho¨hn. (anamorph C. leucostoma [Pers.] Sacc.) on several fruit trees, L. cinctum (Fr.) Ho¨hn. (anamorph C. cincta Sacc.; synonym C. rubescens Fr.) on Malus spp. and Valsa iranica Ashkan & Hedjaroude on Juglans regia L. Ashkan and Hedjaroude (1981, 1982) studied the taxonomy of five Cytospora and one Valsa species on fruit trees on the basis of morphological and pathological characteristics. They also reported on new records of C. juglandina Sacc. and C. juglandicola Ellis & Barth. on Juglans regia and of C. rubescens on Malus pumila Mill. from Iran. Ashkan (1997) found C. translucens Sacc. a new pathogen of the willow Salix zygostemon Boiss. in Karaj region of Tehran province. Razzaz-Hashemi and Zakeri (2000) reported C. fuckelii Sacc. on hazelnut from Qazvin province and Taher-Khani et al. (2004) C. sacchari E.J. Butler, the causal agent of sheath rot disease of sugarcane, on Saccharum officinarum L. from southwestern Iran (Khuzestan province). Ahmadi and Banihashemi (2006) provided new records of C. ocellata Fuckel, C. platani Fuckel and C. atra (Bonord.) Sacc. on hazelnut, plane tree and mulberry respectively from Fars province of Iran. Jamali and

Banihashemi (2008) isolated C. therryana Thu¨m. from diseased plane trees in Shiraz. Finally in their studies on Iranian Cytospora species Fotouhifar et al. (2007, 2008) gave accounts on nine species that were new for the mycobiota of Iran based on morphological investigations, including C. atrocirrhata Gvrit., C. carbonacea Fr., C. gutnerae Gvrit., C. intermedia Sacc., C. kantschavelii Gvrit., C. leucosperma (Pers.) Fr., C. nivea (Hoffm.) Sacc., C. ribis Ehrenb. and C. rosarum Grev., as well as some other previously reported species. Species delimitation in Cytospora and associated teleomorphs traditionally has been difficult due to plasticity of morphological characteristics (Gvritishvili 1982, Adams et al. 2006). Also the study of relationships among species and their correct taxonomic placement remain challenging based on available features. Additional informative morphological characters are needed to establish a stable morphological species concept (MSC). On the other hand a phylogenetic species concept (PSC) could be used, for which DNA sequences that allow statistical testing of relationships among isolates and species are required (Adams and Taylor 1993, Harrington and Rizzo 1999, Adams et al. 2002). The objectives of the present study were (i) to identify Cytospora and associated teleomorphic species from Iran, (ii) investigate the phylogenetic relationships of these species based on sequences of the internal transcribed spacers and 5.8S gene (ITS15.8S-ITS2) of the nuclear ribosomal DNA (rDNA) and (iii) test whether morphological species correspond to phylogenetically distinct clades. In this study we obtained new sequences of 114 isolates representing 20 Cytospora, one Leucostoma and five Valsa species from 38 host species. MATERIALS AND METHODS

Material examined.—Isolates included in this study were obtained from various substrates of diseased and dead plants collected by the first author in Iran, 2003–2005. From most collections single-spore isolates were made with the technique of Adams et al. (2006). Fungi of a few collections did not grow on common culture media and spore mass from the host plant had to be used for DNA extraction. All studied specimens are kept in the herbarium of Department of Plant Pathology, University of Tehran, Karaj, Iran, and will be deposited in KAR when the study is finished. In addition a representative number of isolates will be sent to the CBS culture collection in Utrecht, the Netherlands. Morphological identification of species.—In most cases, due to the absence of sexual states, species were identified on naturally infected host substrates based on morphological features of conidiomata, including presence or absence of conceptacle, disks and ostiole(s), arrangement of locules,

FOTOUHIFAR ET AL.: PHYLOGENY OF IRANIAN CYTOSPORA and morphology of conidiophores and conidia with the descriptions provided by Grove (1935), Gutner (1935), Gvritishvili (1982) and others. When ascomata were present species were identified based on the morphology of perithecia, asci and ascospores in addition to the features mentioned above with descriptions provided by Togashi (1930), De´fago (1944), Gilman et al. (1957), Urban (1957), Kobayashi (1970), Spielman (1985), Hayova and Minter (1998a–h) and Sivanesan (1983). Identities of corresponding anamorphic and teleomorphic species were confirmed based on ITS sequence homology or if available to selected reference sequences from GenBank (National Center for Biotechnology Information, Bethesda, Maryland). DNA extraction.—Genomic DNA was extracted from mycelium of pure cultures. Isolates were grown in 125 mL flasks containing 25 mL V8 juice (Campbell Soup Co., USA) medium (Leuchtmann 1994) on a rotary shaker at 120 rpm 7–10 d at room temperature. Mycelia then were harvested and washed by vacuum filtration on No. 1 Whatman filter paper (Whatman International Ltd., Maidstone, England), lyophilized and stored at 220 C. DNA was extracted from lyophilized mycelium by the cetyltrimethyl-ammonium bromide (CTAB) method of Doyle and Doyle (1990) with some modifications. Approximately 25 mg lyophilized mycelium was placed in 2 mL tubes containing one glass bead (Merck, Darmstadt, Germany) and ground in a Retsch MM2000 mill 3–5 min at maximum speed. A total of 400 mL CTAB extraction buffer (1% v/w CTAB, 50 mM Tris-HCl [pH 8], 250 mM EDTA, 1% 2-mercaptoethanol, 0.7 M NaCl) was added and mixed well. The tube was incubated at 65 C for 30 min and cooled to room temperature. A total of 400 mL phenol : chloroform : isoamylalcohol (24 : 24 : 1) was added. The mixture was shaken 5 min and centrifuged at 20 800 g 7 min to remove solid materials. A total of 300 mL supernatant was transferred to 1.5 mL tube and purified twice with equal amount of chloroform:isoamylalcohol (24:1) and centrifugation at 20 800 g 7 min. A total of 200 mL supernatant was precipitated by 180 mL isopropanol and centrifugation at 20 800 g 20 min. DNA was washed with 70% ethanol and air dried in Eppendorf concentrator 5301 (Eppendorf, Hamburg, Germany) 5–10 min. Finally DNA was dissolved in 30 mL distilled water. For specimens that could not be isolated in pure culture, genomic DNA was extracted directly from spore mass produced on host material with the procedures described by Ferreira and Glass (1996) and Tendulkar et al. (2003). For the latter 5 mL supernatant containing genomic DNA was used as a template for PCR amplification. PCR amplification and purification.—The amplification of the ribosomal RNA 5.8S gene and two flanking regions, ITS1 and ITS2, was performed with primers ITS1 or ITS5 combined with ITS4 (White et al. 1990). PCR reactions were carried out in 25 mL final volumes containing approximately 1–50 ng total genomic DNA, 1 U GoTaqH Flexi DNA polymerase (Promega, Madison, Wisconsin), 1.5 mM MgCl2, 60 mM dNTP, 0.4 mM of each primers and 5 mL 53 GoTaqH FLEXI buffer. PCR amplifications were done in a GeneAmpH PCR system 9700 (Applied Biosystems, Foster City, California) with this program: an initial denaturation

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step at 94 C for 2 min, 35 cycles of 1 min at 94 C, 70 s at 50 C and 90 s at 72 C, followed by a final extension step at 72 C for 7 min. PCR products were purified with the GFXTM PCR DNA and gel band Purification Kit (Amersham Bioscience, England) following the manufacturer’s protocol and eluted with 30 mL distilled water. Sequencing.—PCR products of the ITS1-5.8S-ITS2 region were directly sequenced in one direction with ITS4 primer, except C. sacchari isolate 125, which was sequenced in both directions with ITS4 and ITS5 primers. Sequencing reactions were carried out with ABI PRISMH BigDyeHTerminator 3.1 Cycle Sequencing Kit (PE Applied Biosystems, Foster City, California). Reactions were performed in 10 mL final volume containing 0.8 mL BigDyeH, 1.5 mL BigDyeH sequencing buffer at 53 concentration, 1.5 mL primer at 1 mM concentration and 6.2 mL purified PCR product. The sequencing reactions were run on the same cycler as the PCR reactions with this program: an initial denaturation step at 94 C for 2 min, 60 cycles of 10 s at 94 C, 5 s at 50 C and 3 min at 60 C. Sequencing PCR products were cleaned with G50 fine DNA grade Sephadex columns (Amersham Bioscience, England), following the manufacturer’s protocol and separated and analyzed on ABI PRISMH 3130xl Genetic Analyzer (Applied Biosystems, Foster City, California). ITS1-5.8S-ITS2 rDNA sequences were edited manually with Sequencher 4.6 software (Gene Codes Corp., Ann Arbor, Michigan). Only high quality sequences allowing for unambiguous reading of chromatograms were used. The sequences of C. sacchari isolate 125 were aligned after editing, and the consensus sequence determined. Sequences of all isolates were aligned with Clustal X 1.83 (Thompson et al. 1997) and the alignment adjusted by eye in MacClade 4.08 (Maddison and Maddison 2005) where necessary. All sequences of Cytospora spp. and associated teleomorphs from Iran obtained in this study have been deposited in GenBank under accession numbers beginning with EF447305 (TABLE I). Phylogenetic analyses.— They were performed on the aligned ITS sequences of the 114 Iranian isolates to which were added 13 reference sequences of the homologous regions from species of Cytospora, Leucostoma and Valsa obtained from GenBank. In addition ITS sequences of Diaporthe vaccinii Shear and Phomopsis vaccinii Shear, N.E. Stevens & H.F. Bain were included as outgroup taxa (Adams et al. 2002). Details on origin of the examined isolates from Iran and the GenBank accessions are provided (TABLE I). Maximum parsimony (MP) analysis was performed by heuristic search with PAUP* 4.0b10 (Swofford 2003) on Mac OS 9.2. Character changes were unweighted and unordered with gaps treated as missing data. Trees were built by 100 iterations of random stepwise taxon addition with tree bisection reconnection (TBR) branch swapping. Confidence of individual clades were assessed by MP bootstrap analyses (Felsenstein 1985) with 1000 heuristic replicates (Hedges 1992). Bayesian posterior probabilities of nodes were calculated with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the GTR + I + G model selected by AIC in Modeltest 3.6 (Posada and Crandall 1998). Searches were run 1 000 000

1372 TABLE I.

MYCOLOGIA Examined isolates from Iran with GenBank accession numbers of ITS sequences and reference sequences used Taxon

Cytospora atrocirrhata Gvrit. Cytospora aurora Mont. & Fr. Cytospora carbonacea Fr. Cytospora chrysosperma (Pers.) Fr.

Cytospora cincta Sacc.

Isolate number 22 81 50 174 102

Host Salix excelsa S.G.Gmelin Populus nigra L. Salix aegyptiaca L. Ulmus minor Mill. Populus alba L.

103 109 184 200 203 215 225 226 268 282 293 300 304 305 308 316 321 331 332 334 335 150-1

Platanus orientalis L. Platanus orientalis Salix sp. Fraxinus excelsior L. Fraxinus excelsior Ligustrum latifolium Hook.F. Robinia pseudoacacia L. Armeniaca vulgaris Lam. Salix excelsa Crataegus azarolus L. Populus deltoides Marsh. Morus alba L. Malus pumila Mill. Tamarix sp. Populus nigra Populus nigra Platanus orientalis Olea sativa Lam. Ficus carica L. Salix excelsa Juglans regia L. Prunus domestica L.

216-1 233-1 279-1 284-1 314-1 317-1 54-1 58-1 69-1 69-2 19 36 37 96 134 136 147 155 190 191 248 281 329 154-1 156-1 209-1

Armeniaca vulgaris Malus pumila Juglans regia Thuja orientalis L. Populus alba Thuja orientalis Salix aegyptiaca Salix aegyptiaca Persica vulgaris Mill. Persica vulgaris Rosa sp. Amygdalus communis L. Amygdalus communis Armeniaca vulgaris Malus pumila Malus pumila Malus pumila Juglans regia Malus pumila Cerasus avium (L.) Moench Cydonia oblonga Mill. Prunus domestica Crataegus azarolus Malus pumila Malus pumila Vitis vinifera L.

Geographic origina Ardabil Zanjan Western Azarbaijan Fars Chaharmahaal and Bakhtiari Tehran Tehran Fras Markazi Markazi Lorestan Lorestan Lorestan Kurdistan Kurdistan Hamadan Hamadan Hamadan Qazvin Semnan Razavi Khorasan Razavi Khorasan Mazandaran Fras Tehran Tehran Kohkiluyeh and Buyer Ahmad Lorestan Lorestan Kurdistan Kurdistan Razavi Khorasan Razavi Khorasan Western Azarbaijan Western Azarbaijan Western Azarbaijan Western Azarbaijan Ardabil Eastern Azarbaijan Eastern Azarbaijan Markazi Isfahan Isfahan Fars Fars Fars Fars Kermanshah Kurdistan Semnan Fras Fars Markazi

Year

GenBank number

2004 2004 2004 2005 2004

EF447305 EF447306 EF447307 EF447308 EF447313

2004 2004 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2003 2005 2005

EF447314 EF447315 EF447317 EF447318 EF447319 EF447320 EF447322 EF447323 EF447325 EF447327 EF447329 EF447330 EF447331 EF447332 EF447333 EF447335 EF447337 EF447338 EF447339 EF447340 EF447341 EF447316

2005 2005 2005 2005 2005 2005 2004 2004 2004 2004 2004 2004 2004 2004 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005

EF447321 EF447324 EF447326 EF447328 EF447334 EF447336 EF447309 EF447310 EF447311 EF447312 EF447342 EF447343 EF447344 EF447345 EF447346 EF447347 EF447348 EF447350 EF447352 EF447353 EF447355 EF447359 EF447362 EF447349 EF447351 EF447354

FOTOUHIFAR ET AL.: PHYLOGENY OF IRANIAN CYTOSPORA TABLE I.

Continued Taxon

Cytospora fugax (Bull.) Fr.b Cytospora Cytospora Cytospora Cytospora

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gutnerae Gvrit. intermedia Sacc. kantschavelii Gvrit. leucosperma (Pers.) Fr.

Cytospora leucostoma (Pers.) Sacc.

Cytospora nivea (Hoffm.) Sacc. Cytospora pruinosa (Fr.) Sacc.

Cytospora ribis Ehrenb.

Cytospora rosarum Grev. Cytospora sacchari E.J. Butler Cytospora sacculus (Schwein.) Gvrit. Cytospora schulzeri Sacc. & P. Syd.

Cytospora terebinthi Bres. Cytospora translucens Sacc. Leucostoma cinctum (Fr.) Ho¨hn.

Valsa ceratosperma (Tode) Maire Valsa cypri (Tul.) Tul. & C. Tul.

Isolate number

Host

250-1 250-2 270-1 292-1 299-1 138-1 257-1 214 171 287-2 222 264 295 279-2 198 261 313 209-2 216-2 99

Cydonia oblonga Cydonia oblonga Cydonia oblonga Armeniaca vulgaris Armeniaca vulgaris Populus alba Salix excelsa Platanus orientalis Quercus brantii Lindl. Populus deltoides Platanus orientalis Amygdalus communis Robinia pseudoacacia Juglans regia Persica vulgaris Armeniaca vulgaris Rosa canina L. Vitis vinifera Armeniaca vulgaris Populus deltoides

239 271 163-1 201 327 330 214-1 284-2 218 125 246-1

Fraxinus excelsior Fraxinus excelsior Morus alba Elaeagnus angustifolia L. Elaeagnus angustifolia Lepidium latifolium L. Platanus orientalis Thuja orientalis Rosa canina Saccharum officinarum L. Pyrus communis L.

56 132 143 319 336 154-2 167-1

Malus pumila Malus pumila Colutea sp. Malus pumila Cerasus vulgaris Mill. Malus pumila Crataegus pseudoheterophylla Pojack Malus pumila Malus pumila Thuja orientalis Malus pumila Pistacia khinjuk Stocks Celtis australis L. Populus alba Malus pumila Cydonia oblonga Armeniaca vulgaris Armeniaca vulgaris Pyrus communis Populus deltoides Morus alba

233-2 256-1 317-2 57-1 227 35 138-2 156 250 292 299 246 287 163

Geographic origina

Year

GenBank number

Kermanshah Kermanshah Kurdistan Hamadan Hamadan Fars Kurdistan Lorestan Fars Hamadan Lorestan Kurdistan Hamadan Kurdistan Markazi Kurdistan Razavi Khorasan Markazi Lorestan Chaharmahaal and Bakhtiari Kermanshah Kurdistan Fars Markazi Razavi Khorasan Tehran Lorestan Kurdistan Lorestan Khuzestan Kermanshah

2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2004

EF447356 EF447357 EF447358 EF447360 EF447361d EF447363 EF447364 EF447365 EF447366 EF447367 EF447368 EF447369 EF447371 EF447370 EF447372 EF447375 EF447376 EF447373 EF447374 EF447377

2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005

EF447380 EF447381 EF447379 EF447382 EF447385 EF447386 EF447383 EF447384 EF447387 EF447388 EF447389

Western Azarbaijan Isfahan Fars Razavi Khorasan Tehran Fars Fars

2004 2005 2005 2005 2004 2005 2005

EF447390 EF447392 EF447393 EF447400 EF447401 EF447394 EF447395

Lorestan Kurdistan Razavi Khorasan Western Azarbaijan Lorestan Eastern Azarbaijan Fars Fars Kermanshah Hamadan Hamadan Kermanshah Hamadan Fars

2005 2005 2005 2004 2005 2004 2005 2005 2005 2005 2005 2005 2005 2005

EF447396 EF447397 EF447399 EF447391 EF447402 EF447403 EF447404 EF447405 EF447406 EF447407 EF447408 EF447409 EF447410 EF447411

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MYCOLOGIA Continued Isolate number

Taxon

57 167 212 256 257 54 58 279 314

Valsa malicola Z. Urb.

Valsa salicina (Pers.) Fr. Valsa sordida Nitschke

Reference sequencesc Cytospora carbonacea Cytospora rhodophila Sacc. Cytospora ribis Cytospora sacchari Diaporthe vaccinii Shear Leucostoma cinctum Leucostoma cinctum Leucostoma persoonii (Nitschke) Ho¨hn. Leucostoma persoonii Leucostoma translucens (de Not.) Ho¨hn. Phomopsis vaccinii Shear, N.E. Stevens & H.F. Bain Valsa cypri Valsa malicola Valsa salicina Valsa sordida

Geographic origina

Host

Year

GenBank number

2004 2005 2005 2005 2005 2004 2004 2005 2005

EF447412 EF447414 EF447415 EF447416 EF447417 EF447418 EF447419 EF447420 EF447421

Malus pumila Crataegus pseudoheterophylla Malus pumila Malus pumila Salix excelsa Salix aegyptiaca Salix aegyptiaca Juglans regia Populus alba

Western Azarbaijan Fars Lorestan Kurdistan Kurdistan Western Azarbaijan Western Azarbaijan Hamadan Razavi Khorasan

Ulmus campestris Rosa sp. Ribes rubrum Saccharum officinarum Vaccinium corymbosum Armeniaca vulgaris Prunus domestica Prunus persica

Germany Germany the Netherlands Taiwan Michigan, USA Michigan, USA Michigan, USA South Africa

DQ243805 DQ243809 DQ243810 DQ996044 AF191166 AF191169 AF191171 AF191180

Prunus persica Salix sp.

Michigan, USA Switzerland

AF191181 AF191182

Vaccinium macrocarpon

Wisconsin, USA

AF317579

Olea europaea Malus domestica Salix sp. Populus tremula

South Africa Michigan, USA Switzerland United Kingdom

DQ243790 DQ243802 AY347323 AY347322

a

Provinces of Iran or country of isolate origin. Taxa newly reported from Iran are indicated in boldface. c Sequences published by Adams et al. (2002, 2005, 2006), Singh et al. (2007) and Castlebury and Farr (direct submission). d Sequence identical to L. cinctum 299 and not included in phylograms of FIGS. 1 and 2. b

generations with sampling one out of every 100 trees. The first 2500 trees were discarded as burn-in, while the remaining trees were used to determine posterior probabilities of clades. The heating scheme used default settings in MrBayes. Neighbor joining analysis (Saitou and Nei 1987) also was applied to the same sequence alignment with PAUP*. The GTR model was used as distance model with proportions of invariable sites 5 0.5321, gamma distribution shape 5 0.4576 and the remaining parameters set to default. No sequence characters were excluded and break tie was treated as random initial seed. Bootstrap analysis of the NJ tree was performed on 1000 replicates. RESULTS

Identification of species.—In this study a total of 26 species in the genera Cytospora, Leucostoma and Valsa were identified from naturally infected specimens of host plants collected in Iran. Of these, nine taxa were new for the mycobiota of Iran (TABLE I, in boldface).

Identifications of the species are based on current morphological concepts of the published literature. They were verified by ITS sequence homology to trusted reference sequences from GenBank, if available (TABLE I). Phylogenetic analyses.—Analyses included a total of 126 ITS sequences from isolates of Iran and from GenBank deposited by other authors (TABLE I). The ITS sequences of Cytospora, Leucostoma and Valsa species were similar in length across the isolates studied, with 535–550 nucleotides except C. sacchari isolate 125, which had 576 nucleotides. The aligned dataset had 637 characters. Of these 491 characters were constant, 32 were uninformative and 114 were parsimony informative. No sequence characters were excluded. In maximum parsimony analysis of aligned sequences 1000 MPTs were saved, each of 345 steps with consistency index (CI) of 0.5623, retention index (RI) of 0.9148, rescaled consistency index (RC) of

FOTOUHIFAR ET AL.: PHYLOGENY OF IRANIAN CYTOSPORA 0.5144 and a homoplasy index (HI) of 0.4377. A strict consensus tree of the 1000 MPTs was constructed and its branches indicated by thickened lines on a phylogram of the MPT (FIG. 1). The MP tree revealed five major clades. Of these only two clades received bootstrap support greater than 50%. One included isolates of L. cinctum and its C. cincta anamorph and the other isolates of Valsa sordida Nitschke and its anamorph, C. chrysosperma (Pers.) Fries. Within major clades sequences were grouped into 12 subclades, eight of which were supported by moderate to high bootstrap values (clades 1, 2, 3, 4, 6, 10, 11 and 12). Nine clades (1, 2, 5, 6, 7, 8, 9, 11 and 12) consisted exclusively of members of the same anamorphic or/and teleomorphic taxa, while the remaining three clades included more than one morphospecies. The most basal branch in the tree was separated into two clades, clade 1 with two C. sacchari isolates and 2 with two C. atrocirrhata isolates, which respectively received bootstrap support of 100% and 68%. The second major clade comprised three subclades, clade 3 (100% bootstrap support) including C. nivea on Populus deltoides L. and C. cincta Sacc. on Malus pumila, clade 4 including L. persoonii and L. translucens (de Not.) Ho¨hn. and associated anamorphs, C. leucostoma and C. translucens Sacc. respectively, clade 5 including Valsa cypri (Tul.) Tul. & C. Tul. and its associated anamorph, C. pruinosa (Fr.) Sacc. isolates. The third major clade consisted of clade 6 (85% bootstrap support) including L. cinctum and its anamorph, C. cincta. In the fourth major clade a basal branch separated clade 7 consisting of a single isolate of C. kantschavelii. Clade 8 contained two V. salicina (Pers.) Fr. isolates and two associated anamorphic isolates of C. fugax (Bull.) Fr. obtained from Populus alba L. and Salix excelsa S.G. Gmelin. Tentative clade 9 consisted of V. malicola Z. Urb. and its anamorph, C. schulzeri Sacc. & P. Syd. isolates. Clade 10 (99% bootstrap support) appeared to be the most heterogeneous. It included C. carbonacea, C. gutnerae, C. intermedia, C. leucosperma, C. rhodophila Sacc., C. ribis, C. rosarum and C. terebinthi Bres. isolates. V. ceratosperma (Tode) Maire and its anamorphic state, C. sacculus (Schwein.) Gvrit isolates, clustered in clade 11 (100% bootstrap support). The fifth major clade composed of well resolved clade 12 (69% bootstrap support) was the largest clade consisting mainly of V. sordida and its anamorph, C. chrysosperma, with one isolate each of V. ceratosperma and C. aurora Mont. & Fr. Neighbor joining analysis of the same set of aligned sequences resulted in a single tree with four major clades arranged in different topology compared to the MP tree and with some of the subclades identified

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in the MP analysis grouping disparately (FIG. 2). The most striking difference was that clade 10 moved to the tree base, while clades 1–6 formed a major clade higher within the tree. Eight clades (1, 2, 3, 4, 6, 10, 11 and 12) were resolved with moderate to high bootstrap support (62–100%), the same as in the MP analysis. By contrast clades 5, 8 and 9 (as determined with MP analysis) remained unresolved with its isolates positioned at two branches within a major clade. DISCUSSION

Molecular approaches such as DNA sequencing have become indispensable to taxonomy and for species delimitation in many fungal groups. This particularly applies to taxa that have a limited number of, but often extremely variable, morphological features, such as species and genera of Valsaceae (Gvritishvili 1982; Spielman 1985; Adams et al. 2005, 2006). For this study we collected many specimens of Cytospora, Leucostoma and Valsa, representing 26 taxa occurring on native and non-native plants of Iran. Nine of these taxa are new records for the mycobiota of the country, and none has been studied with molecular techniques based on material from the country before this investigation. In addition sexual states of six Cytospora species representing six of the 12 clades were identified. Sexual reproduction of heterothallic Valsa/Cytospora species is rare in Iran and anamorphs are more commonly found in nature (Fotouhifar et al. 2007, 2008). Phylogenetic analyses based on nucleotide sequences of ITS1-5.8S-ITS2 region of ribosomal DNA grouped the Iranian strains of Cytospora, Leucostoma and Valsa into 12 distinguishable clades. We consider these clades, with the exception of clade 9, as phylogenetic lineages representing one or several related species. Relationships among clades however are not conclusively resolved based on our sequence data because bootstrap support of deeper branches often was low. We will use the descriptive terms proposed by Adams et al. (2005) to characterize conidiomatal and ascomatal types of Cytospora, Leucostoma and Valsa species in each detected clade. Clade 1 consists of C. sacchari represented by an isolate from southwestern Iran (Khuzestan province) and a reference isolate from Taiwan. The species is characterized by black multiloculate, convoluted cytosporoid conidiomata with a single, long ostiolar beak and without any distinguishable disk, that is immersed in stem and sheath tissues of infected sugarcane. Taher-Khani et al. (2004) first reported C. sacchari as causal agent of sheath rot disease of sugarcane, especially on commercial cultivars, from

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FIG. 1. One of 1000 equally most parsimonious trees based on aligned sequences of ITS1-5.8S-ITS2 region of 126 isolates of Cytospora, Leucostoma and Valsa generated from maximum parsimony analysis (MP) in PAUP*. Numbers separated by a slash (or below and above branches) represent MP bootstrap values greater than 50% from 1000 heuristic replicates and Bayesian posterior probabilities greater than 70%, respectively (2 designates a value lower than 50% or 70%). Thickened lines indicate branches that appeared in the strict consensus of the 1000 trees. Tree is rooted with outgroup taxa Diaporthe vaccinii and Phomopsis vaccinii.

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FIG. 2. Neighbor joining tree based on aligned sequences of ITS1-5.8S-ITS2 region of 126 isolates of Cytospora, Leucostoma and Valsa. Bootstrap values greater than 50% from 1000 replicates are indicated above branches. The tree is rooted with outgroup taxa Diaporthe vaccinii and Phomopsis vaccinii. Clade numbers correspond to clades identified in the MP tree (FIG. 1).

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the same area in Iran. The distinct phylogenetic position at the root of the phylogenetic tree also was noticed by Adams et al. (2006) and might be explained by the unusual host and arid geographic origin of this species. Clade 2 is represented by two isolates of C. atrocirrhata from Populus nigra L. and Salix excelsa collected from central and northwestern Iran. This species was described by Gvritishvili (1973a, 1982) to accommodate Cytospora isolates with well developed conceptacles, conidiomata with single undivided locule and black tendril of conidia, and was included in his subgenus and section Leucocytospora. The Iranian specimens of C. atrocirrhata, first reported by Fotouhifar et al. (2007), corresponded in most morphological characters with Gvritishvili’s description except for the conidiomata, which had only one big wrinkled locule. Clade 3 consists of two leucocytosporoid species, C. cincta and C. nivea on Malus pumila and Populus deltoides respectively, collected in southern Iran. Despite obvious differences in morphology and host preference, the species appear to be close genetically. However Adams et al. (2006) reported C. nivea on Malus domestica (Borkh.) Mansf. from South Africa and proposed that its teleomorph, Valsa nivea (Hoffm.) Fr., has moved from a Populus host to Malus in South Africa. Genetic similarity and the potential for host jumps might suggest conspecificity of the two taxa. Clade 4 included isolates of C. leucostoma and C. translucens from Iran with leucocytosporoid conidiomata and the associated teleomorphs L. persoonii and L. translucens with leucostomoid ascomata from South Africa, Switzerland and United States. We did not encounter teleomorphs of the two species in Iran. C. translucens was reported first from Iran by Ashkan (1997) as a pathogen of the willow Salix zygostemon in the Shahrestanak area of Karaj region of Tehran province. In this study the species was found on Celtis australis L. and Populus alba collected respectively from northwestern and southern Iran. L. persoonii and its anamorph C. leucostoma have been reported on stone and pome fruit trees by Esfandiari (1946), Scharif and Ershad (1966) and Ashkan and Hedjaroude (1981) from different parts of Iran. Here we found C. leucostoma also on Rosa canina L. and Vitis vinifera L., which are new hosts for this species. The grouping of C. translucens and C. leucostoma in a common clade is in agreement with Gvritishvili (1982) who synonymized the two species. Clade 5 accommodates isolates of V. cypri and its anamorph C. pruinosa with cyprious ascomata and cytophomoid conidiomata respectively that often occur mixed on infected plant tissues. Our isolates

were identified on Fraxinus excelsior L. and Morus alba L. collected in western and southern Iran, while Gvritishvili (1982) and Hayova and Minter (1998e) reported the species on Fraxinus, Ligustrum, Olea, Syringa (Oleaceae) and Celtis (Celtidaceae) and in the teleomorphic state only on Broussonetia (Moraceae) worldwide. V. cypri/C. pruinosa are new for the mycoflora of Iran, and Morus alba is a new host. Clustering of clade 5 with clades 3 and 4 is interesting because clade 5 species do not share the common character of having a dark conceptacle around the ascomata or conidiomata. Clade 6 accommodates all isolates of L. cinctum and its anamorph C. cincta with leucostomoid ascomata or leucocytosporoid conidiomata. Among the anamorphic specimens the conceptacle was variable, ranging from absent to forming a pale brown or well developed black layer around the conidiomata, while a conceptacle was always present around the teleomorphic ascomata. This species usually infects members of family Rosaceae (Hayova and Minter 1998a), but Gvritishvili (1982), using the synonym C. rubescens Fr. for the anamorph, listed many host genera of other plant families. We found the anamorph on two additional new host plants, Juglans regia and Vitis vinifera. L. cinctum (misspelled as L. cincta) was reported first from Iran by Ashkan (1994) as causal agent of perennial canker of apple trees in Tehran province. The species was reported from stone and pome fruit trees in various parts of Iran (Ashkan and Hedjaroude 1981, 1993). Clade 7 is represented only by one isolate of C. kantschavelii from Populus deltoides collected in western Iran. Gvritishvili (1973b) described this species for Cytospora on Populus nigra, having small cytosporoid conidiomata with two-layered endostromatic walls and cinnamon-brown or black surface layer composed of large paraplectenchymatous cells up to 8 mm diam. C. kantschavelii has been reported from Iran by Fotouhifar et al. (2008) on the new host Populus deltoides. Considering this species as its own phylogenetic lineage is supported by the distinct morphology. Isolates of V. salicina and its anamorph C. fugax with euvalsoid ascomata and cytosporoid conidiomata are placed in clade 8. They occur mainly on Salix spp. worldwide, but Urban (1958) and Hayova and Minter (1998g) listed also Alnus glutinosa (L.) Gaertn., Carpinus betulus L., Ligustrum vulgare, Populus nigra, Rosa canina and Ulmus carpinifolia Borkh. as hosts. V. salicina and C. fugax are reported here for the first time from Iran. The anamorph was detected on Populus alba and Salix excelsa from western and southern Iran, and the teleomorphic state on S. excelsa from western Iran.

FOTOUHIFAR ET AL.: PHYLOGENY OF IRANIAN CYTOSPORA Clade 9 consists of isolates of V. malicola with euvalsoid ascomata and isolates of its anamorph C. schulzeri with well developed cytosporoid conidiomata. This clade is not well resolved and overlaps clade 11 based on sequences of the ITS regions. V. malicola and C. schulzeri are new for the mycoflora of Iran and were found on the new hosts, Cerasus vulgaris Mill. and Colutea sp., and on species of Malus, Crataegus and Thuja collected from all over Iran. These taxa predominantly infect members of Rosaceae (mainly apple trees) throughout the world (Hayova and Minter 1998f), and additional hosts are listed by Gvritishvili (1982). Eight closely related species of Cytospora are grouped in well resolved clade 10. They were isolated from 10 host species from all over Iran, except C. rhodophila, which originated from Germany and was added as reference sequence from GenBank. C. carbonacea, C. gutnerae, C. intermedia, C. leucosperma, C. ribis and C. rosarum first were reported from Iran by Fotouhifar et al. (2007, 2008) including a new host Pistacia khinjuk Stocks for C. terebinthi. C. terebinthi had been reported also on P. mutica Fisch. et Mey. and P. vera L. from Iran (Esfandiari 1948, Scharif and Ershad 1966). The species of this clade are morphologically similar and share characteristics of cytosporoid conidiomata with 1–4 ostioles and large allantoid conidia forming yellowish tendrils or droplets. In his monograph Gvritishvili (1982) considered C. intermedia and C. rosarum synonyms of C. leucosperma, which is consistent with our findings. Clade 11 consists of isolates of V. ceratosperma and its anamorph C. sacculus that originated from a specimen of Pyrus communis L. collected in western Iran. A second isolate of V. ceratosperma from Populus deltoides collected in the same part of Iran was placed in a different clade (12), suggesting that this species is genetically diverse, although all isolates are morphologically similar with euvalsoid ascomata and torsellioid conidiomata. V. ceratosperma/C. sacculus is reported here for the first time from Iran. The species occurs on dead or dying twigs and branches of many angiosperms throughout the world (Gvritishvili 1982, Hayova and Minter 1998d). A similar phylogenetic diversity among V. ceratosperma isolates was observed by Adams et al. (2005) who proposed to narrow the scope of this species to one distinct phylogenetic lineage that is in agreement with the morphological features of the original description. Clade 11 in our study could serve as such a monophyletic lineage representing the typical morphological characteristics of the species. But clade 11 does not correspond to the V. ceratosperma s. str. clade of Adams et al. (2005) and appears to be genetically close to V. ceratosperma isolate AF192324.

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Clade 12 includes all isolates of V. sordida with euvalsoid ascomata and its anamorphic state, C. chrysosperma, with cytosporoid conidiomata, plus one isolate of C. aurora, which has been synonymized with C. chrysosperma by Gvritishvili (1982), and the disparate isolate of V. ceratosperma mentioned above (clade 11). The teleomorphic state, V. sordida, was found on Salix aegyptiaca L., Populus alba and Juglans regia and is reported here for the first time from Iran. Its anamorph, C. chrysosperma, had been reported repeatedly from Iran on Populus sp., P. alba and P. nigra (Petrak and Esfandiari 1941, Esfandiari 1946, Steyaert 1953, Viennot-Bourgin 1958, Eskandari 1964, Scharif and Ershad 1966). The species usually occurs on Salicaceae and more rarely on other woody angiosperms throughout the world (Gvritishvili 1982, Hayova and Minter 1998h). Genera Crataegus, Ficus, Fraxinus, Ligustrum, Olea, Persica, Platanus, Prunus, Robinia, Tamarix and Thuja are reported as new hosts for C. chrysosperma in this study. Despite the wide host range and distant geographic origins within Iran, isolates of V. sordida/C. chrysosperma appear to be genetically uniform compared to other species of Valsa and Cytospora, suggesting more recent speciation and host range expansion. Overall clustering of Cytospora, Leucostoma and Valsa isolates into 12 distinguishable clades based on sequence data is mostly consistent with morphological groups based on locule type and other morphological characteristics. In most cases clades correspond to single species (clades 1, 2, 5, 6, 7, 8, 11 and 12), confirming morphological species recognition (MSR) criteria suggested by Gvritishvili (1982) and others. Species of Cytospora and associated teleomorphs cause annual or perennial cankers in wild and domesticated fruit trees and other woody or rarely herbacous plants. Infected plants usually are weakened, stressed or injured and may show symptoms throughout the growing season (Agrios 2005). In the present study many strains of different species were isolated from infected twigs or branches of even vigorously growing plants, suggesting that pathogenicity of strains of a species might vary considerably on different hosts or in different environments. Conversely under the same environmental conditions and on the same host strains of a species obviously can vary in virulence, which might reflect genetic diversity among strains. Thus inoculation tests using different hosts should be performed to better understand pathogenicity of certain strains and to evaluate the probability of host jumps. Conclusions.—This study presents a preliminary assessment of the phylogenetic relationships among species of Cytospora, Leucostoma and Valsa that were

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identified on host plants collected in Iran. In addition it provides new information on the geographic distribution and host preferences of these species in Iran. Species and genera of Valsaceae are difficult to define morphologically because only a limited set of characters is available and these are often variable even within taxa. Nucleotide sequences of the ITS1-5.8SITS2 region might help delimit more precisely the different taxa. Moreover sequences represent an important data resource that can be used as reference to compare and identify unknown isolates of this important group of fungi. The morphological species concept may need to be reviewed and adjusted so that it matches biological or phylogenetic concepts. Future studies should include sequences of ITS regions and other genes from two additional teleomorphic genera, Valsella and Valseutypella. A phylogenetic account of the entire group will serve as the basis for determining morphological characters that allow reliable identification of genera and species of Valsaceae. ACKNOWLEDGMENTS

We are grateful for the help of Edith Lang, Hans-Heini Vogel and Karsten Rohweder who provided technical assistance, Alex Kocyan who provided advice on data analyses, University of Tehran, Iran, which provided financial support, and Swiss Federal Institute of Technology (ETH Zurich), which hosted Kh.-B. Fotouhifar and provided technical support. We also are indebted to the curator of the herbarium ETH (Z + ZT), Reinhard Berndt, who provided helpful assistance.

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