Phylogenetic structure of the genus Leveillula (Erysiphales: Erysiphaceae) inferred from the nucleotide sequences of the rDNA ITS region with special reference to the L. taurica species complex

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Mycol. Res. 105 (8) : 909–918 (August 2001). Printed in the United Kingdom.

Phylogenetic structure of the genus Leveillula (Erysiphales : Erysiphaceae) inferred from the nucleotide sequences of the rDNA ITS region with special reference to the L. taurica species complex

Seyed Akbar KHODAPARAST1, Susumu TAKAMATSU2* and Ghorban-Ali HEDJAROUDE3 " Department of Plant Protection, College of Agriculture, Gilan University, Rasht, Iran. # Laboratory of Plant Pathology, Faculty of Bioresources, Mie University, Tsu 514-8507, Japan. $ Department of Plant Pathology, College of Agriculture, Tehran University, Karadj, 31585, Iran. E-mail : takamatu!bio.mie-u.ac.jp Received 19 September 2001 ; accepted 13 March 2001.

The nucleotide sequences of the nuclear ribosomal DNA, including ITS1, ITS2 and the 5n8S rDNA, were determined for 54 specimens representing 13 Leveillula species on 50 different host plant species. The maximum nucleotide sequence diversity among taxa in ITS1-5n8S-ITS2 was 7n1 %. Phylogenetic analyses revealed that Leveillula species formed six clades and three basal taxa. The taxonomic positions of several species that are well characterized by morphology of conidia, especially primary conidia, are supported by the present molecular analyses. This result shows that the morphology of primary conidia mostly provides a good criterion to identify Leveillula species. Twenty-six collections of Leveillula taurica recovered from 14 different plant families formed a distinct clade with L. chrozophorae, L. duriaei and L. elaeagni, and showed high homology in the ITS regions. Eight isolates of L. taurica recovered from the Asteraceae, Balsaminaceae, Fabaceae and Campanulaceae showed high sequence diversity, in contrast with other L. taurica specimens, and clustered separately or with other taxa. The result of Kishino-Hasegawa and Templeton tests demonstrated that the possibility of monophyly of L. taurica s. lat. could be significantly rejected, indicating that L. taurica s. lat. is a species complex composed of several biological species.

INTRODUCTION Of the 14 genera of Erysiphaceae (Braun 1987, 1999, Braun & Takamatsu 2000), 11 genera are ectoparasitic on plants, whereas only three genera, Leveillula, Phyllactinia and Pleochaeta, are endoparasitic. The three endophytic genera form a distinct clade, which suggests that the endophytic nature is a synapomorphy of these genera that occurred as an adaptation to xerophytic conditions (Mori, Sato & Takamatsu 2000). Of these genera, Phyllactinia and Pleochaeta are strictly confined to woody plants, whereas Leveillula mostly infects herbaceous plants. Leveillula is mainly distributed in arid and warmer areas, from the Mediterranean to Central Asia, and is considered to have expanded its distribution from these areas to the world (Amano 1986). The first important taxonomic treatment of powdery mildews belonging to Leveillula dates back to Salmon (1900), the first monographer of the powdery mildew fungi. Salmon (1900) referred the whole complex of taxa to a single species, viz. Erysiphe taurica. In 1906, he discovered the endophytic habit of this fungus, and pointed out relationships between the anamorphic genus Oidiopsis and E. taurica. Arnaud (1921) confirmed Salmon’s observations and introduced the new generic name Leveillula for this species. According to Durrieu * Corresponding author.

& Rostam (1984), Braun (1987, 1995) and Palti (1988) Leveillula comprises about 20 species. Golovin (1956) proposed a new species concept with one species for collections on hosts of each host family. However, his taxonomic system, based on the assumption that each host plant family has its own Leveillula species, has not been generally accepted. Most of his ‘ new species ’ are not validly published but they have also been rejected because the taxa are morphologically indistinguishable. The genus Leveillula is morphologically very uniform, especially with regard to the characters of the teleomorphs. Most taxa are hardly distinguishable from each other by ascoma characters, such as ascoma size, appendages, and shape of asci and ascospores. Almost all taxonomic treatments have been mainly based on L. taurica s. lat. which is agriculturally the most important species. This species has been reported on hosts belonging to more than 50 plant families that are distantly related to each other (Braun 1987, Palti 1988). Leveillula taurica is considered a complex species that urgently needs further investigation. During the past two decades, several taxonomists have debated this problem, and some attempts have been made to split L. taurica s. lat. into more natural units (Geljuta 1979, Braun 1980, Durrieu & Rostam 1984, Simonjan 1985, Geljuta & Simonjan 1987, 1988, Simonjan & Geljuta 1987, 1989) mainly based on examinations of conidial surfaces by scanning electron

Phylogenetics of Leveillula microscopy (SEM), morphology of conidia (especially primary conidia), and some host infectivity tests. Following these studies, new taxa such as L. chrozophorae, L. contractirostris, L. duriaei, L. elaeagni, L. lactucarum and L. picridis have been proposed (Braun 1980, Durrieu & Rostam 1984, Simonjan & Geljuta 1987, Geljuta & Simonjan 1988), but many problems are not yet solved. Comprehensive investigations and new approaches to solve taxonomic problems of Leveillula spp. are necessary. The rDNA diversity has recently been used for phylogenetic analysis of several powdery mildew fungi (Saenz, Taylor & Gargas 1994, Hirata & Takamatsu 1996, Takamatsu, Hirata & Sato 1998, Takamatsu et al. 1999, Saenz & Taylor 1999, Mori et al. 2000). According to these studies, the nucleotide sequences of the 5n8S rDNA region were highly conserved but the ITS regions were variable and useful for phylogenetic studies of closely related genera, species and intraspecies (Hirata & Takamatsu 1996, Takamatsu et al. 1998, Saenz & Taylor 1999). In the present study, we have determined the nucleotide sequences of about 600 nucleotides of the ITS regions including the 5n8S rDNA, and identified the nucleotide sequence diversity of the nuclear rDNA gene for 54 specimens of Leveillula, including 34 specimens of L. taurica s. lat., on hosts from 21 host plant families. The main purposes of this study are : (1) to determine if L. taurica isolates from different host plant families are genetically uniform ; (2) to reconstruct phylogenetic relationships among Leveillula species ; and (3) to prove the current species concept in the genus. This is the first comprehensive study of the molecular phylogeny of the genus Leveillula. MATERIALS AND METHODS Sample sources Fungi used in this study, their original hosts, localities, and accession numbers of the nucleotide sequence databases (DDBJ, EMBL, and GenBank) are given in Table 1. Fifty-four isolates were studied, with two outgroup species from the genus Phyllactinia, and, among these isolates, 34 belonged to Leveillula taurica. L. taurica was collected on 33 plant species belonging to 17 families. Species were identified by morphological characters based on the monographs of Braun (1987, 1995) and some additional papers (Durrieu & Rostam 1984, Khodaparast, Braun & Hamzeh Zarghani 2000). DNA extraction and PCR amplification The total DNA has been isolated from dried and herbarium specimens by either 20–30 ascomata or mycelia by the chelex method (Walsh, Metzger & Higuchi 1991, Hirata & Takamatsu 1996). The age of the samples ranged from six months to 60 yr, but most were about 10 yr old. Best results were generally obtained in the specimens collected after 1990 and stored in good conditions. In old specimens, DNA bands were usually not found in the first PCR. Thus, the first PCR product was reamplified by using a nested primer set. A region spanning ITS1, 5n8S, and ITS2 of the rDNA was amplified. A total of 50 µl PCR reaction mixture consisted of 5 µl of 10X

910 PCR buffer (0n5 M KCl, 0n1 M Tris-HCl, pH 8n3, 0n015 M MgCl ), 4 µl of 10X dNTPs (2n5 mM each), 1 µl each of two # primers (20 pmol\µl each), one unit of Taq DNA polymerase (Takara), 10 µl of extracted DNA solution and sterile water to bring the volume to 50 µl. For the first PCR, regions spanning the 3h-end of the 18S rDNA, ITS1-5n8S-ITS2 and the 5h-end of the 28S rDNA were amplified using the primers ITS5 and P3 (Hirata & Takamatsu 1996). For the amplification, an initial denaturing of 95 mC for 1n5 min was followed by 30 PCR cycles consisting of 95 m for 30 s, 52 m for 30 s, 72 m for 30 s, followed by one extension period of 72 m for 6n5 min. The second PCR was conducted by using 1 µl of the first amplification mixture and the nested primer set ITS1 and P3 (Hirata & Takamatsu 1996) with the same component and thermal cycles as for the first PCR. Gel electrophoresis of the second amplification product was performed using 1n5 % agarose gel including 0n5 µg ml−" ethidium bromide in 1X TAE buffer. The DNA band was excised from the gel and purified using a JETSORB kit (Genomed) following the manufacturer’s protocol. DNA sequencing 5 µl of purified DNA was diluted in 45 µl sterile water to determine the concentration of DNA using DU 530 Life science spectrophotometer (Beckman). It was necessary to optimize the concentration of DNA for cycle sequencing. DNA was cycle sequenced using PRISM Dye Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems) following the manufacturer’s protocol. Sequence reaction products were precipitated using 74 µl 70 % ethanol. Four primers [ITS1, T4 ,T3 (Hirata & Takamatsu 1996) and ITS4 (White et al. 1990)] were used for the sequencing in both directions using direct sequencing in an Applied Biosystem 373A DNA sequencer. Data analysis The obtained sequences were initially inspected manually and visually and aligned using the Clustal V package (Higgins, Bleaby & Fuchs 1992). The alignment was then refined with a word processing program to remove unalignable regions from the analysis. The data were analysed using the distance, parsimony and maximum likelihood methods. For distance and parsimony methods, PAUP* version 4n0b4a (Swofford 2000) was used. In the distance method, a neighbour-joining (NJ) tree was obtained using Kimura’s two-parameter distances (Kimura 1980). The most parsimonious (MP) trees were found with a heuristic search. All nucleotide substitutions were equally weighted and unordered. Alignment gaps were treated as missing information. For maximum likelihood (ML) analysis, we used the computer program package MOLPHY version 2n3 (Adachi & Hasegawa 1996). The strength of the internal branches from the resulting trees was statistically tested by bootstrap analysis from 1000 replications (Felsenstein 1985) and decay analysis (Bremer 1988, Donoghue et al. 1992). We constructed constraint trees from the data matrix using MacClade (Maddison & Maddison 1992) and PAUP* according to various possible hypotheses. The best

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Table 1. Sources of fungal materials and sequence database accession numbers Original fungal name

Host plant

Collector and Location

Voucher Materiala

GenBank Accession no.b

Leveillula chrozophorae L. chrozophorae L. cylindrospora L. cylindrospora L. cylindrospora L. duriaei L. duriaei L. elaeagni L. elaeagni L. lanuginosa L. lanuginosa L. lanuginosa L. loranthi L. picridis L. rubiae L. saxaouli L. simonianii L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. taurica L. verbasci Leveillula sp. Leveillula sp.

Chrozophora tinctoria (Euphorbiaceae) C. tinctoria (Euphorbiaceae) Anthochlamys polygoides (Chenopodiaceae) Noaea mucronata (Chenopodiaceae) Salsola kali (Chenopodiaceae) Salvia nemorosa (Lamiaceae) S. nemorosa (Lamiaceae) Elaeagnus angustifolia (Elaeagnaceae) E. orientalis (Elaeagnaceae) Daucus carrota (Apiaceae) Echinophora sibthorpiana (Apiaceae) Heracleum persicum (Apiaceae) Loranthus europeus (Loranthaceae) Picris stigosa (Asteraceae) Rubia tinctorum (Rubiaceae) Haloxylon sp. (Chenopodiaceae) Thevenotia persica (Asteraceae) Artemisia annua (Asteraceae) Helianthus annuus (Asteraceae) Lactuca serriola (Asteraceae) Acroptilon repens (Asteraceae) Cirsium arvense (Asteraceae) Medicago sativa (Fabaceae) Medicago sp. (Fabaceae) Astragallus sp. (Fabaceae) Onobrychis viciafolia (Fabaceae) Glycyrrhiza glabra (Fabaceae) Vicia variabilis (Fabaceae) Lotus cornicolata (Fabaceae) Ononis spinosa (Fabaceae) Ammodendron connolyi (Fabaceae) Psoralea drupaeca (Fabaceae) Alhagi sp. (Fabaceae) Zygophyllum fabago (Zygophyllaceae) Z. attriplicoides (Zygophyllaceae) Peganum harmala (Zygophyllaceae) Eringium sp. (Apiaceae) Capparis spinosa (Capparidaceae) Impatiens sp. (Balsaminaceae) Celosia sp. (Amaranthaceae) Haplophyllum perphoratum (Rutaceae) Euphorbia petiolata (Euphorbiaceae) E. heterophylla (Euphorbiaceae) Clematis orientale (Ranunculaceae) Alcea cf. popovii (Malvaceae) Alcea sp. (Malvaceae) Mindium laevigatum (Campanulaceae) Lepidium lotifolium (Brassicaceae) Epilobium sp. (Onagraceae) Capsicum annuum (Solanaceae) Glaucium oxylobum (Papaveraceae) Verbascum sp. (Scrophulariaceae) Chondrilla juncea (Asteraceae) C. juncea (Asteraceae)

S. A. Khodaparast ; Iran, Gilan S. A. Khodaparast ; Iran, Tehran S. A. Khodaparast ; Iran, Tehran S. A. Khodaparast ; Iran, Gilan S. A. Khodaparast ; Iran, Gilan S. A. Khodaparast ; Iran, Gilan S. A. Khodaparast ; Iran, Tehran S. A. Khodaparast ; Iran, Gilan O. Nasyrov ; Turkmenia S. A. Khodaparast ; Iran, Gilan S. A. Khodaparast ; Iran, Gilan Abbasi ; Iran, Mashhad A. Mohareri ; Iran, Eilam Tajick ; Iran, Golestan Hamzeh ; Iran, Yazd Anonymous ; Iran, Mashhad Mesbah ; Iran, Esfehan S. A. Khodaparast ; Iran, Gilan Mesbah ; Iran, Tehran S. A. Khodaparast ; Iran, Tehran S. A. Khodaparast ; Iran, Tehran S. A. Khodaparast ; Iran, Tehran Mesbah ; Iran, Esfehan S. A. Khodaparast ; Iran, Gilan S. A. Khodaparast ; Iran, Tehran S. A. Khodaparast ; Iran, Tehran S. A. Khodaparast ; Iran, Gilan Tajick ; Iran, Golestan Sharif ; Iran, Lorestan Moosavi ; Iran, Chaharmahal O. Nasyrov ; Turkmenia V. Mel’nik ; Turkmenia Mesbah ; Iran, Esphahan Khodaparast ; Iran, Gilan Hamzeh ; Iran, Fars S. A. Khodaparast ; Iran, Guilan Ershad ; Iran, Qazvin S. A. Khodaparast ; Iran, Gilan Javaheri ; Iran, Tehran S. A. Khodaparast ; Iran, Tehran Tajick ; Iran, Golestan S. A. Khodaparast ; Iran, Tehran S. Takamatsu ; Thailand, Chiangmai O. Nasyrov ; Turkmenia S. A. Khodaparast ; Iran, Tehran Hamzeh ; Iran, Fars Moosavi ; Iran, Karadj O. Nasyrov ; Turkmenia Abbasi ; Iran, Tehran T. Kobayashi : Japan, Kochi S. A. Khodaparast ; Iran, Gilan M. Abbasi ; Iran, Tehran S. A. Khodaparast ; Iran, Karaj S. A. Khodaparast ; Iran, Gilan

IRAN11141 IRAN11140 IRAN11114 IRAN11117 IRAN11118 IRAN11142 IRAN11143 IRAN11138 LE192668 IRAN11119 IRAN11120 IRAN10546 KAR501 IRAN9121 IRAN10575 IRAN11121 IRAN11145 IRAN11124 IRAN11134 IRAN11144 IRAN11116 IRAN11129 IRAN11115 IRAN11132 IRAN11127 IRAN11131 IRAN11133 IRAN10502 IRAN2238 IRAN10551 LE192677 LE192683 IRAN11125 IRAN11136 IRAN11135 IRAN11122 IRAN2287 IRAN11130 IRAN11137 IRAN11123 IRAN9119 IRAN11128 MUMH807 LE192686 IRAN11126 IRAN11113 IRAN10909 LE192665 IRAN10501 MUMH124 IRAN111139 IRAN11019 MUMH806 MUMH805

AB044346 AB045147 AB044350 AB044352 AB044372 AB044373 AB044374 AB048350 AB042642 AB042641 AB044376 AB045153 AB044377 AB044380 AB044381 AB044382 AB044383 AB044384 AB044378 AB044375 AB044379 AB044991 AB044994 AB044995 AB044992 AB044993 AB045114 AB045154 AB044996 AB044997 AB045115 AB044998 AB044999 AB045000 AB045148 AB045149 AB045001 AB045002 AB045003 AB045150 AB045151 AB045004 AB045005 AB045152 AB045006 AB045105 AB045106 AB044349 AB045107 AB000940 AB045108 AB045155 AB045109 AB045156

a IRAN l Herbarium of Iran, Department of Botany, Plant Pests and Diseases Institute, Evin, Tehran ; KAR l Karaj Agricultural College Mycological Herbarium ; LE l Herbarium of Komarov Botanical Inistitute ; MUMH l Mie University Mycological Herbarium. b The nucleotide sequence data will appear in the DDBJ, EMBL, and GenBank database under the respective accession number.

trees were found using the NJ method, and then, the likelihood of the constraint and best trees were determined using PAUP*. Hypothetical trees were tested against best trees by the Kishino-Hasegawa (Kishino & Hasegawa 1989) and Templeton tests (Templeton 1983).

RESULTS AND DISCUSSION Length and GC content of ITS1-5n8S-ITS2 region Whole nucleotide lengths for the Leveillula species sequenced in our study (except for L. rubiae) ranged from 556 to 575 base

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Table 2. Matrix of percentage distance among ITS1-5n8S-ITS2 region from 12 morphologically defined Leveillula species and eight genetically diverse L. taurica isolates.a

Du Im Mi On Ar As Ac Ci La Ca Cs Cn Sx Pi Si Ln Lo Ru Lv Lc

El

Du

Im

Mi

On

Ar

As

Ac

Ci

La

Ca

Cs

Cn

Sx

Pi

Si

Ln

Lo

Ru

Lv

0n7 3n2 1n6 3n2 5n1 5n1 4n0 0n9 1n2 2n3 2n3 2n8 2n8 3n5 3n2 1n6 2n1 3n2 3n2 4n7

3n4 2n1 3n4 5n6 5n7 4n0 1n6 1n9 2n8 2n8 3n4 3n2 3n7 3n4 1n8 2n3 4n0 3n5 5n0

4n1 5n4 6n5 6n9 4n9 3n7 4n1 4n2 4n2 4n8 4n8 0n4 0n4 3n7 4n2 4n9 5n5 6n1

3n0 5n1 4n6 3n6 1n8 2n1 2n5 2n5 3n4 2n8 4n1 3n7 0n5 1n1 1n4 2n7 4n5

6n0 4n6 4n2 3n8 4n1 4n3 4n3 5n2 5n0 5n4 5n0 3n2 3n9 4n4 4n5 4n9

6n7 4n7 5n4 5n3 5n8 5n6 6n4 6n2 6n4 6n4 5n1 5n3 5n8 6n7 5n3

6n3 5n3 5n7 5n9 5n9 6n0 6n6 7n1 6n7 5n1 5n7 5n5 6n9 6n7

4n0 4n1 4n5 4n3 5n2 5n4 5n1 4n7 3n4 3n6 5n0 5n1 4n0

1n1 2n5 2n5 3n0 2n8 4n1 3n7 1n8 2n3 3n4 3n2 4n9

2n8 2n8 3n7 3n2 4n4 4n1 2n1 2n3 3n5 3n7 4n9

0n4 1n6 2n8 4n2 3n9 2n5 3n0 3n9 4n4 5n4

1n6 2n8 4n2 3n9 2n5 3n0 3n9 4n4 5n4

3n7 4n8 4n8 3n4 3n9 4n6 4n6 6n0

4n8 4n4 2n8 3n4 4n4 4n8 6n3

0n4 3n7 4n2 5n1 5n5 6n5

3n4 3n9 4n8 5n1 6n1

0n5 2n3 2n5 4n1

2n7 3n0 4n3

4n2 5n7

5n6

a Abbreviations for taxa name are as follow : El l L. elaeagni (also for L. chrozophorae) ; Du l L. duriaei ; Im, Mi, On, Ar, As, Ac, Ci, and La for L. taurica on Impatiens, Mindium, Onobrychis, Artemisia, Astragallus, Acroptilon, Cirsium, and Lactuca ; Ca, Cs and Cn for L. cylindrospora on Anthochlamys, Salsola and Noaea, respectively ; Sx l L. saxouli ; PI l L. picridis ; Si l L. simonianii ; ln l L. lanuginosa ; Lo l L. loranthi ; Ru l L. rubiae ; Lv l L. verbasci and Lc l Leveillula sp. on Chondrilla.

Table 3. Matrix of percentage sequence divergence among ITS1-5n8S-ITS2 from 32 isolates of Leveillula taurica s. lat.a

Ca Zf Ah Cs Lo Ei Et Ps On Vi Me Gl Ep Er Al Ci Im Mi As Ob Ac Ar

Leb

Ca

Zf

Ah

Cs

Lo

Ei

Et

Ps

On

Vic

Me

Gl

Ep

Er

Al

Ci

Im

Mi

As

Ob

Ac

0n2 0n2 0n2 0n4 0n4 0n2 0n0 0n2 0n4 1n1 0n9 0n9 1n2 0n9 0n5 0n9 3n2 1n6 5n1 3n2 4n0 5n1

0n4 0n4 0n5 0n5 0n4 0n2 0n4 0n5 1n1 0n9 0n9 1n4 1n1 0n7 1n1 3n4 1n8 5n3 3n2 4n2 5n1

0n4 0n5 0n5 0n4 0n2 0n4 0n5 1n3 1n1 1n1 1n4 1n1 0n7 1n1 3n4 1n8 5n3 3n4 4n1 5n3

0n5 0n5 0n4 0n2 0n4 0n5 1n3 1n1 1n1 1n4 1n1 0n7 1n1 3n4 1n8 5n3 3n4 4n1 5n3

0n7 0n5 0n4 0n5 0n7 1n4 1n3 1n2 1n6 1n2 0n9 1n2 3n5 2n0 5n5 3n6 4n3 5n5

0n5 0n4 0n5 0n7 1n4 1n3 1n2 1n6 1n2 0n9 1n2 3n5 2n0 5n5 3n6 4n3 5n4

0n2 0n4 0n5 1n3 1n1 1n1 1n4 1n1 0n7 1n1 3n4 1n8 5n3 3n4 4n0 5n3

0n2 0n4 1n1 0n9 0n9 1n2 0n9 0n5 0n9 3n2 1n6 5n1 3n2 4n0 5n1

0n5 1n3 1n1 1n1 1n4 1n1 0n7 1n1 3n0 1n8 5n3 3n4 4n1 5n1

1n3 1n1 1n1 1n6 1n2 0n9 1n2 3n5 2n0 5n5 3n6 4n1 5n3

0n2 0n5 0n9 1n6 1n6 1n8 3n6 2n2 5n4 3n6 4n4 5n5

0n4 0n7 1n4 1n4 1n8 3n6 2n2 5n4 3n6 4n4 5n5

1n1 1n6 1n4 1n8 3n9 2n3 5n9 3n9 4n5 5n6

1n8 1n8 2n1 3n9 2n5 5n8 3n9 4n5 6n0

1n4 1n8 4n1 2n5 5n3 3n6 4n7 5n6

1n4 3n0 2n1 5n3 3n8 4n5 5n6

3n7 1n8 5n3 3n8 4n0 5n4

4n1 6n9 5n4 4n9 6n5

4n6 3n0 3n6 5n1

4n6 6n3 6n7

4n2 6n0

4n7

a Abbreviations for host name are as follows : Le l Lepidium, Ca l Capparis, Zf l Zygophyllum fabago, Ah l Alhagi, Cs l Capsicum, Lo l Lotus, Ei l Euphorbia from Iran, Et l Euphorbia from Thailand, Ps l Psoralea, On l Ononis, Vi l Vicia, Me l Medicago, Gl l Glaucium, Ep l Epilobium, Er l Eringium, Al l Alcea, Ci l Cirsium, Im l Impatiens, Mi l Mindium, As l Astragallus, Ob l Onobrychis, Ac l Acroptilon, Ar l Artemisia. b Leveillula taurica on Ammodendron, Celosia, Clematis, Glycyrrhiza, Haplophyllum, Peganum, and Z. attriplicoides, L. chrozophorae and L. elaeagni showed nucleotide sequences identical to L. taurica on Lepidium. c L. taurica on Helianthus showed identical nucleotide sequence to L. taurica on Vicia.

pairs (bp), spanning 212–224 bp for ITS1 (261 bp in L. rubiae), 154 bp for the 5n8S rDNA, and 183–202 bp for the ITS2 region. L. rubiae has a distinct insertion site including 23 nucleotides (5h-TTTTTGTTTTTGTTTTTGTTCTT-3h) in the ITS1 region, resulting in 607 bp in total length. The insertion

was excluded from the phylogenetic analyses. The shortest size of ITS1-5n8S-ITS2 was found in L. taurica on Artemisia (556 bp). The total GC contents of the ITS1-5n8S-ITS2 region ranged from 55n1 % to 59n2 % (53n2 % in L. rubiae), which suggests a lack of appreciable effects of base composition bias.

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Fig. 1. Adams consensus tree of more than 40,000 equally most parsimonious trees. Numbers above branches indicate bootstrap values based on 1000 replications. Decay indices are shown below the respective branches. The tree length is 235, the consistency index (CI) is 0n779, the retention index (RI) is 0n839, and the rescaled consistency index (RC) is 0n653. Leveillula taurica is highlighted by solid circles.

The GC content of L. taurica on Artemisia was higher than those of other Leveillula specimens. Multiple alignment and sequence divergence The data set includes 56 taxa, of which 54 are members of the genus Leveillula. The 54 members of the genus Leveillula include 13 species on 50 different plant species belonging to 22 plant families. Two species of the genus Phyllactinia were selected as outgroup taxa, as it has been known that Phyllactinia is the closest relative of Leveillula in both morphological and molecular studies (Braun 1987, Saenz & Talor 1999, Mori et al. 2000). The data set consisted of 635 characters including 28 gaps of which 48 sites were removed because of ambiguous alignment. Of the 587 remaining

characters, 152 sites were variable and 96 sites were phylogenetically informative. The ITS1 region, with 81 variable sites, showed the highest variation among taxa. The 5n8S rDNA was highly constant and only two variable sites were found among three taxa. Pair-wise percentages of sequence divergence of the ITS15n8S-ITS2 region were calculated using PAUP* for 54 Leveillula isolates. The nucleotide divergence for the ITS region between morphologically defined species ranged from 2n5 % to 6n5 % (Table 2). Two exceptions were found : between L. picridis and L. simonianii with 0n4 % divergence, and between L. lorantii and L. lanuginosa with 0n5 % divergence. Of the 34 isolates of L. taurica s. lat., the divergence between 26 isolates from 14 plant families did not exceed 1n8 % (Table 3). However, the remaining eight isolates of L. taurica from Acroptilon, Artemisia,

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Fig. 2. Neighbour-joining tree based on distances derived from the 54 taxa of the genus Leveillula plus two outgroup taxa. The bar indicates a distance of 0n5 % (0n5 base change per 100 nucleotide position). The numbers above the branches represent branch support using 1000 bootstrap replications (Bootstrap values below 50 % are not shown). Leveillula taurica isolates belonging to the groups 1, 2, and 3 in clade 1 (see text) were shown by solid circles, squares, and triangles at the left of the fungal names.

Astragallus, Circium, Impatiens, Lactuca, Mindium and Onobrychis spp. showed 1n1–6n0 % diversity with other L. taurica isolates, which suggests a distant relationship to other L. taurica isolates (Table 3). Leveillula taurica on Astragallus (4n6–7n1 %), Artemisia (4n7–6n7 %) and Leveillula sp. on Chondrilla (4n0–6n7 %) showed the highest divergence from other species.

analysis using Kimura’s two-parameter distance yielded a tree topology, which is shown in Fig. 2. After removing the taxa with identical nucleotide sequences, the ML tree was constructed using MOLPHY package v.2n3, and yielded similar tree topology (tree not shown). Leveillula specimens used in this study were divided into six clades and four basal isolates in all the tree topologies.

Phylogenetic analysis

Clade 1

Parsimony analysis yielded more than 40 000 trees. An Adams consensus of the 40 000 trees is shown in Fig. 1. The NJ

This the largest clade, and comprised 32 isolates from 28 plant species belonging to 16 plant families and four Leveillula

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f

d

c

b

g

h

l

e

i

m

n

j

o

k

p

q

r s

t

u

v

w x

y

Fig. 3. Conidia of Leveillula species : (a) L. taurica on Peganum, (b) L. taurica on Euphorbia, (c) L. taurica on Capparis, (d) L. elaeagni on Elaeagnus, (e) L. chrozophorae on Chrozophora, (f ) L. taurica on Cirsium, (g) L. taurica on Helianthus, (h) L. taurica on Medicago, (i) L. taurica on Glaucium, (j) L. duriaei on Salvia, (k) L. taurica on Lactuca, (l) L. taurica on Alcea, (m) L. taurica on Impatiens, (n) L. saxaouli, (o) L. cylindrospora, (p) L. lanuginosa, (q) L. loranthi, (r) L. verbasci, (s) L. taurica on Mindium, (t) L. rubiae, (u) L. taurica on Onobrychis, (v) L. taurica on Acroptilon, (w) L. taurica on Artemisia, (x) Leveillula sp. on Chondrilla, (y) L. taurica on Eringium. (a-w, y) primary conidia. Bar l 50 µm.

species : L. chrozophorae, L. duriaei, L. elaeagni and L. taurica. Although the bootstrap analyses did not strongly support this clade in the MP and NJ analyses (Figs 1–2), the ML analysis strongly supported this group with 93 % bootstrap value. In this clade, 26 L. taurica isolates were included. Isolates of L. taurica s. lat. belonging to clade 1 shared high sequence similarity in ITS1-5n8S-ITS2 with the recently described species, L. chrozophorae, L. elaeagni and L. duriaei. In particular, the nucleotide sequences of L. chrozophorae and L. elaeagni were identical to eight L. taurica isolates recovered from the Amaranthaceae, Brassicaceae, Fabaceae, Ranunculaceae, Rutaceae and Zygophyllaceae. Leveillula chrozophorae have been reported on Chrozophora (Braun 1984, 1995), whereas other Euphorbiaceae are colonized by L. taurica (Braun 1987, 1995).

As shown in Table 3, L. taurica on Euphorbia from Iran showed only one base difference with L. chrozophorae, and only one base gap was found between the former taxon and L. taurica on Euphorbia sp. collected in Thailand. L. chrozophorae and L. elaeagni are mainly characterized by primary conidia that are broadly lanceolate (Fig. 3d, e). The present result suggests that the character is not stable enough to distinguish these species from L. taurica, and that L. chrozophorae and L. elaeagni are conspecific with L. taurica. L. duriaei, and L. taurica on Vicia, Helianthus, Medicago and Glaucium spp. formed subclade 1a with a moderate bootstrap support. All of these taxa are morphologically similar to L. taurica, which was included in subclade 1c. However, L. duriaei is differentiated from L. taurica s. lat. by its rather broad

Phylogenetics of Leveillula

916

Table 4. Kishino-Hasegawa and Templeton tests.a Kishino-Hasegawa test

Templeton test

Constraint tree

Ln L

Difference Ln Lb

Standard deviationc

T-valued

Tree length (steps)

Rank sums

N

Pe

Significantly worse?f

Best unconstrainted tree (Fig. 2) L. taurica s. lat. monophyletic Fabaceous taxa monophyletic Asteraceous taxa monophyletic Leveillula on Cirsium mono-phyletic with L. taurica

k2177 k2313 k2230 k2292 k2180

(Best) k135 k52 k115 k2n5

24 18 22 3n0

5n6 2n9 5n2 0n8

235 273 248 266 236

(Best) 580 73 457 1

35 12 31 1

0n0001 0n005 0n0001 0n30

Yes Yes Yes No

Implemented in PAUP 4n0b4a (Swofford 2000). Difference in log-likelihood compared to that of the best tree. c The standard deviation in log-likelihood. d The T-value is determined by dividing the difference in log-likelihood by the standard deviation. e Probability of getting a more extreme test statistic under the null hypothesis of no differences between the two trees (two-tailed test). f The constraint tree is considered significantly worse if the difference in log-likelihood is more than twice the standard deviation in Kishino-Hasegawa test, or if the P-value is less than 0n05 in Templeton test. a

b

conidia (Fig. 3j) and the occurrence of a low net of ridges on the conidial surface (Durrieu & Rostam 1984, Braun 1984, 1987, 1995). In our preliminary morphological study, L. taurica on Glaucium, Helianthus, and Medicago can be differentiated from other L. taurica specimens of clade 1 by its cylindrical conidia with parallel sides that are pointed towards the apex (Fig. 3g, h, i). Further morphological and molecular investigations are necessary to clarify the taxonomic treatment of the taxa included in this subclade. The two L. taurica isolates from Alcea (Malvaceae) showed identical sequences, and formed a distinct subclade (1b). The isolates on Alcea have ovoid-lanceolate conidia that differ somewhat from other L. taurica. Recently, L. contractirostris was proposed for isolates from the family Malvaceae (Geljuta & Simonjan 1988). It seems that our specimens on Alcea are near to or identical to L. contractirostris. Subclade 1b, however, showed only 0n5–0n9 % sequence diversity with L. taurica isolates comprising clade 1c.

Clade 2 Clade 2 consists of L. taurica on Cirsium and Lactuca. The former fungus showed 0n9–2n1 % and 1n1 % nucleotide divergence with L. taurica isolates comprising clade 1 and L. taurica on Lactuca, respectively. Grouping of these two specimens is supported at the 66 %, 84 % and 67 % bootstrap values in the MP, NJ and ML trees, respectively. Moderate supports of bootstrap analyses, intermediate nucleotide divergence, and morphological differences of L. taurica on Cirsium may be interpreted to indicate the intermediate position of this fungus. Therefore, we conducted KishinoHasegawa and Templeton tests based on the hypothesis that L. taurica on Cirsium sp. could be monophyletic with L. taurica isolates comprising clade 1. We were not able to reject this hypothesis (Table 4). Morphological studies of L. taurica on Cirsium showed that it differs from L. taurica on Lactuca in conidial shape (Fig. 3f, k) and seems to be near to L. taurica belonging to clade 1.

Clade 3 Clade 3 consists of L. lanuginosa, L. loranthi, L. rubiae, L. verbasci and L. taurica on Mindium sp. This group was supported by 63 %, 68 % and 84 % bootstrap values for the MP, NJ and ML analyses, respectively. L. loranthi showed 0n5 % nucleotide divergence with L. lanuginosa and these two species clustered together. This species is closely allied to L. lanuginosa ; both taxa coincide well by having cylindrical conidia with cingulum-like rings near the ends (Fig. 3p, q). Leveillula loranthi differs from L. lanuginosa only by having narrower ascospores and unrelated hosts (Hajian, Moharrery & Hedjaroude 1999). Leveillula rubiae is well characterized by having cylindrical conidia that are often somewhat wider in the ends (Fig. 3t), and distinct insertion in ITS region, which is peculiar to this species. Leveillula taurica on Mindium also possess distinct cylindrical conidia (Fig. 3s). Therefore, the taxonomic position of L. taurica on Mindium in this clade needs more morphological studies. Clade 4 Clade 4 consists of two morphologically distinct species : L. cylindrospora and L. saxaouli. Both species have commonly been reported on Chenopodiaceae. L. cylindrospora on Anthochlamys and Salsola spp. showed 0n4 % nucleotide divergence with each other, and 1n6 % diversity was found between these taxa and L. cylindrospora on Noaea. The MP, NJ and ML trees strongly supported the grouping of L. cylindrospora on three different host genera at the 97 %, 94 % and 99 % bootstrap levels, respectively. However, grouping of L. saxaouli with L. cylindrospora is weakly supported by MP, NJ and ML bootstrap analyses (Figs 1, 2). Clade 5 Clade 5 comprises L. picridis, L. simonianii and L. taurica on Impatiens and is strongly supported by 99 % bootstrap values in the MP, NJ trees and 100 % in the ML tree. The decay index also strongly supported this clade (d l 9). Picridis and

S. A. Khodaparast, S. Takamatsu and G.-A. Hedjaroude Thevonotia belong to the Asteraceae, and Impatiens is a member of Balsaminaceae. Bootstrap support of the grouping of L. taurica on Impatiens with L. picridis was weak, with 55 %, 59 % and 81 % in the MP, NJ and ML trees, respectively. Leveillula taurica on Impatiens is morphologically near to L. picridis, both of which are characterized by cylindric primary conidia (Fig. 3m). Clade 6 Clade 6 consists of two L. taurica isolates on two fabaceous hosts, Astragallus and Onobrychis, and is strongly supported by bootstrap analysis both for the MP (86 %) and ML (90 %) trees. Basal group Leveillula on Acroptilon, Artemisia and Chondrilla (Asteraceae) are included in the basal group. These taxa occupy the primitive base of the phylogenetic tree and do not form a clade. Two isolates of Leveillula sp. on Chondrilla sp. formed a clade near the base of the phylogenetic trees. The present two specimens on Chondrilla sp. have cylindrical conidia that are usually wider at the ends and never pointed (Fig. 3x). Numerous asci, usually more than 40 (and up to 70 asci), are included in an ascoma. These morphological characters are clearly different from those of L. taurica, and different from L. lactucarum, which has been reported on Lactuca sp. and some other Asteraceae including Chondrilla juncea. The present molecular analysis confirmed our morphological observation, which suggests that the two Leveillula isolates on Chondrilla juncea are independent species. Leveillula taurica s. lat. Of the 54 Leveillula isolates investigated in this study, 34 were originally identified as L. taurica s. lat. Of these, 26 L. taurica isolates on hosts belonging to 14 distantly related plant families were included in clade 1. The remaining eight isolates of L. taurica were scattered throughout other clades ; i.e. clades 2, 3, 5, 6 and the basal group. The 26 isolates included in clade 1 could be assumed to be L. taurica s. str. These 26 isolates could be further divided into three groups. Group 1 (solid circles in Fig. 2) includes isolates with identical nucleotide sequences. This group comprises collections on hosts belonging to unrelated plant families, including Amaranthaceae (Celosia), Brassicaceae (Lepidium), Fabaceae (Glycyrrhiza, Ammodendron), Ranunculaceae (Clematis), Rutaceae (Haplophyllum), and Zygophyllaceae (Zygophyllum, Peganum). Group 2 (solid squares in Fig. 2) includes isolates with 0n2–0n7 % diversity between them, and comprises L. taurica collected on host plants of various families such as Capparidaceae (Capparis), Euphorbiaceae (Euphorbia), Solanaceae (Capsicum), and some members of the Fabaceae (Alhagi, Lotus, Ononis, Psoralea). Group 3 (solid triangles in Fig. 2) includes isolates having moderately high nucleotide sequence diversity with each other (0n2–1n8 %) and with the former groups (0n5–1n6 %). This group is classified into subclades 1a and 1b. From these results, we could infer that L. taurica is a complex of more or less genetically divergent isolates. To test the monophyly of

917 L. taurica s. lat., we constructed a constraint tree based on the hypothesis that all L. taurica s. lat. could be monophyletic. We then compared this constraint with the best unconstrained tree topology (Fig. 2) by the Kishino-Hasegawa and Templeton tests. In the Kishino-Hasegawa test, the differences of loglikelihood of the phylogenetic trees increased to 5n5 times that of the standard deviation and we could reject this hypothesis (Table 4). The results of the Templeton test also demonstrated that the hypothesis could be significantly rejected (Table 4). These results clearly show that L. taurica s. lat. is a complex species comprising several biological species. Host plant relations The host range of Leveillula fungi extends to 69 families, 378 genera and 1042 species of angiosperm plants (Amano 1986). In this study, we analysed 54 Leveillula isolates from 50 plant species covering 21 families. Isolates from a single plant family were usually confined to a single clade. On the other hand, isolates from the Asteraceae and Fabaceae were found in more than one clade. To test the strength of this result, we constructed constraint trees based on the following hypotheses : 1. Leveillula species on the Asteraceae are monophyletic, 2. Leveillula species on the Fabaceae are monophyletic. Constraint trees based on the above hypotheses were compared with the best NJ tree shown in Fig. 1, by the Kishino-Hasegawa and Templeton tests. Our data indicated that both of the hypothesis trees should be significantly rejected (Table 4). It is likely that Leveillula species have colonized the Asteraceae and Fabaceae several times during evolution. The Asteraceae and Fabaceae are the two biggest plant families in dicotyledons. Leveillula spp. have been recorded on 221 species in 78 genera of the Asteraceae and 155 species in 56 genera of the Fabaceae s. lat. (Palti 1988). These two families are the largest host plant families for the genus Leveillula. As a result, genetic diversity is reasonable in Leveillula species on hosts belonging to these host plant families. In fact, several species, viz, L. lactucarum, L. picridis, L. simonianii and L. taurica, have previously been recorded on the Asteraceae (Braun 1987). In addition, three Leveillula isolates on the Asteraceae occupied a basal position in our phylogenetic trees (Figs 1–2). This result suggests the alternative hypothesis that the composites are associated with an early evolution of Leveillula followed by host expansion from the Asteraceae to other plant families. CONCLUSION The taxonomic and phylogenetic structures of the genus Leveillula have been unclear for a long time, because of the rather uniform morphological characters. In this study, we have shown that the nucleotide sequence diversity of the rDNA ITS region is useful for clarifying the phylogenetic structure of the genus. The taxonomic position of several species (L. cylindrospora, L. lanuginosa, L. loranthi, L. picridis, L. rubiae, L. saxaouli, and L. simonianii) are supported by the present molecular analyses. These species are mostly well characterized by morphology of conidia, especially primary conidia. As a result, the present study showed that morphology of primary conidia mostly provides a good criterion to

Phylogenetics of Leveillula identify Leveillula species. This study also clearly showed that L. taurica s. lat. is a species complex composed of several biological species. Further morphological studies are required to define these species. Isolates from distantly related plant families have been revealed to be closely related to each other and formed a clade in the present phylogenetic trees, whereas isolates from the Asteraceae and Fabaceae included genetically divergent taxa comprising several independent lineages. Thus, the species concept of Golovin (1956), that confined species to hosts of a single plant family, is significantly rejected. In the present study, most of the specimens have been collected from Iran, which is one of the most important regions for Leveillula species. Except for one species, L. clavata, all generally accepted species of the genus Leveillula have been reported to occur in Iran (Ershad 1971, 1995 ; data not shown). However, nucleotide sequence data of L. taurica from other parts of the world may also help us understand more precisely the phylogenetic relationships within this species. A C K N O W L E D G E M E N TS The authors wish to thank Uwe Braun (Martin-Luther University, Halle) for critical reviewing the manuscript and identifying some Leveillula specimens ; F. Termeh (Plant Pests and Diseases Research Institute, Ministry of Agriculture of Iran) for identifying plant species ; D. Ershad for valuable suggestions ; M. Inagaki (Mie University) for providing unpublished nucleotide sequences of Phyllactina species ; M. Abbasi (curator of the Herbarium of Iran, IRAN), for providing valuable herbarium specimens ; V. A. Melnik (Komarov Botanical Institute, St Petersburg) for providing specimens. This work was partially supported by a Grant-in-Aid for Scientific Research (Nos 09660048 and 10306004) from Ministry of Education, Science, Sports and Culture of Japan. Article No. 156 from the Laboratory of Plant Pathology, Mie University.

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