Genetic diversity within the Albugo candida complex (Peronosporales, Oomycota) inferred from phylogenetic analysis of ITS rDNA and COX2 mtDNA sequences

Molecular Phylogenetics and Evolution 40 (2006) 400–409 www.elsevier.com/locate/ympev

Genetic diversity within the Albugo candida complex (Peronosporales, Oomycota) inferred from phylogenetic analysis of ITS rDNA and COX2 mtDNA sequences Young-Joon Choi a, Seung-Beom Hong b, Hyeon-Dong Shin a,¤ a

Division of Environmental Science and Ecological Engineering, College of Life and Environmental Sciences, Korea University, Seoul 136-701, Republic of Korea b Korean Agricultural Culture Collection, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Republic of Korea Received 19 March 2005; revised 6 March 2006; accepted 16 March 2006 Available online 27 April 2006

Abstract Albugo candida is a destructive fungus infecting brassicaceous hosts. The genetic diversity within the A. candida complex from various host plants was investigated by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and the cytochrome c oxidase subunit II (COX2) region of mtDNA. The aligned nucleotide sequences of A. candida shared signiWcantly high distances, up to 20.4 and 8.9%, in two genes. The phylogenetic trees, obtained using the Bayesian method and maximum parsimony analysis, showed two separate groups that corresponded to the host genera. Group I included A. candida isolates infecting Arabis, Autrieta, Berteroa, Biscutella, Brassica, Cardaminopsis, Diplotaxis, Eruca, Erysimum, Heliophila, Iberis, Lunaria, Raphanus, Sinapis, Sisymbrium, and Thlaspi. Group II contained all isolates from Capsella, Descurainia, Diptychocarpus, Draba, and Lepidium. The genetic similarities between the two genes among isolates within Group I were 99.0–100% and 99.6–100%, while those within Group II were 90.4–100% and 91.1–100%, respectively, showing considerably lower values than for Group I. The A. candida isolates from Capsella bursa-pastoris in Korea are clearly separated by sequence analysis for the two genes compared to those from Wales, England, and the USA. Based on the molecular data from the two genes, we suggest the high degree of genetic diversity exhibited within A. candida complexes warrants their division into several distinct species. © 2006 Elsevier Inc. All rights reserved. Keywords: Albugo; Species complex; Brassicaceae; ITS rDNA; COX2 mtDNA

1. Introduction Albugo candida (Pers.) Kuntze (Peronosporales, Oomycota), among the 40 species of the genus Albugo (Choi and Priest, 1995), is an obligate parasitic fungus responsible for white rust disease in brassicaceous hosts over widely diVerent geographical areas throughout the world. The pathogen causes signiWcant damage in economically important agricultural crops and common weeds (Farr et al., 1989), and its

*

Corresponding author. Fax: +82 2 921 1715. E-mail address: [email protected] (H.-D. Shin).

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hosts have been reported to include as many as 63 genera and 241 species (Biga, 1955; Saharan and Verma, 1992), of which Brassica and Raphanus species are the most important cultivated hosts. A. candida has been separated into diVerent forms by S8vulescu and Rayss (1930), and divided into two varieties by Biga (1955) based on the sporangial size and host ranges, viz. var. candida and var. macrospora, which were partly supported by Pound and Williams (1963) and Makinen and Hietajarvi (1965). Although, A. candida var. macrospora has been reported from some species of the genera Brassica and Raphanus in the Brassicaceae (Yu et al., 1998; Zhang and Wang, 1981), the species is not widely accepted. Albugo lepidii recorded from the Lepidium

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(Rao, 1979), another genus within the Brassicaceae was also regarded by Choi and Priest (1995) as a synonym. Therefore, the name A. candida currently refers to the pathogenic fungus causing white rust disease on all brassicaceous hosts. The classiWcation of species within the genus Albugo, which infects the same host family, has been often impossible base on morphological characteristics. The host speciWcity of A. candida has been demonstrated by Hiura (1930) and Eberhardt (1904), and these studies, which examined predominately crop species, resulted in A. candida being classiWed into 10 races based on their speciWcity to diVerent brassicaceous hosts (Hill et al., 1988; Pound and Williams, 1963). The degree of host speciWcity within A. candida, however, has not been clearly deWned. For example, Khunti et al. (2000) showed that A. candida isolates from Brassica can infect Amaranthus viridis (Amaranthaceae) and Cleome viscosa (Capparaceae, now included in Brassicaceae; APG, 2003), as well as Brassica campestris var. rapa. The complexity of the minor morphological characters and physiological specializations found among isolates infecting the Brassicaceae has resulted in problems for taxonomic studies. However, no molecular evidence which supports the homogeneity of A. candida isolates from diVerent host genera and/or species has yet been provided, because the methodology of both obtaining good sample and sequences out of the samples is diYcult for the obligate parasitic fungi. Recently, two molecular studies, based on ampliWed fragment length polymorphism (AFLP) and ITS1 sequencing (Rehmany et al., 2000) and 28S rDNA sequencing (Riethmüller et al., 2002), partially showed that there was both diversity and similarity within A. candida isolates. However, these studies included relatively small numbers of specimens of the species; and therefore, further molecular studies are needed to conWrm whether A. candida is a homogenous taxon. In the present study, a sequence analysis of the ITS rDNA was carried out to better understand the interrelationships of this complex from various host plants. Many studies have already proved the sequence analysis of the ITS region of rDNA to be a very powerful tool for the comparison of closely related species or isolates within the Oomycota (Choi et al., 2003; Constantinescu and Fatehi, 2002; Cooke et al., 2000; Rehmany et al., 2000; Voglmayr, 2003). The phylogenetic study based on the mitochondrial COX2 gene (also known as COXII), which encodes subunit II of the cytochrome c oxidase complex, was also carried out with Albugo specimens from various host plants, and has been previously used in phylogenetic studies of species of Phytophthora (Martin and Tooley, 2003), Pythium (Martin, 2000), and Peronosporales (Hudspeth et al., 2003) within the Oomycota. Despite the powerful utility for phylogenetic analysis of the two genes, ITS rDNA and COX2 mtDNA, these tools remain to be applied to resolving the taxonomic and phylogenetic problems of Albugo species. The objective of this work was to investigate the genetic diversity occurring within A. candida isolates infecting various plants of Brassicaceae sensu stricto hosts, by analyzing the ITS rDNA and COX2 mtDNA sequences.

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2. Materials and methods 2.1. Fungal isolates The ITS and COX2 genes of 43 isolates of A. candida and one Albugo bliti were sequenced in this study. Of these, 26 dried herbarium specimens, including 19 of BPI (National Fungus Collections, Beltsville, Maryland, USA), two of the CUP (Plant Pathology Herbarium, Cornell University, New York, USA), and Wve of the SOMF (Mycological Collection Herbarium, Institute of Botany, SoWa, Bulgaria) were also included. For comparison, seven ITS1 sequences and one COX2 sequence of A. candida were obtained from GenBank: two from Capsella bursa-pastoris, and one each, respectively, from Arabidopsis thaliana, Brassica oleracea, Erysimum menziesii, Lepidium campestre, and Thlaspi arvense for ITS rDNA and one from Capsella bursa-pastoris for COX2 mtDNA. Voucher specimens of the newly collected specimens are preserved at the SMK (Herbarium of Systematic Mycology of Korea, Korea University, Seoul, Korea). Information on the 51 isolates is shown in Table 1. 2.2. DNA extraction Genomic DNA was extracted using the sporangiophores and sporangia formed on the upper or under surface of infected leaves, or the host tissue of herbarium specimens. The presence of the sporangiophores and sporangia of white rusts was conWrmed under the dissecting microscope (Olympus SZ), which were then scraped with a dissecting blade into 1.5 ml tubes. Plant samples (0.5 g) from the herbarium specimens were ground in liquid nitrogen, and the frozen powder used to one-third Wll the 1.5 ml tubes. Four hundred microliter of lysis buVer (50 mM Tris–HCl, pH 7.5, 50 mM EDTA, 3% SDS, and 1% 2-mercaptoethanol) was added to each tube, which were then incubated at 65 °C for 1 h. An equal volume of phenol/chloroform/isoamyl alcohol (25/24/1, v/v/v) was added, and the tubes centrifuged for 15 min at 13,000g. The supernatant was removed to a clean tube, and the DNA precipitated by the addition of 10 l of 3 M sodium acetate and 162 l isopropanol. The DNA pellet was washed twice with 70% ethanol, dried, and resuspended in TE buVer (10 mM Tris–HCl, 1 mM EDTA). One microliter RNase (500 g ml¡1) was added to each sample, and incubated at 37 °C for 30 min. 2.3. AmpliWcation and sequencing For the selective ampliWcation of the complete ITS region of rDNA, primers DC6 (5⬘-GAG-GGA-CTT-TTGGGT-AAT-CA-3⬘) and LR0 (5⬘-GCT-TAA-GTT-CAGCGG-GT-3⬘) were used. PCRs were conducted in 50 l reaction volumes, with each reaction tube containing 1.2 l of template DNA solution (approximately 100 ng), as prepared above, 5 l of 10£ buVer (50 mM KCl, 100 mM

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Table 1 Summary of information about Albugo specimens used in this study Species

Albugo bliti A. candida

Host

Amaranthus spinosus Arabidopsis thaliana Arabis turrita Aubrieta deltoidea A. deltoidea Berteroa incana B. incana B. incana Biscutella laevigata B. laevigata Brassica juncea B. juncea B. juncea B. oleracea Capsella bursa-pastoris C. bursa-pastoris C. bursa-pastoris C. bursa-pastoris C. bursa-pastoris C. bursa-pastoris Cardaminopsis halleri subsp. ovirensis Descurainia sophia Diptychocarpus strictus Diplotaxis erucoides D. tenuifolia Draba nemorosa Erysimum menziesii Eruca sativa Erysimum cuspidatum E. cheiranthoides E. cheiranthoides Heliophila meyeri H. meyeri Iberis amara I. umbellata Lepidium apetalum L. apetalum L. campestre L. campestre L. virginicum L. virginicum Lunaria sp. Raphanus sativus R. sativus R. sativus Sinapis arvensis Sisymbrium leteum S. leteum S. loeselii Thlaspi arvense T. arvense

Source or herbarium number/geographical origin

SMK19835, Chunchon, Korea Acem1, Kent, England SOMF00337, Bulgaria BPI184659, Oberhessen, Germany BPI796090, Oberhessen, Germany BPI184200, Krems, Austria BPI184678, Krems, Austria BPI184679, Riga, Latvia BPI184686, Switzerland BPI796093, Romania SMK15570, Namyangju, Korea SMK20024, Namyangju, Korea SMK21051, Jeju, Korea A001, Warwickshire, England SMK13752, Namyangju, Korea SMK15670, Seoul, Korea SMK17254, Yongin, Korea Acaps, Warwickshire, England CAN1, Wales — BPI199991, Suceava District, Rominia SOMF19655, URSSa SOMF19659, URSSa BPI184862, Palestine BPI184865, Spain SMK15732, Gapyong, Korea California, USA BPI184870, Daudkhel, Pakistan BPI199988, Romania BPI184874, Moravia, Czech BPI184875, Bohemia, Czech BPI184888, Vanrhynsdorp, S. Africa BPI184889, Vanrhynsdorp, S. Africa BPI184897, California, USA BPI184898, Paget West, Bermuda SMK13747, Seoul, Korea SMK14479, Seoul, Korea Michigan, USA SOMF18525, Bulgaria SMK17251, Seoul, Korea SMK17312, Seoul, Korea CUP065639, OR, USA SMK10614, Seoul, Korea SMK13330, Kangnung, Korea SMK19419, Busan, Korea BPI185100, Velvary, Czech SMK19086, Pyongchang, Korea SMK19577, Pyongchang, Korea SOMF12295, Bulgaria CUP065777, NY, USA —

Collected year

2003 — 1955 1953 1953 1987 1876 1932 1903 1973 1998 2003 2004 — 1997 1999 2000 — 1997 — 1980 1977 1978 1935 1920 1999 1995 1968 1979 1896 — 1896 1896 1938 1927 1997 1998 1994 1984 2000 2000 2000 1990 1994 2002 1899 2002 2003 — 2002 —

GenBankb ITS

COX2

AY929824 AF241765 AY929825 DQ418500 DQ418501 DQ418495 — — DQ418494 — AY929826 AY929827 AY929828 AF241767 AY929829 AY929830 AY929831 AF241769 AF271231 — DQ418502 AY929832 AY929833 DQ418496 DQ418497 AY929834 AF016836 DQ418503 DQ418498 — — DQ418493 — DQ418499 — AY929835 AY929836 AF016837 AY929837 AY929838 AY929839 AY929840 AY929841 AY929842 AY929843 — AY929844 AY929845 AY929846 AY929847 AF016838

AY913805 — AY913803 DQ418511 DQ418512 DQ418508 DQ418509 DQ418510 DQ418506 DQ418507 AY927046 AY913810 AY927047 — AY927048 AY927049 AY927050 — — AY286229 DQ418513 AY927051 AY927052 DQ418517 DQ418518 AY927053 — DQ418514 DQ418519 DQ418520 DQ418521 DQ418515 DQ418516 DQ418522 DQ418504 AY927054 AY927055 — AY927056 AY927057 AY927058 AY913797 AY927059 AY927060 AY913801 DQ418505 AY913808 AY927061 AY913802 AY913809 —

BPI: National Fungus Collections, Beltsville, Maryland, USA, CUP: Plant Pathology Herbarium, Cornell University, New York, USA, SMK: Herbarium of Systematic Mycology of Korea, Korea University, Seoul, Korea, and SOMF: Mycological Collection of Institute of Botany in SoWa, Bulgaria. a Leg. & det. by B.A. Mel’nik (Komarov Institute of Botany, Russian Academy of Sciences, URSS). b Accession No. for GenBank Database.

Tris–HCl, pH 8.0, 0.1% Triton X-100, and 15 mM MgCl2), 3 l of 2.5 mM dNTP, 0.4 l (each) of 100 M primers, 0.4 l Taq polymerase (5 U l¡1), and 39.6 l ddH2O. The thermal cycling parameters were: denaturation for 1 min at 95 °C, annealing for 1 min at 58 °C, and extension for 2 min at

72 °C. Thirty-Wve cycles were performed with the Wrst denaturation and last extension times extended to 5 and 10 min, respectively. For COX2 ampliWcation, the forward (5⬘-GGC-AAATGG-GTT-TTC-AAG-ATC-C-3⬘) and reverse (5⬘-CCA-

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TGA-TTA-ATA-CCA-CAA-ATT-TCA-CTA-G-3⬘) primers designed by Hudspeth et al. (2000) were employed. Reactions identical to that for the ampliWcation of the ITS region were performed using the following cycling conditions; denaturation for 30 s at 96 °C, annealing for 30 s at 50 °C, and extension for 1 min at 72 °C. Thirty cycles were performed with both the Wrst denaturation and last extension times extended to 4 min. The success of the ampliWcation was monitored by electrophoresis on 1% agarose gels. The PCR products were then subjected to electrophoresis on a 0.8% agarose gel, and puriWed using a QIAquick gel extraction kit (Qiagene, Hilden, Germany). PuriWed DNAs were directly sequenced on an automatic sequencer (ABI Prism TM 377 DNA Sequencer), with the primers ITS1 and ITS3 for ITS rDNA, and primers identical to those used for ampliWcation of the COX2 mtDNA.

data, with all nucleotide substitutions equally weighted and unordered. The consistency index (CI) and retention index (RI) were calculated for all parsimony trees (Farris, 1989; Kluge and Farris, 1969). The relative robustness of the individual branches was estimated by bootstrapping, using 10,000 replicates, with heuristic searches, branch swapping by tree bisection-reconnection (TBR) and MAXTREES set at 100. For a distance analysis of ITS1 sequences, the most appropriate evolutionary model was determined for a given data set, using PAUP* version 4b10 and Modeltest 3.06. The Hasegawa–Kishino–Yano (HKY85) distance model (Hasegawa et al., 1985) was chosen, which was used to calculate the distance matrix and construct a neighbour-joining tree in PAUP* version 4b10. Trees were rooted using the TREEVIEW program version 1.6.6 (Page, 1996) by selecting the A. bliti sequence.

2.4. Sequence alignment and phylogenetic analysis

3. Results

Sequences were edited with the DNASTAR computer package, with alignment of the sequences performed using the CLUSTAL W (Thompson et al., 1994) program. Both the Bayesian inference and maximum parsimony methods were used for the phylogenetic analysis. Bayesian analysis was performed using the computer program MRBAYES, version 2.01 (Huelsenbeck and Ronquist, 2001). This program performs a Bayesian inference of the phylogeny, using Metropolis-coupled Markov chain Monte Carlo (MC3; Geyer, 1991) analyses. The general time reversible model (GTR), with gamma-distributed substitution rates, is selected for both the ITS and COX2 data matrix using Modeltest 3.06 (Posada and Crandall, 1998). Four incrementally heated simultaneous Markov chains were run for one-million generation, saving a tree every 100th generation. Among these, the Wrst 1000 trees were ignored. MRBAYES was used to compute a 50% majority-rule consensus of the remaining trees to obtain estimates for the posterior probabilities of the groups. Branch lengths were computed as the mean values over the sampled trees. To test the reproducibility of results, the analysis was repeated four times, starting with random trees and default parameter values. A maximum parsimony (MP) analysis (Fitch, 1971) was performed with the heuristic search, with random addition sequences, branch swapping by tree bisection-reconnection (TBR), and MAXTREES set at 20,000, using PAUP* version 4b10 (SwoVord, 2002). Gaps were treated as missing

3.1. Sequence alignment PCR products of a length between 1200 and 1350 bp, including a partial 18 S and complete ITS region (ITS1, 5.8 S rDNA, and ITS2), were ampliWed from each isolate using the primers, DC6 and LR0. The sequences of the 35 isolates from A. candida and A. bliti were adjusted to the length of the complete ITS region. Information about the sequence alignment of the ITS rDNA regions of A. candida is given in Table 2. A. candida isolates produced diVerent sizes, ranging from 836 to 846 bp, in the ITS regions, while the length of the ITS fragment from A. bliti was very short, 747 bp. The ITS2 region showed the greatest variation in length (480–492 bp), and was more than twice that of the ITS1 region, ranging from 187 to 191 bp. The aligned data set had 175 variable and 151 informative sites in the ITS2 region, but 29 and 23 in the ITS1 region. In the alignment of partial COX2 sequences, not including the gaps, 79 variable and 62 informative sites were observed (Table 2). In the ITS gene, the rates of the number of variable and informative sites per characters were 24.2 and 20.9%, respectively, which were more than for the COX2 gene (13.9 and 10.9%). Sequence distances within or among isolates were measured using HKY85 correction method, and the average genetic similarities among isolates within the ITS1 and ITS2 regions were more than 87.5 and 67.3%, respectively, while that within the COX2 region is more than 91.1%.

Table 2 Sequence information on the Albugo candida isolates used in this study Genes

Length

Character

ITS1 5.8S ITS2 Complete ITS Partial COX2

187–191 166 480–492 836–846 567

194 166 498 859 567

Variable sites

Informative sites

No.

No./character (%)

No.

No./character (%)

29 1 175 208 79

14.9 0.6 35.1 24.2 13.9

23 0 151 180 62

11.9 — 30.3 20.9 10.9

Similarity (%)

87.5–100 99.4–100 67.3–100 79.8–100 86.8–100

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3.2. Phylogenetic analysis of ITS rDNA The phylogenetic relationships within the A. candida complex were inferred from the Bayesian (MCMC) and heuristic maximum parsimony (MP) analyses of the aligned nucleotide sequences of the ITS rDNA. For the Bayesian inference, all Wve analyses showed the same tree topology and almost identical posterior probability values. After running one-million generations, a 50% major0.98

ity-rule consensus tree was constructed from all the trees, excluding the Wrst 1000 trees discarded, as shown in Fig. 1A. Out of 870 total characters, 187 were parsimonyinformative, and parsimony analysis resulted in 82 most parsimonious trees of 603 steps with a CI of 0.9005 and an RI of 0.9579. A 50% majority-rule consensus tree is shown in Fig. 1B, with no diVerence found between the tree topologies inferred from the MCMC and MP analyses.

SOMF12295 (ex Sisymbrium loeselii) SMK19577 (ex Sisymbrium leteum)

A

0.99

SMK19086 (ex Sisymbrium leteum) BPI184897 (ex Iberis amara)

0.64 B

0.80

BPI199991 (ex Cardaminopsis halleri subsp. ovirensis)

0.81 0.84

BPI184200 (ex Berteroa incana) SMK19419 (ex Raphanus sativus) SMK13330 (ex Raphanus sativus)

0.71 0.58 0.61

SMK10614 (ex Raphanus sativus)

0.81

0.58

BPI199988 (ex Erysimum cuspidatum) BPI184659 (ex Aubrietia deltoidea) BPI796090 (ex Aubrietia deltoidea) BPI184686 (ex Biscutella laevigata)

0.91

BPI184888 (ex Heliophila meyeri) CUP065639 (ex Lunaria sp.)

0.86

1.00 0.54

CUP065777 (ex Thlaspi arvense) BPI184870 (ex Eruca sativa)

Group I 0.57

BPI184862 (ex Diplotaxis erucoides) BPI184865 (ex Diplotaxis tenuifolia)

0.61

SMK21051 (ex Brassica juncea) SMK15570 (ex Brassica juncea) SMK20024 (ex Brassica juncea) SOMF00337 (ex Arabis turrita) SMK17251 (ex Lepidium virginicum)

1.00 1.00 0.99 0.96

Group II

SMK14479 (ex Lepidium apetalum) SMK17312 (ex Lepidium virginicum)

0.74

1.00

SMK13747 (ex Lepidium apetalum)

0.77

SOMF18525 (ex Lepidium campestre)

0.87

SOMF19655 (ex Descurainia sophia) SOMF19659 (ex Diptychocarpus strictus)

1.00

SMK15732 (ex Draba nemorosa)

1.00

SMK13752 (ex Capsella bursa-pastoris)

1.00

SMK17254 (ex Capsella bursa-pastoris) SMK15670 (ex Capsella bursa-pastoris) A. bliti SMK19835 (ex Amaranthus spinosus)

0.05 Fig. 1. Phylogenetic trees for Albugo candida isolates from various hosts based on the complete ITS region (ITS1, 5.8S rDNA, and ITS2). (A) Bayesian analysis showing mean branch lengths of a 50% majority-rule consensus tree calculated from 9000 trees revealed during a MCMC analysis of one-million generations. Numbers above the branches are the posterior probability values. The number of nucleotide changes between taxa is represented by branch length and the scale bar equals the number of nucleotide substitution per site. The branch of A. bliti is scaled down due to long length. (B) Maximum parsimony analysis showing a 50% majority-rule consensus tree constructed from 82 most parsimonious trees found by heuristic search (20,000 rounds of random addition of sequences and subsequent TBR branching swapping). The tree length is 603 steps, the consistency index (CI) D 9005, and the retention index (RI) D 0.9579. Numbers above the branches are the bootstrap values (10,000 replicates, values smaller than 50% not shown).

Y.-J. Choi et al. / Molecular Phylogenetics and Evolution 40 (2006) 400–409

Albugo candida isolates from brassicaceous hosts formed two main groups in the MCMC method and MP analysis. A. candida isolates from Arabis, Autrieta, Berteroa, Biscutella, Brassica, Cardaminopsis, Diplotaxis, Eruca, Erysimum, Heliophila, Iberis, Lunaria, Raphanus, Sinapis, Sisymbrium, and Thlaspi formed the most closely related clade (Group I), with 86 and 100% support in both analyses, and shared high sequence similarity (99.0–100%). Sequences from the same fungus/host combination were identical to each other (100% homology). Group II was supported by values of 96 and 100%, respectively, in the MCMC and MP analyses, which was found to include all isolates from the Capsella, Descurainia, Diptychocarpus, Draba, and Lepidium. Isolates of the A. candida complex infecting Capsella bursa-pastoris formed a subgroup within Group II, with high posterior

405

probability (100% in MCMC and MP trees). A. candida collections from Lepidium spp. were identical to each other in their ITS sequences, and further grouped with Descurainia, Diptychocarpus, and Draba in two trees. Neighbour-joining analysis of the ITS1 region was carried out with the sequences obtained in the present study and those obtained from GenBank. The NJ tree is presented in Fig. 2. The topology of the tree was very similar to those obtained by the MCMC and MP methods. In the ITS1 sequence analysis, A. candida isolates on Brassica, Capsella, Erysimum, and Thlaspi from the GenBank were clustered together with those on Group I, sequenced in this study (bootstrap values of 76%, Fig. 2), and showed little nucleotide diVerence, in spite of diVerent geographical origins. A. candida on Capsella bursa-pastoris of GenBank

SMK10614 (ex Raphanus sativus) BPI184200 (ex Berteroa incana) SMK19419 (ex Raphanus sativus) SMK13330 (ex Raphanus sativus) BPI199991 (ex Cardaminopsis halleri subsp. ovirensis) BPI184686 (ex Biscutella laevigata) AF271231 (ex Capsella bursa-pastoris)* SMK21051 (ex Brassica juncea) SOMF00337 (ex Arabis turrita) AF241769 (ex Capsella bursa-pastoris)* CUP065639 (ex Lunaria sp.) BPI184659 (ex Aubrietia deltoidea) AF016836 (ex Erysimum menziesii)* BPI184870 (ex Eruca sativa) AF016388 (ex Thlaspi arvense)* CUP065777 (ex Thlaspi arvense) SMK20024 (ex Brassica juncea) AF241767 (ex Brassica oleracea)* BPI184888 (ex Heliophila meyeri) BPI796090 (ex Aubrietia deltoidea) BPI184897 (ex Iberis amara) BPI199988 (ex Erysimum cuspidatum) BPI184862 (ex Diplotaxis erucoides) BPI184865 (ex Diplotaxis tenuifolia) SMK15570 (ex Brassica juncea)

0.76

Group I

0.51

SOMF12295 (ex Sisymbrium loeselii) SMK19577 (ex Sisymbrium leteum) SMK19086 (ex Sisymbrium leteum) SMK13752 (ex Capsella bursa-pastoris) 1.00 SMK17254 (ex Capsella bursa-pastoris) SMK15670 (ex Capsella bursa-pastoris) SMK15732 (ex Draba nemorosa) SOMF19659 (ex Diptychocarpus strictus) 0.64 AF241765 (ex Arabidopsis thaliana)* SOMF19655 (ex Descurainia sophia)

0.84

Group II 0.82

SMK17251 (ex Lepidium virginicum) SMK14479 (ex Lepidium apetalum) SMK13747 (ex Lepidium apetalum) SMK17312 (ex Lepidium virginicum) SOMF18525 (ex Lepidium campestre) AF016837 (ex Lepidium campestre)* A. bliti SMK19835 (ex Amaranthus spinosus)

0.05 Fig. 2. Phylogenetic tree for Albugo candida isolates inferred from neighbour-joining analysis of the complete ITS1 region. Numbers above the branches are the bootstrap values (10,000 replicates, values lower than 50% not shown). The number of nucleotide changes between taxa is represented by branch length and the scale bar equals the number of nucleotide substitution per site. An asterisk (¤) denotes sequences obtained from GenBank.

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(Group I) were clearly separated from the Capsella white rust disease of Korea (Group II), sharing a similarity of only 89.4%. A. candida on A. thaliana obtained from GenBank was found to be more closely related to those within clade II. NJ analysis also showed that the A. candida isolates on Lepidium apetalum and L. virginicum, which were sequenced in this study, and that on L. campestre from GenBank, formed a clade with a sequence similarity of 99.5%. 3.3. Phylogenetic analysis of COX2 mtDNA MCMC and MP analyses of the alignment of the COX2 mtDNA were also carried out to study the genetic diversity within the A. candida complex. The setting for the Bayesian inference was identical to that described for the ITS rDNA, with the 50% majority-rule consensus tree constructed from all the trees, excluding the Wrst 1000 trees discarded, shown in Fig. 3A. Out of 567 total characters, 62 were parsimonyinformative, and parsimony analysis resulted in 11 most parsimonious tree of 194 steps, with a CI of 0.8093 and an RI of 0.9100. A 50% majority-rule consensus tree is also shown in Fig. 3B, with little diVerence found between the tree topologies inferred from the MCMC and MP analyses. The trees showed two separate groups, which were identical with the result from the ITS rDNA. Group I showed the most closely related clade with 99% posterior probability and 100% bootstrap support, respectively, also with high sequence similarity (99.6–100%). Group II was clustered with similarities between 93.2 and 100%, and supported by values of 60 and 93% in the MCMC and MP analyses, respectively. A. candida from Lepidium spp. was further grouped with Descurainia, Diptychocarpus, and Draba in both trees. A. candida on Capsella bursa-pastoris of GenBank, clustered together with other isolates within Group I in the MCMC and MP trees, was clearly separated from the Korean Capsella white rust in Group II, and sharing a similarity of only 93.1%. 4. Discussion Traditionally, as exempliWed by several monographs (Kochman and Majewski, 1970; Vanev et al., 1993), the lack of appropriate applicable characters for the taxonomy of species within the genus Albugo, which infects the same host family, makes it diYcult to diVerentiate these fungi, particularly if the plant host from which the fungus was isolated is unknown. In the present study, the phylogenetic analysis of A. candida, based on the ITS rDNA and COX2 mtDNA sequences, was intended to determine the relationship among these morphologically very similar isolates more precisely. The MCMC and MP analyses were performed using sequences of the two genes, but no diVerence was found between the inferred tree topologies. Although, A. candida is commonly regarded as only pathogen causing white rust disease on Brassicaceae, the present study showed that A. candida isolates from various hosts were

clearly divided into two major clades, which are then separated into several branches, with high sequence dissimilarities. Therefore, the possibility of the existence of several Albugo species infecting the brassicaceous host plants is suggested, although, appropriate morphological characters for diVerentiating the complex were not found in this work. Albugo candida isolates infecting Arabis, Autrieta, Berteroa, Biscutella, Brassica, Cardaminopsis, Diplotaxis, Eruca, Erysimum, Heliophila, Iberis, Lunaria, Raphanus, Sinapis, Sisymbrium, and Thlaspi, formed a well-supported clade (Group I) with a high degree of sequence similarity. The NJ tree also showed that the ITS1 sequences of A. candida on Brassica, Erysimum, and Thlaspi (this work) were identical to those on the same plants obtained from GenBank, regardless of other geographical origins. The link between pathogens in Group I supposed a close taxonomic relationship among their host genera. Among their host genera, Brassica, Sisymbrium, and Thlaspi were more homogeneous than the other brassicaceous genera based on the phylogenetic analysis of Adc and ndhF sequences (Galloway et al., 1998). Brassica and Raphanus were closely related in the sequence analysis of the ITS region of the rDNA (Yang et al., 1999), belonging to the tribe Brassicaceae of the Brassicaceae, according to the morphological taxonomy (Heywood, 1993). Interestingly, A. candida sequences from all plants of the tribe Brassicaceae (Brassica, Diplotaxis, Eruca, Raphanus, Sinapis) examined in this study were positioned in Group I. Because of its economic importance, much work on the host speciWcity of A. candida has been reported, but most of the work has revealed no basis for sub-division of A. candida. Pound and Williams (1963) showed that the A. candida isolates from Raphanus sativus, Brassica juncea, Capsella bursa-pastoris, and Sisymbrium oYcinale can cause white rust disease on Brassica hirta, indicating susceptibility to all known races, while the plants of Thlaspi and Raphanus were not infected by all races. Therefore, the phylogeny of A. candida isolates studied in that work did not appear to be strictly related to that of their hosts or within the host ranges. Within Group II, the white rust from Descurainia, Diptychocarpus, Draba, and Lepidium showed greatest nucleotide dissimilarities, suggesting a high level of genetic diversity within Group II, which corresponded to their hosts. Lepidium white rusts are diVerentiated from other isolates in Group II, as well as those in Group I. This result supports the conclusion of Rao (1979), who described Albugo isolates from Lepidium as A. lepidii. However, based on the present study, it is too early to accept it as a separate taxon within A. candida complex. All isolates from Capsella bursa-pastoris in Korea were in Group II, while the group was clearly separated from the Capsella white rust sequences obtained from GenBank in its ITS1 and COX2 sequences. The three GenBank sequences were obtained from A. candida on Capsella bursa-pastoris in Wales, England, and the USA. Therefore, the result suggests that A. candida has two independent lineages on Capsella bursapastoris. However, the apparent relationship between the

Y.-J. Choi et al. / Molecular Phylogenetics and Evolution 40 (2006) 400–409

0.85

A 0.84

0.64

0.99

Group I

BPI184862 (ex Diplotaxis erucoides) SMK19419 (ex Raphanus sativus) SMK13330 (ex Raphanus sativus) SMK10614 (ex Raphanus sativus) SMK21051 (ex Brassica juncea) SMK15570 (ex Brassica juncea) SMK20024 (ex Brassica juncea) SMK19577 (ex Sisymbrium leteum) SMK19086 (ex Sisymbrium leteum) CUP065639 (ex Lunaria sp.) BPI184865 (ex Diplotaxis tenuifolia) BPI184870 (ex Eruca sativa) SOMF00337 (ex Arabis turrita) AY286219 (ex Capsella bursa-pastoris)* BPI184659 (ex Aubrietia deltoidea) BPI796090 (ex Aubrietia deltoidea) BPI199991 (ex Cardaminopsis halleri subsp. ovirensis) BPI184888 (ex Heliophila meyeri) BPI184889 (ex Heliophila meyeri) BPI185100 (ex Sinapis arvensis) BPI184686 (ex Biscutella laevigata) BPI796093 (ex Biscutella laevigata) BPI184200 (ex Berteroa incana) BPI184678 (ex Berteroa incana) BPI184679 (ex Berteroa incana) BPI199988 (ex Erysimum cuspidatum) BPI184874 (ex Erysimum cheiranthoides) BPI184875 (ex Erysimum cheiranthoides) BPI184897 (ex Iberis amara) BPI184898 (ex Iberis umbellata ) CUP065777 (ex Thlaspi arvense) SOMF12295 (ex Sisymbrium loeselii) SMK13752 (ex Capsella bursa-pastoris)

1.00

0.65

B 0.63

1.00

1.00

SMK17254 (ex Capsella bursa-pastoris) SMK15670 (ex Capsella bursa-pastoris)

0.60

0.93

SMK15732 (ex Draba nemorosa)

0.58

Group II

407

SOMF19655 (ex Descurainia sophia) SOMF19659 (ex Diptychocarpus strictus)

0.56

SMK17251 (ex Lepidium virginicum)

0.99

SMK14479 (ex Lepidium apetalum)

1.00

SMK13747 (ex Lepidium apetalum) SMK17312 (ex Lepidium virginicum)

0.61

SOMF18525 (ex Lepidium campestre) A. bliti SMK19835 (ex Amaranthus spinosus)

0.05 Fig. 3. Phylogenetic trees for Albugo candida isolates from various hosts based on the mitochondrial COX2 gene. (A) Bayesian analysis showing mean branch lengths of a 50% majority-rule consensus tree calculated from 9000 trees revealed during a MCMC analysis of one-million generations. Numbers above the branches are the posterior probability values. The number of nucleotide changes between taxa is represented by branch length and the scale bar equals the number of nucleotide substitution per site. The branch of A. bliti is scaled down due to long length. (B) Maximum parsimony analysis showing a 50% majority-rule consensus tree constructed from 11 most parsimonious trees found by heuristic search (20,000 rounds of random addition of sequences and subsequent TBR branching swapping). The tree length is 194 steps, the consistency index (CI) D 0.8093, and the retention index (RI) D 0.9100. Numbers above the branches are the bootstrap values (10,000 replicates, values smaller than 50% not shown). An asterisk (¤) denotes sequences obtained from GenBank.

fungus and the geographic origins of the host, has not been resolved by the results presented in this study, due to the small number of collections used; and therefore, additional work will be required to conWrm the relationship. The present study has proved the ITS region to be phylogenetically useful, giving a large number of informative characters for delineating relationships within the A. candida complex. The results also showed that the ITS2 region

was more variable, with more phylogenetically informative sites than the ITS1 region; and therefore, the former is a more useful tool for classiWcation of the A. candida complex. The mitochondrially encoded COX2 gene also provided good resolution of the complex in this study. The sequence divergence was too low for intraspeciWc comparisons (with the exception of A. candida from Capsella), but was high enough for interspeciWc comparisons to provide

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separation of fungus/host combinations. One advantage in using this gene for phylogenetic analysis is that length mutations have not been observed, which simplify the sequence alignment. In contrast, the alignment of ITS sequences can be more complicated due to the presence of length mutations. Because variation in alignment has been shown to aVect subsequent phylogenetic analysis, much more signiWcantly than the particular algorithm used in tree construction (Morrison and Ellis, 1997), the sequence analysis of the COX2 gene more accurately reXects the phylogenetic divergence among isolates. Although, in Phytophthora and Pythium the genetic diversity of COX2 has been shown to be higher than that of the ITS sequences (Martin, 2000; Martin and Tooley, 2003), in the present data (Table 2) ITS sequences were more variable and had more informative sites than the COX2 sequences. Nevertheless, this study has shown that both genes are useful in separating the A. candida complex. Comparable with the present study, molecular variability within A. candida isolates has been investigated, at least in part, by ampliWed fragment length polymorphism (AFLP) and ITS1 sequencing (Rehmany et al., 2000) and Feulgen image analysis (Voglmayr and Greilhuber, 1998). Sansome and Sansome (1974) also showed that A. candida on Lunaria has larger chromosomes than that on Capsella, indicating a genetic diVerence between the two isolates. Therefore, from a combination with earlier data, we propose that a high degree of genetic diversity is present within the A. candida complex, and therefore the species should be divided into a number of distinct species. It may be diYcult to reveal morphological characters for diVerentiating the genetically distinct lineages. Therefore, only way to diVerentiate these lineages is probably sequence data. Acknowledgments The authors are grateful to Micheal Priest, the curator of Plant Pathology herbarium (DAR), for helpful comments and suggestion, to the curators of BPI (National Fungus Collections, Beltsville, Maryland, USA), CUP (Plant Pathology Herbarium, Cornell University, New York, USA), and SOMF (Mycological Collection of Institute of Botany, Bulgarian Academy of Sciences, SoWa, Bulgaria) for providing the A. candida specimens, and to staV at KACC (Korean Agricultural Culture Collection) for their technical assistance in the DNA sequencing and phylogenetic analysis. This work was Wnancially supported by a research grant from the Korea Research Foundation (KRF-2003-015-C00611). References APG, 2003. An update of the angiosperm phylogeny group classiWcation for the orders and families of Xowering plants: APG II. Bot. J. Linn. Soc. 141, 399–436. Biga, M.L.B., 1955. Review of the species of the genus Albugo based on the morphology of the conidia. Sydowia 9, 339–358.

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