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Phylogenetic relationships in some Melampsora rusts on Salicaceae assessed using rDNA sequence information
Mycol. Res. 109 (4): 401–409 (April 2005). f The British Mycological Society
401
doi:10.1017/S0953756205002479 Printed in the United Kingdom.
Phylogenetic relationships in some Melampsora rusts on Salicaceae assessed using rDNA sequence information
Ming H. PEI*, Carlos BAYON and Carmen RUIZ Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK. E-mail : [email protected] Received 9 July 2004; accepted 5 January 2005.
The 5k end of the large subunit (LSU) region and the entire internal transcribed spacer (ITS) region of ribosomal DNA were sequenced from 11 species or special forms of Melampsora on Salix, and three species on Populus. For all the species, except for M. larici-epitea and M. coleosporioides, the sequences in both the examined regions were identical within a species. Within M. larici-epitea, f. sp. larici-epitea typica and f. sp. larici-retusae shared the same sequences which slightly differed from that of f. sp. larici-daphnoides. In the LSU region, M. larici-epitea, M. capraearum and the stem-infecting form on S. viminalis shared the same sequence and the Far-Eastern species M. epiphylla differed from them only slightly (p distance 0.006), indicating that they may share a common ancestral lineage. M. amygdalinae and M. coleosporioides formed a distict group (bootstrap value 100% for combined ITS and LSU data). M. larici-epitea and M. ribesii-purpurea, both belonging to the M. epitea complex, appeared to be distinct. The molecular data also suggest that the differences in certain characteristics, such as the thickness of teliospore walls and host specificity, may have evolved relatively recently.
INTRODUCTION The genus Melampsora was established by Castagne in 1843 based on a rust on Euphorbia, M. euphorbiae. The main characteristic of the genus is formation of a crust of sessile, laterally adherent single-celled teliospores on the host surface. Melampsora species are either heteroecious or autoecious. Heteroecious species infect taxonomically very different plants to complete their life-cycle. Autoecious species, in contrast, complete their life-cycle on the same hosts. Typically, Melampsora species produce five forms of spores, i.e. basidiospores, spermatia, aeciospores, urediniospores and teliospores, during the life-cycle. In a number of Melampsora species, however, only uredinial and telial stages have been described and it is not known whether and which aecial hosts are involved in their life-cycle. Of some 80–100 species listed under Melampsora (Hiratsuka & Sato 1982, Hawksworth et al. 1995), more than half were described on Salicaceae, which consists of two genera, Salix (willow) and Populus (poplar). Melampsora species on Salicaceae were classified mainly on the basis of their morphology, alternate hosts, and telial host range. Broadly, there are * Corresponding author.
two types of urediniospores in Melampsora on willow and poplar, one having evenly echinulate, relatively small urediniospores, and the other relatively large urediniospores which are echinulate except for a smooth patch. The position of telia and uredinia on host leaves, the thickness of spore walls and, to a lesser extent, morphology of uredinial paraphyses also serve as criteria to distinguish species. Alternate hosts of Melampsora on Salicaceae include conifers, as well as dicotyledonous and monocotyledonous plants. Positive identification of alternate hosts is achieved by inoculating potential alternate hosts using overwintered telia in the spring. Usually, however, not all the potential alternate hosts are readily available and, if they are available, the maturity and conditions for overwintering of the teliospores and the growing conditions of the plants may still affect the outcome of such inoculations. Of some 12–16 species (sensu stricto) of Melampsora described on Populus, the most widespread and frequent is M. larici-populina. The rust is indigenous to Eurasia, but spread to Australasia in 1970s (van Kraayenoord et al. 1974) and, more recently, to North America (Newcombe & Chastagner 1993). M. alliipopulina occurs from northern Africa to western Asia and in much of south-central Europe. M. populnea was
rDNA of Melampsora willow rusts adopted by Wilson & Henderson (1966) as a collective species to include those which cannot be distinguished in their morphology : M. larici-tremulae, M. magnusiana, M. pinitorqua and M. rostrupii. Recently, Bagyanarayana (1998) proposed that M. allii-populina be placed under P. populnea as f. sp. allii-populina. However, M. allii-populina differs from other species placed under the M. populnea complex by having thick urediniospore walls. The taxonomy of Melampsora on willow is notoriously difficult. Numerous species of Melampsora have been described on Salix and, excluding various synonyms, at least 30 species (sensu stricto) may occur on this host. The majority of willow Melampsora species were described in the late 19th and early 20th century. Many species were separated by having different alternate or telial hosts, but without clear morphological distinctions between them. Hylander, Jørstad & Nannfeldt (1953) therefore recognsied M. epitea as a complex including a number of species which are morphologically indistinguishable. Likewise, Wilson & Henderson (1966) adopted M. epitea as a collective species and treated those previously established species as races and groups of races. Hiratsuka & Kaneko (1982), and Pei, Royle & Hunter (1993, 1996), also adopted the name M. epitea following the treatment by Hylander et al. (1953). However, the problem remains that, because M. epitea contains many groups widely different in their host range and life-cycle, the name has little bearing on the biological or genetic identity of the rusts grouped under the complex. In the M. epitea complex, M. larici-epitea (the larch-alternating M. epitea) is the most common and widespread. Within M. larici-epitea, six special forms have been recognised in continental Europe (Sydow & Sydow 1915, Ga¨umann 1959), and two ‘races ’ (out of four reported originally) in Japan (Hiratsuka 1932, Hiratsuka & Kaneko 1982). In the UK, three special forms ff.spp., larici-epitea typica (LET), lariciretusae (LR), and larici-daphnoides (LD), were identified within M. larici-epitea (Pei et al. 1996). There is further variation in pathogenicity within a special form. A number of pathotypes (defined according to their pathogenicity on a certain range of willow clones) were identified within LET and within LR (Pei et al. 1996, Pei, Hunter & Ruiz 1999a). Some pathotypes within a special form appear to be genetically differentiated. For example, when three pathotypes belonging to LET were examined using the amplified fragment length polymorphism (AFLP), the cluster analysis placed them into three distinct groups (Pei et al. 2002). Of other willow Melampsora species included in this study, M. salicis-albae, M. larici-pentandrae, M. amygdalinae and M. coleosporioides occur on willows belonging to subgen. Salix. M. salicis-albae alternates on Allium, and M. larici-pentandrae on Larix. For M. coleosporioides, no alternate host is known and M. amygdalinae is the only known autoecious species
402 on willow. M. epiphylla, M. capraearum and M. ribesiipurpureae occur on willows belonging to subgen. Vetrix, with M. epiphylla and M. capraearum alternating on Larix, and M. ribesii-purpurea on Ribes. The taxonomic status of the stem-infecting form (SIF) of Melampsora is uncertain. It occurs on S. viminalis clones in the British Isles and exists in nature as an asexual (uredinial) population (Pei, Royle & Hunter 1995, Pei & Ruiz 2000). Ribosomal DNA (rDNA) sequence information is now widely been used to study phylogenetic relationships in fungi. The rDNA is a multigene family with nuclear copies arranged in tandem arrays in eukaryotes. Each unit within a single array consists of the genes coding for SSU and LSU rRNAs. The ITS region (comprising ITS1, 5.8S rRNA gene and ITS2) lies embedded between the SSU and LSU regions. The rDNA sequences encoding the SSU and the LSU rRNAs are highly conserved while the sequences of the ITS region evolve much faster. Among the several examined Puccinia species on cereal and grass, while the LSU rDNA had little variation, the sequences from ITS region could be used to identify closely related species (Zambino & Szabo 1993). In analyses of ITS sequences from pine stem rusts (Vogler & Bruns 1998), Cronartium species formed distinct clades that correlate with host families Fagaceae and Santalaceae. This study was conducted to examine the extent of variation among the sequences of nuclear LSU and the ITS regions and to assess phylogenetic relationships in the Melampsora species on Salicaceae.
MATERIALS AND METHODS Rust isolates/collections We obtained 95 Melampsora collections from willows and 35 from poplars (Table 1). The majority of the samples were from various parts of the UK and collected during 1990–2000. The species identity of the collections was determined by morphological examination, and, in a number of cases, confirmed through inoculations of alternate hosts as described in Pei et al. (1993). The collection JT98/01 from S. babylonica in Tokyo, was identified as M. coleosporoides according to the uredinial and telial morphology. Two collections obtained from north-eastern China were identified as M. coleosporoides (CJ01/2/01) and M. epiphylla (CJ01/1/02). Rust isolates containing a hyphenated code name, e.g. RFCR92-2, were obtained from single uredinia developed after inoculations of detached leaves of host plants with the field rust collections. From each isolate, the urediniospores were bulked up on detached leaves or whole plants. Urediniospores were collected from each collection/isolate using a cyclone spore collector, dried at 4 xC for 1–2 wk and stored at x15 x until use.
S.rstipularis S.rstipularis S. viminalis cv. ‘Bowles Hybrid’ S. viminalis cv. ‘Mullatin’ S. viminalis cv. ‘Mullatin’ S. viminalis cv. ‘Mullatin’ S.rcalodendron cv. ‘DeBiardii 445’ S.rcalodendron cv. ‘DeBiardii 445’ S.rcalodendron cv. ‘DeBiardii 445’ S.rmollissima cv. ‘Q83’ S.rmollissima cv. ‘Q83’ S. burjatica ‘Germany’ S. burjatica ‘Germany’
M. larici-epitea f. sp. larici-epitea typica (LET) LET1 STNI91-1 STNWC91-1 VBP892-2 VMCR96-1, 2, 3, 4, 5 (5 isolates) VMLG96-11, 12, 13, 14, 15 (5 isolates) VMP896-1, 2, 3, 4, 5 (5 isolates) LET3 DBCR96-1, 2, 3, 4, 5 (5 isolates) DBLG96-1, 2, 3, 4, 5 (5 isolates) DBP896-1, 2, 3, 4, 5 (5 isolates) LET4 QLG96-1, 2, 3, 4, 5 (5 isolates) QP896-1, 2, 3, 4, 5 (5 isolates)
M. larici-epitea f. sp. larici-retusae (LR) LR1 GCR96-1, 2, 3, 4, 5 (5 isolates) GLG96-1, 2, 3, 4, 5 (5 isolates)
S. viminalis
S. pierotii S. sp.
S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rsericans ‘Dasyclados’ S.rsericans ‘Dasyclados’ S.rsericans ‘Dasyclados’
Salix triandra ‘Black Maul’
Telial host
S. acutifolia S. daphnoides cv. ‘Meikle’ S. daphnoides cv. ‘Meikle’ S. daphnoides cv. ‘Swindon A’ S. daphnoides cv. ‘Meikle’ S. daphnoides cv. ‘Meikle’ S. daphnoides cv. ‘Meikle’
CJ01/2/01
JT98/01 CJ01/1/01
RFCR92-2 RFCR93-19 RFCR95-S9 RFCR96-21 RFLG96 RFP893-15R RFP896 DASLG96 DASMK95 DASP896
BLACKMAULLG96
Code
M. larici-epitea f. sp. larici-daphnoides (LD) ACNI92 DMNWC96 DMNWC99-1 DSNWC99-1 DM90-1 DM91-1 DAPH90/I-II
M. epiphylla
M. coleosporioides
M. capraearum
M. amygdalinae
Rust species
Table 1. Melamspora isolates/collections sequenced.
Larix Larix
Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix
Larix Larix Larix Larix Larix Larix Larix
Larix
Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix
S. triandra
Aecial host
Scotland N Ireland
N Ireland SW England SW England Scotland N Ireland SW England Scotland N Ireland SW England N Ireland SW England
N Ireland SW England SW England SW England SW England SW England SW England
Jilin, China
Tokyo, Japan Jilin, China
Scotland Scotland Scotland Scotland N Ireland SW England SW England N Ireland N England SW England
N Ireland
Collection site
1996 1996
1996 1991 1992 1996 1996 1996 1996 1996 1996 1996 1996
1992 1996 1999 1999 1990 1991 1990
2001
1998 2001
1992 1993 1995 1996 1996 1993 1996 1996 1995 1996
1996
Year
AY444790
AY444789
AY444792
AY652951 AY652950
AY444781
AY444782
LSU
AY444778
AY444777
AY652947
AY652948 AY652949
AY444779
AY444776
ITS
GenBank accession nos.
M. H. Pei, C. Bayon and C. Ruiz 403
–
S. viminalis cv. ‘Bowles Hybrid’ S. alba cv. ‘Caerulea Oath’ S. alba cv. ‘Caerulea Tree 60’ P. deltoidesrP. trichocarpa ‘Beaupre´ ’ P.rberolinensis P. cathayana P. deltoidesrP. nigra ‘Ghoy’ P. deltoidesrP. nigra ‘Ghoy’ P. deltoidesrP. nigra ‘Ghoy’ P. deltoidesrP. nigra cv. ‘Ghoy’ P. deltoidesrP. trichocarpa cv. ‘Boelare’ P. deltoidesrP. trichocarpa cv. ‘Donk’ P. deltoidesrP. trichocarpa cv. ‘Donk’ P. deltoidesrP. trichocarpa cv. ‘Beaupre´ ’ P. deltoidesrP. trichocarpa cv. ‘Beaupre´ ’ P. deltoidesrP. trichocarpa cv. ‘Beaupre´ ’ P. deltoidesrtrichocarpa cv. ‘Boelare ’ P. laurifolia
M. salcis-albae Kleb. ALBANWC96/10 ALBANWC98/23
M. allii- populina Kleb. BEAUPMK95
M. larici-populina Kleb. BEROAH00-1 CATHAAH00-1 GHOYAH00-2 GHOYSM00-1 GHOYSM00-2
M. larici-populina Kleb. GHOYSM00-2 BOEMKP97-1, 2 (2 Isolates) DONKAH00-1 DONKAH00-2 BEAUPMK97-1, 2 (2 Isolate) BEAUPNM98 BEAUPSM00-2 BOEP796-1 LAURIAH00-2
Larix Larix Larix Larix Larix Larix Larix Larix Larix
Larix Larix Larix Larix Larix
Allium
Allium Allium
Larix Ribes Ribes Ribes Ribes Ribes
S. purpurea ‘012’ S. purpurea ‘012’ S. purpurea ‘012’ S. purpurea ‘012’ S. purpurea cv. ‘Uralensis’
Larix Larix Larix Larix Larix
Larix Larix Larix Larix
Aecial host
M. ribesii-purpureae Kleb. PURP897-1 PURP898-1 PURLG96 PURLG99-1 PURNWC91 ‘Stem-infecting form’ (SIF) VBP893-S1
S. pentandra cv. ‘Dark French’ S. pentandra cv. ‘Dark French’ S. pentandra cv. ‘MacMillan Bloedel ’ S. pentandra cv. ‘Murray Forest’ S. pentandra cv. ‘Patent Lumley’
M. larici-pentandrae Kleb. PENTNWC97/093/03 PENTNWC98/093/03 PENTNWC00093/01 PENTNWC97/093/02 PENTNWC00/093/04
Telial host
S. burjatica ‘Germany’ S. burjatica ‘Korso’ S. burjatica ‘Korso’ S. burjatica ‘Korso’
Code
M. larici-epitea f. sp. larici-retusae LR1 GP896-1, 2, 3, 4, 5 (5 isolates) KCR92-1 LR2 KLG92-6 LR3 KMK92-10
Rust species
Table 1. (Cont.)
SW England N England SE England SE England N England SW England SW England SW England SE England
SE England SE England SE England SW England SW England
N England
SW England SW England
SW England
SW England SW England N Ireland N Ireland SW England
SW England SW England SW England SW England SW England
SW England Scotland N Ireland N England
Collection site
2000 1997 2000 2000 1997 1998 2000 1996 2000
2000 2000 2000 2000 2000
1995
1996 1998
1993
1997 1998 1996 1999 1991
1997 1998 2000 1997 2000
1996 1992 1992 1992
Year
AY444785
AY444784
AY444788
AY444787
AY444791
AY444783
LSU
AY444774
AY444773
AY444775
AY444780
AY444770
AY444771
ITS
GenBank accession nos.
rDNA of Melampsora willow rusts 404
M. H. Pei, C. Bayon and C. Ruiz
405
2000 2000 SE England SE England Pinus ? Pinus ? M. populnea (Pers.) Karst. AAH00-1 GrAAH00-1
P. alba P. grandidentatarP. alba
SE England N England N England SE England N England N England SE England SE England SE England SE England SW England SW England MAXIMAH00-3 VERMK96-1 VERMK97-1 ROBUSTAAH00-1 ROBUSTAMK95-1 ROBUSTAMKP95-1 SPIJK AH00-2 SPIJKAHD2 SIMONIIAH00-1 SZECHENICAAH00-2 BLOMNWC98-2 TRICHOSM00-2
P. maximowiczi P. nigra cv. ‘Vereecken ’ P. nigra cv. ‘Vereecken ’ P. nigrarP. deltoids cv. ‘Robusta’ P. nigrarP. deltoides cv. ‘Robusta’ P. nigrarP. deltoides cv. ‘Robusta’ P. nigrarP. Deltoides cv. ‘Spijk’ P. nigra x deltoides cv. ‘Spijk’ P. simonii P. szechuanica P. trichocarpa cv. ‘Blom’ P. trichocarparP. trichocarpa
Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix
2000 1996 1997 2000 1995 1995 2000 2000 2000 2000 1998 2000
AY444786
AY444772
DNA extraction and sequencing Genomic DNA was extracted from 2–5 mg rust urediniospores using the method described in Pei & Ruiz (2000). The LSU rDNA region was amplified using the primer pair 5k-GCATATCAATAAGCGGAGAAAAG, corresponding to the positions 41–63 of the nuclear LSU of Saccharomyces cerevisae (Geogiev et al. 1981), and 5k-GGGTCCGTGTTTCAAGACGG (Gueho et al. 1989). The internal transcribed spacer (ITS) region was amplified using the primer pair ITS1 (5k-TCCGTAGGTGAACCTGCGG) and ITS4 (5kTCCTTCCGCTTATTGATATGC) (White et al. 1990). PCR was performed in a Perkin Elmer 9700 thermocycler using the profile of 65 x for 5 min, then 25 cycles of 94 x for 30 s, 56 x for 30 s and 72 x for 1 min, followed by a final extension period at 72 x for 10 min. PCR products were visualised in 1.5 % agarose gels and cleaned using the Qiaquick PCR Purification Kit (Qiagen, Hilden). Purified DNA was sequenced directly using the two pairs of primers in conjunction with BigDyeTM Terminator Cycle Sequencing Reaction Kit (PE Applied Biosystems, Foster City, CA). Sequencing products were resolved on an ABI (Strech 377) automated sequencer.
Data analyses The data sets for LSU and ITS regions were initially analysed separately using the pine stem rust Cronartium ribicola as outgroup. The LSU sequence information of C. ribicola was provided by Szabo & Bruns (pers. comm., GenBank AF522166), and the ITS by Vogler & Bruns (1998 ; GenBank L76499). Sequences were aligned using ClustalW version 1.8 (http://www.ebi.ac.uk/clustalw/). MEGA version 2.01 (Kumar et al. 2001) was used to calculate the proportion of different nucleotides between two sequences (p distance) (Nei & Kumar 2000) by deleting gaps on a pairwise basis. Phylogenetic trees were constructed using the Kimura 2-parameter model and the neighbour-joining algorithm in PAUP 4.0 beta 10 (Swofford 2002). The robustness of internal branches was tested using bootstrap analyses (1000 replicates). The incongruence length difference (ILD) test (Farris et al. 1994, 1995) was conducted to evaluate whether incongruence between the two nuclear rDNA data sets might be present. The ILD test was performed using partition-homogeneity test in PAUP 4.0 beta 10 by treating gaps as missing data, with branch-and-bound search, tree bisectionreconnection (TBR) branch swapping and MulTrees options and 1000 replicates. Then the data sets for the LSU and the ITS regions were combined. The combined data were analysed using the neighbourjoining method and the resulting tree was tested using bootstrap analysis (1000 replicates) in Paup 4.0 beta 10.
rDNA of Melampsora willow rusts
406 Stem-infecting form 71
63 61
92 99
M. capraearum M. larici-epitea ff. spp. LET & LR M. larici-epitea f.sp. LD M. epiphylla M. amygdalinae M. coleosporioides (CJ01/1/01) M. coleosporioides (JT98/01) M. salicis-albeae
71 77 62 99 98
56
M. allii-populina 53
51
68
M. ribesii-populina 57
M. populnea M. larici-pentantrae M. larici-populina Cronartium ribicola
(a)
(b)
Fig. 1. Bootstrap consensus neighbour-joining trees based on the sequences of LSU (a) and ITS (b) regions of rDNA of Melampsora species/forms on Salicaceae. Bootstrap values >50 % are shown above branches.
RESULTS
ITS sequences
LSU sequences
All samples except for Melampsora larici-populina produced a single 665–667 bp fragment. A part of sequences obtained from the Japanese M. coleosporioides sample was ambiguous and, therefore, only 544 characters were used. With all other samples, all the characters excluding the priming sites (625–643 bp in total) were used. The GC content of the region was 38.6–40.5 %. All M. larici-populina isolates produced a fragment containing a 16 bp insertion in the ITS2 region. Except for M. larici-epitea and M. coleosporioides, sequences among all samples within the same species were identical. Within M. larici-epitea, LET and LR produced identical sequences. The isolates belonging to LD of M. larici-epitea differed from LET and LR by three characters (p distance=0.005). There was a 10-character difference between M. coleosporioides from Japan and that from China (p distance=0.018). Of 703 characters aligned (including C. ribicola), 208 were variable and 49 were parsimony-informative. The average p distance among the Melampsora rust isolates/collections was 0.038. The neighbour-joining consensus tree (Fig. 1b) placed M. larici-epitea, M. capraearum, SIF and M. epiphylla in the same clade (bootstrap value 71 %) and further grouped the three special forms of M. larici-epitea, LET, LR and LD, into one clade (bootstrap value 77%).
All collections and isolates produced a single 585–590 base-pair (bp) long fragment from the nuclear LSU region. Excluding the priming sites, 547 characters were used for Melampsora larici-populina and 542 for the remainder. Parts of the sequences of both M. coleosporioides samples were ambiguous (appeared to be polymorphic within a sample) and, therefore, only 471 clearly resolved characters were used for the analyses. The GC content of the LSU region was 37.1–40.3 %. For all the species, except for M. lariciepitea, LSU sequences were identical among all samples within a species. The LSU sequences were identical in the samples belonging to M. capraearum, LET and LR of M. larici-epitea, and the SIF. Melampsora larici-epitea LD differed from other M. larici-epitea samples only by a single character (p distance=0.002). Of 549 aligned characters, including C. ribicola, 87 were variable and 24 were parsimony-informative. The average p distance for the examined LSU region was 0.019. The largest distance among Melampsora isolates on Salicaceae was found between M. allii-populina and M. coleosporioides (p distance=0.034). There were only three nucleotides differences (p distance=0.006) between the Far-Eastern species M. epiphylla as compared to M. larici-epitea and M. capraearum. The neighbourjoining consensus tree loosely (bootstrap value 63%) placed M. larici-epitea, M. capraearum and SIF in the same clade (Fig. 1a).
Combined data The IDL test gave P=0.37, indicating that it was unlikely that the two sets of data were incongruent. In the
M. H. Pei, C. Bayon and C. Ruiz
407 69 57
M. larici-epitea ff. spp. LET & LR M. larici-epitea f. sp. LD M. capraearum
91
M. epiphylla Stem-infecting form
86
M. amygdalinae
60
M. coleosporioides (CJ01/1/01)
100 59
M. coleosporioides (JT98/01) M. populnea M. ribesii-purpureae
65
M. allii-populina
62 52
M. salicis-albae M. larici-pentandrae M. larici-populina Cronartium ribicola
Fig. 2. Neighbour-joining consensus tree based on the combined data of LSU and Its sequences of Melampsora species/forms on Salicaceae. Bootstrap values >50 % are shown above branches.
neighbour-joining consensus tree derived from the combined data (Fig. 2), Melampsora larici-epitea, M. capraearum, SIF and M. epiphylla formed a distinct cluster (bootstrap value 91 %) and the three formae speciales of M. larici-epitea were further grouped together (bootstrap value 69 %). Melampsora amygdalinae and M. coleospoioides formed a distinct group (bootstrap value 100 %), which was placed in the same clade as M. larici-epitea, M. capraearum, SIF and M. epiphylla. For the rest, the clusters were less well supported (bootstrap values f65%).
DISCUSSION The present results reveal an insight into the littleknown evolutionary relationships between several species and forms of Melampsora on Salicaceae. M. lariciepitea, M. capraearum and the stem-infecting form of Melampsora (SIF) shared the same sequence in the examined LSU region, but slightly differed (p distance=0.010–0.014) in the ITS region. The Far-eastern species M. epiphylla differed from these three species/ special forms only at three bases in the examined LSU region (p distance=0.006). All the four species/special forms were placed in a distinct cluster (bootstrap value 91 %) when the data from both regions were combined. M. larici-epitea, M. capraearum and M. epiphylla are all macrocyclic and alternate on larch, but differ in their telial morphology. M. larici-epitea forms mainly hypophyllous, subepidermal telia and produces teliospores with evenly thin walls, while M. capraearum and M. epiphylla form epiphyllous, subcuticular telia and produce teliospores with a thickened upper wall, up to 10 mm thick in M. capraearum and 3.5–5 mm in M. epiphylla. Of all Melampsora species on willow, M. larici-epitea has the widest host range which overlaps
with that of M. capraearum. Both M. larici-epitea and M. capraearum are widely distributed in Eurasia. Melampsora epiphylla, on the other hand, is confined to the Far East. The present results suggest that M. lariciepitea, M. capraearum and M. epiphylla have a common ancestral lineage. It appears that the differences in certain characteristics, such as the thickness of teliospore wall and host specificity have evolved relatively recently. Our results suggest that SIF is closely related to M. larici-epitea and M. capraearum. The SIF is highly specialised in its pathogenicity, found only on willow genotypes belonging to S. viminalis, which also serves as the telial host of M. larici-epitea (Pei, Royle & Hunter 1995, 1996). M. larici-epitea infects expanded willow leaves and readily completes a full sexual lifecycle, during which five spore stages are produced (Pei et al. 1993, 1996). In contrast, SIF infects shoot tips and young stems, and causes stem cankers (Pei et al. 1995). Although SIF can produce teliospores morphologically similar to M. larici-epitea under laboratory conditions, the AFLP evidence suggested that it is an asexual population and may have a clonal lineage (Pei et al. 1995, Pei & Ruiz 2000). It is known that a heteroecious, macrocyclic rust species may shorten its lifecycle to produce ‘correlated species ’ (Cummins 1959). The present results indicate that SIF is likely to have evolved relatively recently from its heteroecious ancestor by shifting its niche from fully expanded leaves to very young leaves and shoots. Its ability to overwinter as the asexual uredinial stage on the telial host may have facilitated the shortening of its life-cycle. Similar relationships between long- and short-cycled species have also been reported in pine stem rusts (Vogler & Bruns 1998, Hantula et al. 2002). By comparing ITS sequences, Vogler & Bruns (1998) concluded that some autoecious, short-cycled Peridermium species may have
rDNA of Melampsora willow rusts arisen from the ancestors of heteroecious, macrocyclic Cronartium species. Within M. larici-epitea, all samples belonging to LD differed from LET and LR at one character in the LSU region and three characters in the ITS region. Previously, crossing experiments were carried out with ff. spp. LET, LR and LD to determine the likelihood of hybridisation among them (Pei, Royle & Hunter 1999b). No aecia developed when the spermogonia derived from LD isolates were paired with those of either LET or LR. The sequence data from this study provide further evidence that LD is genetically distinct from LET or LR. The sequences of LET and LR were identical in both the examined rDNA regions. However, this may not necessarily indicate the occurrence of gene exchange between them. In previous crossing experiments, aecia did form when LET was used as spermogonial receptor and LR as donor (not vice versa), but the F1 hybrids between LET and LR were only weakly pathogenic to parental hosts and predominantly sterile (Pei et al. 1999b). Such results indicate that LET and LR are genetically disticnt, even if their ribosomal gene sequences obtained in the present study are identical. Tian et al. (2004) recently showed that the ITS sequences were identical among rust collections which appeared to be M. larici-populina from various parts of China. DNA genes evolve cohesively within a single species, and exhibit only limited sequence divergence within the same individual or between individuals of the same species (Arnheim et al. 1980). In contrast, comparisons between species show normal levels of sequence divergence. The combination of these two observations can be referred to as concerted evolution (Dover 1982). The consistency of rDNA sequences within a given Melampsora taxon may provide the opportunity for the development of species-specific PCR primers to identify Melampsora species on Salicaceae. This approach can be particularly valuable in identification of those species or groups which are genetically separate but morphologically indistinguishable. When the ITS sequences of M. larici-populina obtained in this study were compared with those from Chinese specimens (Tian et al. 2004), there was a difference of only 2 bp. A slight variation in ITS regions within a special form has been reported in other rust species. For example, when the ITS regions of a number of Puccinia species on cereals and grasses were examined, strains in several special forms differed at 1–3 bases by substitutions or deletions/insertions (Zambino & Szabo 1993). In this study, the two M. coleosporioides collections, one from north-eastern China and one from Japan, shared the same LSU sequence. However, there was a noticeable difference (p distance=0.018) in the ITS region. The host of the Chinese collection grew on the banks of Shonghua River and appeared to belong to the subgenus Salix. It appears that the rust which can be classified as
408 M. coleosporioides according to morphology may be highly heterogeneous. The three Melampsora species on poplars, M. laricipopulina, M. allii-populina and M. populnea, did not form a group distinct from those on willows, and neither did the analyses separate the species with relatively small, evenly echinulate urediniospores (M. larici-epitea, M. capraearum, SIF, M. epiphylla, M. ribesii-purpureae and M. populnea) from those with relatively large, unevenly echinulate urediniospores (the remainder). Our analyses have placed M. amygdalinae, the only autoecious species among willow Melampsora spp., in the same cluster as the two collections identified as M. coleosporioides, in which only uredinial and telial stages are known (bootstrap value 100 % for the combined data). This group was placed in the same clade as M. larici-epitea, M. capraearum, SIF, and M. epiphylla. M. amygdalinae and M. coleosporioides occur on willows belonging to subgen. Salix, while M. larici-epitea, M. capraearum, SIF, and M. epiphylla, occur on willows classically grouped under subgen. Vetrix. The present data suggest that these two groups are closely related. In this study, no close relationships were found between M. ribesii-purpureae and M. larici-epitea, both of which are currently placed in the M. epitea complex. Melampsora ribesii-purpureae is morphologically indistinguishable from M. larici-epitea but alternates on Ribes to complete its full life-cycle. The present data provide some indication that the speciation between M. ribesii-purpureae and M. larici-epitea may have occurred relatively early. The M. epitea complex contains many heteroecious species (sensu stricto) or groups whose aecial hosts include Abies, Euonymus, Larix, Ribes, Saxifraga, Viola and some Orchidaceae. Among the Melampsora on poplars, the M. populnea complex also contains several species alternating on Larix, Pinus, Mercurialis, Chelidonii and Corydalis. Further studies are necessary to resolve the evolutionary lineage of the Melampsora rusts on Salicaceae and to determine the relationships among those placed under the M. epitea and M. populnea complexes. The aecial stage is considered to be important in studies of rust phylogeny (Leppik 1953). Melampsora species form relatively simple aecia with no or only rudimentary peridia, and occur on a diverse range of plants, including the members of Pinaceae. Usually, aecial hosts of rust genera on Pinaceae are confined to a single host genus. For example, Uredinopsis, Milesina, Hyalospora and Melampsorella occur on Abies, Coleosporium and Cronartium on Pinus, Melampsoridium on Larix and Chryxomyxa on Picea. In contrast, all of the four genera of Pinaceae host one or more species of Melampsora. Therefore, Durrieu (1980) suggested that Melampsora could be the oldest genus attacking Pinaceae. Maier et al. (2003) examined phylogenetic relationships in rusts using the sequence data from the LSU region of rDNA from some 50 rust species belonging to 25 genera. Three Melampsora
M. H. Pei, C. Bayon and C. Ruiz species, M. helioscopiae, M. euphorbiae and M. hypericorum, were included in their study. From their results, the genus Melampsora appeared to be somewhat distant from other rusts examined. Their neighbourjoining analysis weakly supported the separation of Melampsora from all other genera examined (bootstrap value=62 %). Further molecular data from wider regions of the rust genome and from a wider range of species will provide a better understanding of evolutionary relationships among the taxa within Melampsora, and between different groups of rusts in Uredinales. ACKNOWLEDGEMENTS We would like to thank Pascal Frey for his comments on the manuscript. This research was funded by the European Union (project QLK5-1999-01585) and the Department for Environment, Food and Rural Affairs (DEFRA); Rothamsted Research receives grant-aided support from the UK Biotechnology and Biological Sciences Research Council.
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Corresponding Editor: R. W. S. Weber
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doi:10.1017/S0953756205002479 Printed in the United Kingdom.
Phylogenetic relationships in some Melampsora rusts on Salicaceae assessed using rDNA sequence information
Ming H. PEI*, Carlos BAYON and Carmen RUIZ Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK. E-mail : [email protected] Received 9 July 2004; accepted 5 January 2005.
The 5k end of the large subunit (LSU) region and the entire internal transcribed spacer (ITS) region of ribosomal DNA were sequenced from 11 species or special forms of Melampsora on Salix, and three species on Populus. For all the species, except for M. larici-epitea and M. coleosporioides, the sequences in both the examined regions were identical within a species. Within M. larici-epitea, f. sp. larici-epitea typica and f. sp. larici-retusae shared the same sequences which slightly differed from that of f. sp. larici-daphnoides. In the LSU region, M. larici-epitea, M. capraearum and the stem-infecting form on S. viminalis shared the same sequence and the Far-Eastern species M. epiphylla differed from them only slightly (p distance 0.006), indicating that they may share a common ancestral lineage. M. amygdalinae and M. coleosporioides formed a distict group (bootstrap value 100% for combined ITS and LSU data). M. larici-epitea and M. ribesii-purpurea, both belonging to the M. epitea complex, appeared to be distinct. The molecular data also suggest that the differences in certain characteristics, such as the thickness of teliospore walls and host specificity, may have evolved relatively recently.
INTRODUCTION The genus Melampsora was established by Castagne in 1843 based on a rust on Euphorbia, M. euphorbiae. The main characteristic of the genus is formation of a crust of sessile, laterally adherent single-celled teliospores on the host surface. Melampsora species are either heteroecious or autoecious. Heteroecious species infect taxonomically very different plants to complete their life-cycle. Autoecious species, in contrast, complete their life-cycle on the same hosts. Typically, Melampsora species produce five forms of spores, i.e. basidiospores, spermatia, aeciospores, urediniospores and teliospores, during the life-cycle. In a number of Melampsora species, however, only uredinial and telial stages have been described and it is not known whether and which aecial hosts are involved in their life-cycle. Of some 80–100 species listed under Melampsora (Hiratsuka & Sato 1982, Hawksworth et al. 1995), more than half were described on Salicaceae, which consists of two genera, Salix (willow) and Populus (poplar). Melampsora species on Salicaceae were classified mainly on the basis of their morphology, alternate hosts, and telial host range. Broadly, there are * Corresponding author.
two types of urediniospores in Melampsora on willow and poplar, one having evenly echinulate, relatively small urediniospores, and the other relatively large urediniospores which are echinulate except for a smooth patch. The position of telia and uredinia on host leaves, the thickness of spore walls and, to a lesser extent, morphology of uredinial paraphyses also serve as criteria to distinguish species. Alternate hosts of Melampsora on Salicaceae include conifers, as well as dicotyledonous and monocotyledonous plants. Positive identification of alternate hosts is achieved by inoculating potential alternate hosts using overwintered telia in the spring. Usually, however, not all the potential alternate hosts are readily available and, if they are available, the maturity and conditions for overwintering of the teliospores and the growing conditions of the plants may still affect the outcome of such inoculations. Of some 12–16 species (sensu stricto) of Melampsora described on Populus, the most widespread and frequent is M. larici-populina. The rust is indigenous to Eurasia, but spread to Australasia in 1970s (van Kraayenoord et al. 1974) and, more recently, to North America (Newcombe & Chastagner 1993). M. alliipopulina occurs from northern Africa to western Asia and in much of south-central Europe. M. populnea was
rDNA of Melampsora willow rusts adopted by Wilson & Henderson (1966) as a collective species to include those which cannot be distinguished in their morphology : M. larici-tremulae, M. magnusiana, M. pinitorqua and M. rostrupii. Recently, Bagyanarayana (1998) proposed that M. allii-populina be placed under P. populnea as f. sp. allii-populina. However, M. allii-populina differs from other species placed under the M. populnea complex by having thick urediniospore walls. The taxonomy of Melampsora on willow is notoriously difficult. Numerous species of Melampsora have been described on Salix and, excluding various synonyms, at least 30 species (sensu stricto) may occur on this host. The majority of willow Melampsora species were described in the late 19th and early 20th century. Many species were separated by having different alternate or telial hosts, but without clear morphological distinctions between them. Hylander, Jørstad & Nannfeldt (1953) therefore recognsied M. epitea as a complex including a number of species which are morphologically indistinguishable. Likewise, Wilson & Henderson (1966) adopted M. epitea as a collective species and treated those previously established species as races and groups of races. Hiratsuka & Kaneko (1982), and Pei, Royle & Hunter (1993, 1996), also adopted the name M. epitea following the treatment by Hylander et al. (1953). However, the problem remains that, because M. epitea contains many groups widely different in their host range and life-cycle, the name has little bearing on the biological or genetic identity of the rusts grouped under the complex. In the M. epitea complex, M. larici-epitea (the larch-alternating M. epitea) is the most common and widespread. Within M. larici-epitea, six special forms have been recognised in continental Europe (Sydow & Sydow 1915, Ga¨umann 1959), and two ‘races ’ (out of four reported originally) in Japan (Hiratsuka 1932, Hiratsuka & Kaneko 1982). In the UK, three special forms ff.spp., larici-epitea typica (LET), lariciretusae (LR), and larici-daphnoides (LD), were identified within M. larici-epitea (Pei et al. 1996). There is further variation in pathogenicity within a special form. A number of pathotypes (defined according to their pathogenicity on a certain range of willow clones) were identified within LET and within LR (Pei et al. 1996, Pei, Hunter & Ruiz 1999a). Some pathotypes within a special form appear to be genetically differentiated. For example, when three pathotypes belonging to LET were examined using the amplified fragment length polymorphism (AFLP), the cluster analysis placed them into three distinct groups (Pei et al. 2002). Of other willow Melampsora species included in this study, M. salicis-albae, M. larici-pentandrae, M. amygdalinae and M. coleosporioides occur on willows belonging to subgen. Salix. M. salicis-albae alternates on Allium, and M. larici-pentandrae on Larix. For M. coleosporioides, no alternate host is known and M. amygdalinae is the only known autoecious species
402 on willow. M. epiphylla, M. capraearum and M. ribesiipurpureae occur on willows belonging to subgen. Vetrix, with M. epiphylla and M. capraearum alternating on Larix, and M. ribesii-purpurea on Ribes. The taxonomic status of the stem-infecting form (SIF) of Melampsora is uncertain. It occurs on S. viminalis clones in the British Isles and exists in nature as an asexual (uredinial) population (Pei, Royle & Hunter 1995, Pei & Ruiz 2000). Ribosomal DNA (rDNA) sequence information is now widely been used to study phylogenetic relationships in fungi. The rDNA is a multigene family with nuclear copies arranged in tandem arrays in eukaryotes. Each unit within a single array consists of the genes coding for SSU and LSU rRNAs. The ITS region (comprising ITS1, 5.8S rRNA gene and ITS2) lies embedded between the SSU and LSU regions. The rDNA sequences encoding the SSU and the LSU rRNAs are highly conserved while the sequences of the ITS region evolve much faster. Among the several examined Puccinia species on cereal and grass, while the LSU rDNA had little variation, the sequences from ITS region could be used to identify closely related species (Zambino & Szabo 1993). In analyses of ITS sequences from pine stem rusts (Vogler & Bruns 1998), Cronartium species formed distinct clades that correlate with host families Fagaceae and Santalaceae. This study was conducted to examine the extent of variation among the sequences of nuclear LSU and the ITS regions and to assess phylogenetic relationships in the Melampsora species on Salicaceae.
MATERIALS AND METHODS Rust isolates/collections We obtained 95 Melampsora collections from willows and 35 from poplars (Table 1). The majority of the samples were from various parts of the UK and collected during 1990–2000. The species identity of the collections was determined by morphological examination, and, in a number of cases, confirmed through inoculations of alternate hosts as described in Pei et al. (1993). The collection JT98/01 from S. babylonica in Tokyo, was identified as M. coleosporoides according to the uredinial and telial morphology. Two collections obtained from north-eastern China were identified as M. coleosporoides (CJ01/2/01) and M. epiphylla (CJ01/1/02). Rust isolates containing a hyphenated code name, e.g. RFCR92-2, were obtained from single uredinia developed after inoculations of detached leaves of host plants with the field rust collections. From each isolate, the urediniospores were bulked up on detached leaves or whole plants. Urediniospores were collected from each collection/isolate using a cyclone spore collector, dried at 4 xC for 1–2 wk and stored at x15 x until use.
S.rstipularis S.rstipularis S. viminalis cv. ‘Bowles Hybrid’ S. viminalis cv. ‘Mullatin’ S. viminalis cv. ‘Mullatin’ S. viminalis cv. ‘Mullatin’ S.rcalodendron cv. ‘DeBiardii 445’ S.rcalodendron cv. ‘DeBiardii 445’ S.rcalodendron cv. ‘DeBiardii 445’ S.rmollissima cv. ‘Q83’ S.rmollissima cv. ‘Q83’ S. burjatica ‘Germany’ S. burjatica ‘Germany’
M. larici-epitea f. sp. larici-epitea typica (LET) LET1 STNI91-1 STNWC91-1 VBP892-2 VMCR96-1, 2, 3, 4, 5 (5 isolates) VMLG96-11, 12, 13, 14, 15 (5 isolates) VMP896-1, 2, 3, 4, 5 (5 isolates) LET3 DBCR96-1, 2, 3, 4, 5 (5 isolates) DBLG96-1, 2, 3, 4, 5 (5 isolates) DBP896-1, 2, 3, 4, 5 (5 isolates) LET4 QLG96-1, 2, 3, 4, 5 (5 isolates) QP896-1, 2, 3, 4, 5 (5 isolates)
M. larici-epitea f. sp. larici-retusae (LR) LR1 GCR96-1, 2, 3, 4, 5 (5 isolates) GLG96-1, 2, 3, 4, 5 (5 isolates)
S. viminalis
S. pierotii S. sp.
S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rhirtei ‘Reifenweide’ S.rsericans ‘Dasyclados’ S.rsericans ‘Dasyclados’ S.rsericans ‘Dasyclados’
Salix triandra ‘Black Maul’
Telial host
S. acutifolia S. daphnoides cv. ‘Meikle’ S. daphnoides cv. ‘Meikle’ S. daphnoides cv. ‘Swindon A’ S. daphnoides cv. ‘Meikle’ S. daphnoides cv. ‘Meikle’ S. daphnoides cv. ‘Meikle’
CJ01/2/01
JT98/01 CJ01/1/01
RFCR92-2 RFCR93-19 RFCR95-S9 RFCR96-21 RFLG96 RFP893-15R RFP896 DASLG96 DASMK95 DASP896
BLACKMAULLG96
Code
M. larici-epitea f. sp. larici-daphnoides (LD) ACNI92 DMNWC96 DMNWC99-1 DSNWC99-1 DM90-1 DM91-1 DAPH90/I-II
M. epiphylla
M. coleosporioides
M. capraearum
M. amygdalinae
Rust species
Table 1. Melamspora isolates/collections sequenced.
Larix Larix
Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix
Larix Larix Larix Larix Larix Larix Larix
Larix
Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix
S. triandra
Aecial host
Scotland N Ireland
N Ireland SW England SW England Scotland N Ireland SW England Scotland N Ireland SW England N Ireland SW England
N Ireland SW England SW England SW England SW England SW England SW England
Jilin, China
Tokyo, Japan Jilin, China
Scotland Scotland Scotland Scotland N Ireland SW England SW England N Ireland N England SW England
N Ireland
Collection site
1996 1996
1996 1991 1992 1996 1996 1996 1996 1996 1996 1996 1996
1992 1996 1999 1999 1990 1991 1990
2001
1998 2001
1992 1993 1995 1996 1996 1993 1996 1996 1995 1996
1996
Year
AY444790
AY444789
AY444792
AY652951 AY652950
AY444781
AY444782
LSU
AY444778
AY444777
AY652947
AY652948 AY652949
AY444779
AY444776
ITS
GenBank accession nos.
M. H. Pei, C. Bayon and C. Ruiz 403
–
S. viminalis cv. ‘Bowles Hybrid’ S. alba cv. ‘Caerulea Oath’ S. alba cv. ‘Caerulea Tree 60’ P. deltoidesrP. trichocarpa ‘Beaupre´ ’ P.rberolinensis P. cathayana P. deltoidesrP. nigra ‘Ghoy’ P. deltoidesrP. nigra ‘Ghoy’ P. deltoidesrP. nigra ‘Ghoy’ P. deltoidesrP. nigra cv. ‘Ghoy’ P. deltoidesrP. trichocarpa cv. ‘Boelare’ P. deltoidesrP. trichocarpa cv. ‘Donk’ P. deltoidesrP. trichocarpa cv. ‘Donk’ P. deltoidesrP. trichocarpa cv. ‘Beaupre´ ’ P. deltoidesrP. trichocarpa cv. ‘Beaupre´ ’ P. deltoidesrP. trichocarpa cv. ‘Beaupre´ ’ P. deltoidesrtrichocarpa cv. ‘Boelare ’ P. laurifolia
M. salcis-albae Kleb. ALBANWC96/10 ALBANWC98/23
M. allii- populina Kleb. BEAUPMK95
M. larici-populina Kleb. BEROAH00-1 CATHAAH00-1 GHOYAH00-2 GHOYSM00-1 GHOYSM00-2
M. larici-populina Kleb. GHOYSM00-2 BOEMKP97-1, 2 (2 Isolates) DONKAH00-1 DONKAH00-2 BEAUPMK97-1, 2 (2 Isolate) BEAUPNM98 BEAUPSM00-2 BOEP796-1 LAURIAH00-2
Larix Larix Larix Larix Larix Larix Larix Larix Larix
Larix Larix Larix Larix Larix
Allium
Allium Allium
Larix Ribes Ribes Ribes Ribes Ribes
S. purpurea ‘012’ S. purpurea ‘012’ S. purpurea ‘012’ S. purpurea ‘012’ S. purpurea cv. ‘Uralensis’
Larix Larix Larix Larix Larix
Larix Larix Larix Larix
Aecial host
M. ribesii-purpureae Kleb. PURP897-1 PURP898-1 PURLG96 PURLG99-1 PURNWC91 ‘Stem-infecting form’ (SIF) VBP893-S1
S. pentandra cv. ‘Dark French’ S. pentandra cv. ‘Dark French’ S. pentandra cv. ‘MacMillan Bloedel ’ S. pentandra cv. ‘Murray Forest’ S. pentandra cv. ‘Patent Lumley’
M. larici-pentandrae Kleb. PENTNWC97/093/03 PENTNWC98/093/03 PENTNWC00093/01 PENTNWC97/093/02 PENTNWC00/093/04
Telial host
S. burjatica ‘Germany’ S. burjatica ‘Korso’ S. burjatica ‘Korso’ S. burjatica ‘Korso’
Code
M. larici-epitea f. sp. larici-retusae LR1 GP896-1, 2, 3, 4, 5 (5 isolates) KCR92-1 LR2 KLG92-6 LR3 KMK92-10
Rust species
Table 1. (Cont.)
SW England N England SE England SE England N England SW England SW England SW England SE England
SE England SE England SE England SW England SW England
N England
SW England SW England
SW England
SW England SW England N Ireland N Ireland SW England
SW England SW England SW England SW England SW England
SW England Scotland N Ireland N England
Collection site
2000 1997 2000 2000 1997 1998 2000 1996 2000
2000 2000 2000 2000 2000
1995
1996 1998
1993
1997 1998 1996 1999 1991
1997 1998 2000 1997 2000
1996 1992 1992 1992
Year
AY444785
AY444784
AY444788
AY444787
AY444791
AY444783
LSU
AY444774
AY444773
AY444775
AY444780
AY444770
AY444771
ITS
GenBank accession nos.
rDNA of Melampsora willow rusts 404
M. H. Pei, C. Bayon and C. Ruiz
405
2000 2000 SE England SE England Pinus ? Pinus ? M. populnea (Pers.) Karst. AAH00-1 GrAAH00-1
P. alba P. grandidentatarP. alba
SE England N England N England SE England N England N England SE England SE England SE England SE England SW England SW England MAXIMAH00-3 VERMK96-1 VERMK97-1 ROBUSTAAH00-1 ROBUSTAMK95-1 ROBUSTAMKP95-1 SPIJK AH00-2 SPIJKAHD2 SIMONIIAH00-1 SZECHENICAAH00-2 BLOMNWC98-2 TRICHOSM00-2
P. maximowiczi P. nigra cv. ‘Vereecken ’ P. nigra cv. ‘Vereecken ’ P. nigrarP. deltoids cv. ‘Robusta’ P. nigrarP. deltoides cv. ‘Robusta’ P. nigrarP. deltoides cv. ‘Robusta’ P. nigrarP. Deltoides cv. ‘Spijk’ P. nigra x deltoides cv. ‘Spijk’ P. simonii P. szechuanica P. trichocarpa cv. ‘Blom’ P. trichocarparP. trichocarpa
Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix Larix
2000 1996 1997 2000 1995 1995 2000 2000 2000 2000 1998 2000
AY444786
AY444772
DNA extraction and sequencing Genomic DNA was extracted from 2–5 mg rust urediniospores using the method described in Pei & Ruiz (2000). The LSU rDNA region was amplified using the primer pair 5k-GCATATCAATAAGCGGAGAAAAG, corresponding to the positions 41–63 of the nuclear LSU of Saccharomyces cerevisae (Geogiev et al. 1981), and 5k-GGGTCCGTGTTTCAAGACGG (Gueho et al. 1989). The internal transcribed spacer (ITS) region was amplified using the primer pair ITS1 (5k-TCCGTAGGTGAACCTGCGG) and ITS4 (5kTCCTTCCGCTTATTGATATGC) (White et al. 1990). PCR was performed in a Perkin Elmer 9700 thermocycler using the profile of 65 x for 5 min, then 25 cycles of 94 x for 30 s, 56 x for 30 s and 72 x for 1 min, followed by a final extension period at 72 x for 10 min. PCR products were visualised in 1.5 % agarose gels and cleaned using the Qiaquick PCR Purification Kit (Qiagen, Hilden). Purified DNA was sequenced directly using the two pairs of primers in conjunction with BigDyeTM Terminator Cycle Sequencing Reaction Kit (PE Applied Biosystems, Foster City, CA). Sequencing products were resolved on an ABI (Strech 377) automated sequencer.
Data analyses The data sets for LSU and ITS regions were initially analysed separately using the pine stem rust Cronartium ribicola as outgroup. The LSU sequence information of C. ribicola was provided by Szabo & Bruns (pers. comm., GenBank AF522166), and the ITS by Vogler & Bruns (1998 ; GenBank L76499). Sequences were aligned using ClustalW version 1.8 (http://www.ebi.ac.uk/clustalw/). MEGA version 2.01 (Kumar et al. 2001) was used to calculate the proportion of different nucleotides between two sequences (p distance) (Nei & Kumar 2000) by deleting gaps on a pairwise basis. Phylogenetic trees were constructed using the Kimura 2-parameter model and the neighbour-joining algorithm in PAUP 4.0 beta 10 (Swofford 2002). The robustness of internal branches was tested using bootstrap analyses (1000 replicates). The incongruence length difference (ILD) test (Farris et al. 1994, 1995) was conducted to evaluate whether incongruence between the two nuclear rDNA data sets might be present. The ILD test was performed using partition-homogeneity test in PAUP 4.0 beta 10 by treating gaps as missing data, with branch-and-bound search, tree bisectionreconnection (TBR) branch swapping and MulTrees options and 1000 replicates. Then the data sets for the LSU and the ITS regions were combined. The combined data were analysed using the neighbourjoining method and the resulting tree was tested using bootstrap analysis (1000 replicates) in Paup 4.0 beta 10.
rDNA of Melampsora willow rusts
406 Stem-infecting form 71
63 61
92 99
M. capraearum M. larici-epitea ff. spp. LET & LR M. larici-epitea f.sp. LD M. epiphylla M. amygdalinae M. coleosporioides (CJ01/1/01) M. coleosporioides (JT98/01) M. salicis-albeae
71 77 62 99 98
56
M. allii-populina 53
51
68
M. ribesii-populina 57
M. populnea M. larici-pentantrae M. larici-populina Cronartium ribicola
(a)
(b)
Fig. 1. Bootstrap consensus neighbour-joining trees based on the sequences of LSU (a) and ITS (b) regions of rDNA of Melampsora species/forms on Salicaceae. Bootstrap values >50 % are shown above branches.
RESULTS
ITS sequences
LSU sequences
All samples except for Melampsora larici-populina produced a single 665–667 bp fragment. A part of sequences obtained from the Japanese M. coleosporioides sample was ambiguous and, therefore, only 544 characters were used. With all other samples, all the characters excluding the priming sites (625–643 bp in total) were used. The GC content of the region was 38.6–40.5 %. All M. larici-populina isolates produced a fragment containing a 16 bp insertion in the ITS2 region. Except for M. larici-epitea and M. coleosporioides, sequences among all samples within the same species were identical. Within M. larici-epitea, LET and LR produced identical sequences. The isolates belonging to LD of M. larici-epitea differed from LET and LR by three characters (p distance=0.005). There was a 10-character difference between M. coleosporioides from Japan and that from China (p distance=0.018). Of 703 characters aligned (including C. ribicola), 208 were variable and 49 were parsimony-informative. The average p distance among the Melampsora rust isolates/collections was 0.038. The neighbour-joining consensus tree (Fig. 1b) placed M. larici-epitea, M. capraearum, SIF and M. epiphylla in the same clade (bootstrap value 71 %) and further grouped the three special forms of M. larici-epitea, LET, LR and LD, into one clade (bootstrap value 77%).
All collections and isolates produced a single 585–590 base-pair (bp) long fragment from the nuclear LSU region. Excluding the priming sites, 547 characters were used for Melampsora larici-populina and 542 for the remainder. Parts of the sequences of both M. coleosporioides samples were ambiguous (appeared to be polymorphic within a sample) and, therefore, only 471 clearly resolved characters were used for the analyses. The GC content of the LSU region was 37.1–40.3 %. For all the species, except for M. lariciepitea, LSU sequences were identical among all samples within a species. The LSU sequences were identical in the samples belonging to M. capraearum, LET and LR of M. larici-epitea, and the SIF. Melampsora larici-epitea LD differed from other M. larici-epitea samples only by a single character (p distance=0.002). Of 549 aligned characters, including C. ribicola, 87 were variable and 24 were parsimony-informative. The average p distance for the examined LSU region was 0.019. The largest distance among Melampsora isolates on Salicaceae was found between M. allii-populina and M. coleosporioides (p distance=0.034). There were only three nucleotides differences (p distance=0.006) between the Far-Eastern species M. epiphylla as compared to M. larici-epitea and M. capraearum. The neighbourjoining consensus tree loosely (bootstrap value 63%) placed M. larici-epitea, M. capraearum and SIF in the same clade (Fig. 1a).
Combined data The IDL test gave P=0.37, indicating that it was unlikely that the two sets of data were incongruent. In the
M. H. Pei, C. Bayon and C. Ruiz
407 69 57
M. larici-epitea ff. spp. LET & LR M. larici-epitea f. sp. LD M. capraearum
91
M. epiphylla Stem-infecting form
86
M. amygdalinae
60
M. coleosporioides (CJ01/1/01)
100 59
M. coleosporioides (JT98/01) M. populnea M. ribesii-purpureae
65
M. allii-populina
62 52
M. salicis-albae M. larici-pentandrae M. larici-populina Cronartium ribicola
Fig. 2. Neighbour-joining consensus tree based on the combined data of LSU and Its sequences of Melampsora species/forms on Salicaceae. Bootstrap values >50 % are shown above branches.
neighbour-joining consensus tree derived from the combined data (Fig. 2), Melampsora larici-epitea, M. capraearum, SIF and M. epiphylla formed a distinct cluster (bootstrap value 91 %) and the three formae speciales of M. larici-epitea were further grouped together (bootstrap value 69 %). Melampsora amygdalinae and M. coleospoioides formed a distinct group (bootstrap value 100 %), which was placed in the same clade as M. larici-epitea, M. capraearum, SIF and M. epiphylla. For the rest, the clusters were less well supported (bootstrap values f65%).
DISCUSSION The present results reveal an insight into the littleknown evolutionary relationships between several species and forms of Melampsora on Salicaceae. M. lariciepitea, M. capraearum and the stem-infecting form of Melampsora (SIF) shared the same sequence in the examined LSU region, but slightly differed (p distance=0.010–0.014) in the ITS region. The Far-eastern species M. epiphylla differed from these three species/ special forms only at three bases in the examined LSU region (p distance=0.006). All the four species/special forms were placed in a distinct cluster (bootstrap value 91 %) when the data from both regions were combined. M. larici-epitea, M. capraearum and M. epiphylla are all macrocyclic and alternate on larch, but differ in their telial morphology. M. larici-epitea forms mainly hypophyllous, subepidermal telia and produces teliospores with evenly thin walls, while M. capraearum and M. epiphylla form epiphyllous, subcuticular telia and produce teliospores with a thickened upper wall, up to 10 mm thick in M. capraearum and 3.5–5 mm in M. epiphylla. Of all Melampsora species on willow, M. larici-epitea has the widest host range which overlaps
with that of M. capraearum. Both M. larici-epitea and M. capraearum are widely distributed in Eurasia. Melampsora epiphylla, on the other hand, is confined to the Far East. The present results suggest that M. lariciepitea, M. capraearum and M. epiphylla have a common ancestral lineage. It appears that the differences in certain characteristics, such as the thickness of teliospore wall and host specificity have evolved relatively recently. Our results suggest that SIF is closely related to M. larici-epitea and M. capraearum. The SIF is highly specialised in its pathogenicity, found only on willow genotypes belonging to S. viminalis, which also serves as the telial host of M. larici-epitea (Pei, Royle & Hunter 1995, 1996). M. larici-epitea infects expanded willow leaves and readily completes a full sexual lifecycle, during which five spore stages are produced (Pei et al. 1993, 1996). In contrast, SIF infects shoot tips and young stems, and causes stem cankers (Pei et al. 1995). Although SIF can produce teliospores morphologically similar to M. larici-epitea under laboratory conditions, the AFLP evidence suggested that it is an asexual population and may have a clonal lineage (Pei et al. 1995, Pei & Ruiz 2000). It is known that a heteroecious, macrocyclic rust species may shorten its lifecycle to produce ‘correlated species ’ (Cummins 1959). The present results indicate that SIF is likely to have evolved relatively recently from its heteroecious ancestor by shifting its niche from fully expanded leaves to very young leaves and shoots. Its ability to overwinter as the asexual uredinial stage on the telial host may have facilitated the shortening of its life-cycle. Similar relationships between long- and short-cycled species have also been reported in pine stem rusts (Vogler & Bruns 1998, Hantula et al. 2002). By comparing ITS sequences, Vogler & Bruns (1998) concluded that some autoecious, short-cycled Peridermium species may have
rDNA of Melampsora willow rusts arisen from the ancestors of heteroecious, macrocyclic Cronartium species. Within M. larici-epitea, all samples belonging to LD differed from LET and LR at one character in the LSU region and three characters in the ITS region. Previously, crossing experiments were carried out with ff. spp. LET, LR and LD to determine the likelihood of hybridisation among them (Pei, Royle & Hunter 1999b). No aecia developed when the spermogonia derived from LD isolates were paired with those of either LET or LR. The sequence data from this study provide further evidence that LD is genetically distinct from LET or LR. The sequences of LET and LR were identical in both the examined rDNA regions. However, this may not necessarily indicate the occurrence of gene exchange between them. In previous crossing experiments, aecia did form when LET was used as spermogonial receptor and LR as donor (not vice versa), but the F1 hybrids between LET and LR were only weakly pathogenic to parental hosts and predominantly sterile (Pei et al. 1999b). Such results indicate that LET and LR are genetically disticnt, even if their ribosomal gene sequences obtained in the present study are identical. Tian et al. (2004) recently showed that the ITS sequences were identical among rust collections which appeared to be M. larici-populina from various parts of China. DNA genes evolve cohesively within a single species, and exhibit only limited sequence divergence within the same individual or between individuals of the same species (Arnheim et al. 1980). In contrast, comparisons between species show normal levels of sequence divergence. The combination of these two observations can be referred to as concerted evolution (Dover 1982). The consistency of rDNA sequences within a given Melampsora taxon may provide the opportunity for the development of species-specific PCR primers to identify Melampsora species on Salicaceae. This approach can be particularly valuable in identification of those species or groups which are genetically separate but morphologically indistinguishable. When the ITS sequences of M. larici-populina obtained in this study were compared with those from Chinese specimens (Tian et al. 2004), there was a difference of only 2 bp. A slight variation in ITS regions within a special form has been reported in other rust species. For example, when the ITS regions of a number of Puccinia species on cereals and grasses were examined, strains in several special forms differed at 1–3 bases by substitutions or deletions/insertions (Zambino & Szabo 1993). In this study, the two M. coleosporioides collections, one from north-eastern China and one from Japan, shared the same LSU sequence. However, there was a noticeable difference (p distance=0.018) in the ITS region. The host of the Chinese collection grew on the banks of Shonghua River and appeared to belong to the subgenus Salix. It appears that the rust which can be classified as
408 M. coleosporioides according to morphology may be highly heterogeneous. The three Melampsora species on poplars, M. laricipopulina, M. allii-populina and M. populnea, did not form a group distinct from those on willows, and neither did the analyses separate the species with relatively small, evenly echinulate urediniospores (M. larici-epitea, M. capraearum, SIF, M. epiphylla, M. ribesii-purpureae and M. populnea) from those with relatively large, unevenly echinulate urediniospores (the remainder). Our analyses have placed M. amygdalinae, the only autoecious species among willow Melampsora spp., in the same cluster as the two collections identified as M. coleosporioides, in which only uredinial and telial stages are known (bootstrap value 100 % for the combined data). This group was placed in the same clade as M. larici-epitea, M. capraearum, SIF, and M. epiphylla. M. amygdalinae and M. coleosporioides occur on willows belonging to subgen. Salix, while M. larici-epitea, M. capraearum, SIF, and M. epiphylla, occur on willows classically grouped under subgen. Vetrix. The present data suggest that these two groups are closely related. In this study, no close relationships were found between M. ribesii-purpureae and M. larici-epitea, both of which are currently placed in the M. epitea complex. Melampsora ribesii-purpureae is morphologically indistinguishable from M. larici-epitea but alternates on Ribes to complete its full life-cycle. The present data provide some indication that the speciation between M. ribesii-purpureae and M. larici-epitea may have occurred relatively early. The M. epitea complex contains many heteroecious species (sensu stricto) or groups whose aecial hosts include Abies, Euonymus, Larix, Ribes, Saxifraga, Viola and some Orchidaceae. Among the Melampsora on poplars, the M. populnea complex also contains several species alternating on Larix, Pinus, Mercurialis, Chelidonii and Corydalis. Further studies are necessary to resolve the evolutionary lineage of the Melampsora rusts on Salicaceae and to determine the relationships among those placed under the M. epitea and M. populnea complexes. The aecial stage is considered to be important in studies of rust phylogeny (Leppik 1953). Melampsora species form relatively simple aecia with no or only rudimentary peridia, and occur on a diverse range of plants, including the members of Pinaceae. Usually, aecial hosts of rust genera on Pinaceae are confined to a single host genus. For example, Uredinopsis, Milesina, Hyalospora and Melampsorella occur on Abies, Coleosporium and Cronartium on Pinus, Melampsoridium on Larix and Chryxomyxa on Picea. In contrast, all of the four genera of Pinaceae host one or more species of Melampsora. Therefore, Durrieu (1980) suggested that Melampsora could be the oldest genus attacking Pinaceae. Maier et al. (2003) examined phylogenetic relationships in rusts using the sequence data from the LSU region of rDNA from some 50 rust species belonging to 25 genera. Three Melampsora
M. H. Pei, C. Bayon and C. Ruiz species, M. helioscopiae, M. euphorbiae and M. hypericorum, were included in their study. From their results, the genus Melampsora appeared to be somewhat distant from other rusts examined. Their neighbourjoining analysis weakly supported the separation of Melampsora from all other genera examined (bootstrap value=62 %). Further molecular data from wider regions of the rust genome and from a wider range of species will provide a better understanding of evolutionary relationships among the taxa within Melampsora, and between different groups of rusts in Uredinales. ACKNOWLEDGEMENTS We would like to thank Pascal Frey for his comments on the manuscript. This research was funded by the European Union (project QLK5-1999-01585) and the Department for Environment, Food and Rural Affairs (DEFRA); Rothamsted Research receives grant-aided support from the UK Biotechnology and Biological Sciences Research Council.
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Corresponding Editor: R. W. S. Weber