Mycol Progress (2011) 10:175–187 DOI 10.1007/s11557-010-0687-0 ORIGINAL ARTICLE Taxonomy and phylogeny of Puccinia lagenophorae: a study using rDNA sequence data, morphological and host range features Markus Scholler & Matthias Lutz & Alan R. Wood & Gregor Hagedorn & Mechthilde Mennicken Received: 15 January 2010 / Revised: 28 May 2010 / Accepted: 16 June 2010 / Published online: 6 July 2010 # German Mycological Society and Springer 2010 Abstract Puccinia lagenophorae is a rust fungus originating from Australasia which has spread throughout the world. A phylogenetic analysis of taxa related to this species was performed using rDNA (LSU, ITS) sequence data. The analyses revealed a well-supported cluster Electronic supplementary material The online version of this article (doi:10.1007/s11557-010-0687-0) contains supplementary material, which is available to authorized users. M. Scholler (*) Staatliches Museum f. Naturkunde, Abt. Biowissenschaften, Erbprinzenstr. 13, 76133 Karlsruhe, Germany e-mail: scholler@naturkundeka-bw.de M. Lutz Lehrstuhl f. Organismische Botanik, Institut f. Evolution und Ökologie, Universität Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany A. R. Wood ARC-Plant Protection Research Institute, P. Bag X5017, Stellenbosch 7599, South Africa G. Hagedorn Julius Kühn-Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Königin-Luise-Str. 19, 14195 Berlin, Germany M. Mennicken Lehrstuhl Organismische Botanik, Institut für Evolution und Ökologie, Universität Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany including all specimens of P. lagenophorae. By evaluating morphological and sequence data, the species is taxonomically re-defined and a list of synonyms is provided. Puccinia distincta on Bellis perennis, a species recently separated from P. lagenophorae, P. saccardoi, a species on the Goodeniaceae, and P. byliana, a species so far only known from South Africa, are reduced to synonymy in P. lagenophorae, as are several other species. Our analysis indicates that P. lagenophorae is likely not derived from the northern hemisphere species P. obscura, but from a species from Australia host-alternating between Asteraceae (aecial host) and Cyperaceae/Juncaceae (telial host). Another related species, P. stylidii (on Stylidium sp., Stylidiaceae) may have been derived from the same parental species as P. lagenophorae. From ontogenetical and morphological studies, the presence of pycnia could not be confirmed in the life cycle of this species, and the width of the pedicel of teliospores at the point of attachment was found to be highly variable and not a taxonomic character. The number of known host species is approximately 150, including 41 new host plants recorded herein. Keywords Molecular phylogeny . Morphology . Puccinia lagenophorae and Puccinia distincta and Puccinia saccardoi . Neomycete Introduction Puccinia lagenophorae Cooke (Pucciniales; Pucciniaceae) is an autoecious rust fungus which is native to Australasia (Wilson et al. 1965). It is one of a complex of species that have radiated in Australia on the Asterales (van der Merwe et al. 2008). Today, this neomycete species has an 176 almost worldwide distribution (Europe, South America, North America, North Africa, Southwest Asia). So far, about 60 host species are known (Farr and Rossman 2009), all belonging to the subfamily Asterioideae (family Asteraceae). Puccinia lagenophorae is characterized by a demicyclic life cycle with repeating aeciospores which have hyaline pore plugs (type 5 aeciospores of Savile 1973) and telia with smooth apically thickened telio- and mesospores and no paraphyses. McAlpine (1906) considered all taxa with more or less identical morphology and life cycle but occurring on different host plant genera in Australia as different species. Based on biometrical studies and inoculation experiments, Viennot-Bourgin (1964) and Wilson et al. (1965) indicated that the species can neither be split into “morphological” nor into “biological” species. Consequently, Wilson et al. (1965) combined them under the oldest name, Puccinia lagenophorae (type host species: Lagenophora billardierei Cass., tribe Asterae) within which were included P. allanii G. Cunn., P. calendulae McAlpine, P. calocephali McAlpine, P. cinerariae McAlpine, P. distincta McAlpine, P. erechtitis McAlpine, P. hypochoeridis McAlpine (non P. hypochoeridis Oudem.), P. macalpinei P. Syd. & Syd., P. tasmanica Dietel, and P. terrieriana Mayor (Cunningham 1931; Wilson et al. 1965). Wilson et al. (1965) also indicated that other rust species on other genera of the Asteraceae from Australasia with identical life cycle and spore morphology, such as P. brachycomes McAlpine, would likely prove to also belong to this species. Later, this species concept was confirmed by other authors such as Walker (1983) and Scholler (1993, 1997) after detailed morphological studies. Walker (1983) also indicated that P. byliana Dippenaar, a species from South Africa, may be related or even conspecific with P. lagenophorae. In several publications, Weber and coauthors (Weber and Tilston 1999; Weber et al. 1998, 2003; Preece et al. 2000) differentiated the rust on Bellis perennis L. in Europe from P. lagenophorae (on Senecio vulgaris L.) and re-established P. distincta (Weber et al. 1998), a species described from Australia on B. perennis. According to the authors, P. distincta, in contrast to P. lagenophorae on Senecio vulgaris, is not hosted by S. vulgaris, does not form pycnia, three-celled teliospores, or average pedicel widths above 7 μm. Later, Weber et al. (2003) confirmed their findings by presenting differences between the Bellis and the Senecio rust in three positions of the ITS sequence. This differentiation, however, was not generally accepted (e.g., Koike and Scholler 2001; Bruckart 2003; Henricot and Denton 2005). A phylogenetic analysis using rDNA sequence data (LSU, ITS1-5.8 S-ITS2) and comparative morphological studies of P. lagenophorae and related species were performed to resolve these open questions and conflicting views. Mycol Progress (2011) 10:175–187 Materials and methods Morphology Spores from dried herbarium specimens were mounted in 50% lactic acid aqueous solution, gently heated, and examined using a Nikon Eclipse E600 (specimens from South African) or a Zeiss Axioskop 2 Plus Microscope (all other specimens) at ×1,000 magnification. Generally, measurements of 25–100 spores of each spore stage were made of selected specimens. For determining the percentage of three-celled versus two-celled teliospores, 100 spores were evaluated per specimen. The width of the pedicel of teliospores at the point of attachment was measured for 10 teliospores from each of five telia on the holotype of P. lagenophorae and from other specimens on miscellanous host plants. The data were rounded to the nearest 0.5 μm. A multiple range test (Ryan-Einot-Gabriel-Welsch, alpha= 0.05) was performed on these data. The presence of loosely adherent chains of aeciospores with hyaline (pore) plugs was taken as diagnostic of this species when no telia were observed on the specimens (Baka 1996; Littlefield et al. 2005). For comparison with P. lagenophorae, published descriptions were evaluated (Cunningham 1931; Littlefield et al. 2005; McAlpine 1906; Scholler 1993, 1997; Viennot-Bourgin 1964; Wilson et al. 1965), and material from K, KR, PDD, PUR and VPRI were examined. Herbarium acronyms are from Holmgren et al. (1981). Specimens examined Selected specimens studied for morphological characters and host range are listed in the electronic supplementary material, Appendix A. Molecular analyses Details of herbarium specimens from which genomic DNA was isolated are listed in Table 1. For methods of isolation and crushing of fungal material, DNA extraction, amplification, purification of PCR products, sequencing, and processing of the raw data, see Lutz et al. (2004). For all specimens, we determined sequences of the 5′-end of the nuc-LSU rDNA including the domains D1/D2 (LSU). Since only the ITS of the closest relatives of the P. lagenophorae complex are available in GenBank, we additionally determined base sequences of the ITS1– 5.8 S–ITS2 region of the nuc-rDNA for selected specimens (see Table 1). The LSU was amplified using the primer pair NL1 and NL4 (O’Donnell 1992, 1993); the ITS was amplified using the primer pair ITS1f/ITS5 and ITS4 (Gardes and Bruns 1993; White et al. 1990). For amplifi- Mycol Progress (2011) 10:175–187 177 Table 1 List of sequenced specimens with host plants, DNA isolation numbers, GenBank accession numbers, and reference specimens (acronyms of public herbaria according to Index Herbariorum) Species Host DNA isolation no. GenBank acc. no. (ITS/LSU) Reference specimens Aecidium sp. Ammobium alatum R. Br. ml1280 FJ655859/FJ669219 Australia, New South Wales, Australian Capital Territory, Canberra, Acton, courtyard of the Botany and Zoology building; 25.V.2006, leg. M. van der Merwe; KR15599, duplicate: DAR 77116 Puccinia lagenophorae Cooke Amellus microglossus DC. ml999 -/FJ669235 Puccinia lagenophorae Cooke Amellus nanus DC. ml1000 -/FJ669236 Puccinia lagenophorae Cooke Arctotis fastuosa Jacq. ml1001 -/FJ669237 Puccinia lagenophorae Cooke Bellis perennis L. ml867 FJ655863/FJ669223 Puccinia lagenophorae Cooke Bellis perennis L. ml868 FJ655862/FJ669222 Puccinia lagenophorae Cooke Bellis perennis L. ml870 FJ655861/FJ669221 Puccinia lagenophorae Cooke Puccinia lagenophorae Cooke Bellis perennis L. ml1139 FJ655860/FJ669220 Bellis perennis L. ml1274 FJ655871/FJ669231 South Africa, Western Cape Province, BIOTA-observatory at Flaminkvlakte; 06. IX.2002, leg. M. Mennicken No. RSA 114; PREM 59333 South Africa, Northern Cape Province, Richtersveld National Park, BIOTAobservatory at Koeroegapvlakte; 15. IX.2002, leg. M. Mennicken No. RSA 160; PREM 59350 South Africa, Western Cape Province, BIOTA-observatory at Luiperskop 211, 07.IX.2002, leg. M. Mennicken No. RSA 124; PREM 59338 USA, California, Monterey County, Salinas; 26.III.2001, leg. S. Koike; KR 8475, duplicate: PUR N1126 Germany, Schleswig-Holstein, LübeckIsraelsdorf; 09.VIII.1998, leg. A. Schmidt; KR 8454, duplicate: PUR N2501 Germany, Baden-Württemberg, nördliches Oberrhein-Tiefland, Karlsruhe, Nymphengarten; 17.VII.2003, leg. M. Scholler; KR 12156 Germany, Rheinland-Pfalz, Kaiserslautern; 19.X.1999, leg. R.W.S. Weber; K(M) 92033 New Zealand, Mid Canterbury, Christchurch, Riccarton; 24.III.1998, leg. A. J. Healy 98/10; PDD 70633 Puccinia lagenophorae Cooke Calendula officinalis L. ml1191 -/FJ669250 Puccinia lagenophorae Cooke Dimorphotheca pluvialis (L.) Moench ml869g FJ655864/FJ669224 Puccinia lagenophorae Cooke Dimorphotheca sinuata DC. ml866 FJ655865/FJ669225 Puccinia lagenophorae Cooke Felicia merxmuelleri Grau. ml1004 -/FJ669238 Puccinia lagenophorae Cooke Felicia tenella (L.) Nees. ml1005 FJ655866/FJ669226 Germany, Mecklenburg-Vorpommern, Greifswald; 22.VII.1992, leg. M. Scholler; KR 8443, duplicate: PUR N1174 South Africa, Western Cape, between Clanwilliam and Calvinia, near Brandewyn River, Traveller’s Rest guest farm, Agterpakhuis; 07.XI.2001, leg. A. R. Wood 300; PREM 57975, duplicate: KR8441, PUR N2518 South Africa, Western Cape, between Clanwilliam and Calvinia, near Brandewyn River, Traveller’s Rest guest farm, Agterpakhuis; 13.VII.2001, leg. A. R. Wood 302; PREM 57977, duplicate: KR 8444, PUR N2516 South Africa, Northern Cape Province, BIOTA-observatory at Quaggafontein 478, 11.IX.2002, leg. M. Mennicken No. RSA 143; PREM 59346 South Africa, Western Cape Province, BIOTA-observatory at Riverlands, 20. IX.2002, leg. M. Mennicken No. RSA 206; PREM 59356 178 Mycol Progress (2011) 10:175–187 Table 1 (continued) Species Host DNA isolation no. GenBank acc. no. (ITS/LSU) Reference specimens Puccinia lagenophorae Cooke Gazania krebsiana Less. ml1006 FJ655867/FJ669227 Puccinia lagenophorae Cooke Lagenophora lanata A. Cunn. ml1275 -/FJ669239 Puccinia lagenophorae Cooke Lagenophora stipitata (Labill.) Druce ml1279 -/FJ669251 Puccinia lagenophorae Cooke Nestlera biennis (Jacq.) Spreng. ml1007 -/FJ669240 Puccinia lagenophorae Cooke Osteospermum pinnatum (Thunb.) T. Norl. ml1011 FJ655868/FJ669228 Puccinia lagenophorae Cooke Senecio arenarius Thunb. ml1012 FJ655869/FJ669229 Puccinia lagenophorae Cooke Senecio glaucus L. ssp. coronopifolius (Maire) Alex. Senecio inaequidens DC. ml1192 -/FJ669246 ml988 -/FJ669244 Puccinia lagenophorae Cooke Puccinia lagenophorae Cooke Senecio inaequidens DC. ml1198 -/FJ669247 Senecio niveus (Thunb.) Willd. ml1013 -/FJ669245 Puccinia lagenophorae Cooke Puccinia lagenophorae Cooke Senecio rodriguezzi Willk. ex Rodrig. Senecio vulgaris L. ml1194 -/FJ669248 ml872 FJ655870/FJ669230 Puccinia lagenophorae Cooke Senecio vulgaris L. ml1277 FJ655872/FJ669232 Puccinia lagenophorae Cooke Tripteris amplectens Harv. ml1008 -/FJ669241 Puccinia lagenophorae Cooke Tripteris clandestina Less. ml1009 -/FJ669242 Puccinia lagenophorae Cooke Tripteris microcarpa Harv. ml1010 -/FJ669243 South Africa, Western Cape Province, BIOTA-observatory at Flaminkvlakte 111, 06.IX.2002, leg. M. Mennicken No. RSA 121; PREM 59336 New Zealand, Auckland, Kawau Island, North Cove; 18.X.2003, leg. R.E. Beever; PDD 78352 Australia, New South Wales, Druce, Budwang National Park; 28.XII.2006, leg. M. van der Merwe; KR15600, duplicate: DAR 77117 South Africa, Western Cape Province, BIOTA-observatory at Rocherpan Nature Reserve, 21 XI 2001, leg. M. Mennicken No. RSA 19, PREM. South Africa, Western Cape Province, BIOTA-observatory at Moedverloren 208, 05.IX.2002, leg. M. Mennicken No. RSA 110; PREM 59332 South Africa, Northern Cape Province, Richtersveld National Park, BIOTAobservatory at Numees, 03.IX.2002, leg. M. Mennicken No. RSA 150; PREM 59349 Germany, Berlin, Berlin-Dahlem, testing ground of the Biologische Bundesanstalt; 04.VIII.1997, leg. M. Scholler; KR 14873 Germany, Nordrhein-Westfalen, Siegburg; 09.X.1998, leg. G. Schmitz; KR 8467, duplicate: PUR N2511 Germany, Nordrhein-Westfalen, Bonn; 19. IX.1997, leg. M. Scholler; KR 14883 South Africa, Northern Cape Province, BIOTA-observatory at Remhoogte 416, 08.IX.2002, leg. M. Mennicken No. RSA 130; PREM 59341 Spain, Mallorca, Cap Formentor; 28. IV.1997, leg. A. Rubner; KR 14636 USA, Oklahoma, Wagoner County, SE Tulsa, nursery; 03.VI.2004, leg. L. Littlefield; KR 8500 New Zealand, Mid Canterbury, Christchurch, Sumner, Kinsey Terrace; 11.IX.2005, leg. E. H. C. McKenzie; PDD 83803 South Africa, Northern Cape Province, BIOTA-observatory at Quaggafontein 478, 11.IX.2002, leg. M. Mennicken No. RSA 140; PREM 59345 South Africa, Western Cape Province, BIOTA-observatory at Moedverloren 208, 05.IX.2002, leg. M. Mennicken No. RSA 109; PREM 59331 South Africa, Northern Cape Province, Richtersveld National Park, BIOTAobservatory at Numees, 13.IX.2002, leg. M. Mennicken No. RSA 149; PREM 59348 Puccinia lagenophorae Cooke Mycol Progress (2011) 10:175–187 179 Table 1 (continued) Species Host DNA isolation no. GenBank acc. no. (ITS/LSU) Reference specimens Puccinia lagenophorae Cooke Vernonia cinerea (L.) Less. ml1201 -/FJ669249 Puccinia obscura J. Schröt. Luzula campestris (L.) DC. ml1140 FJ655873/FJ669233 Puccinia obscura J. Schröt. Luzula sylvatica (Huds.) Gaudin ml1137 FJ655874/FJ669234 Germany, Berlin, Berlin-Dahlem, 19. IX.1997, leg. M. Scholler; KR 14878 (artificial inoculation with inoculum from Senecio) England, Devon, Liddecombe, Guinspound, Challacombe Farm; 16.IX.1999, leg. J. Webster; K(M) 92036 Germany, Baden-Württemberg, SSW Oppenau, Hohbruch, Ibacher Holzplatz, 18.VII.2005, leg. M. Scholler; KR 14322 cation of both regions, we adjusted the annealing temperature to 45°C. DNA sequences prepared in the course of this study were deposited in GenBank; accession numbers are given in Table 1. To infer phylogenetic relationships among the analyzed rust specimens, we also analyzed the most similar sequences available in GenBank according to a blast search (Altschul et al. 1997): Aecidium brachycomes Petr. EF635896 (Morin et al. 2009); Puccinia dioicae Magnus EF635897 (Morin et al. 2009); P. lagenophorae EF212446, EF212447 (Bruckart et al. 2007); AY808060 (Henricot and Denton 2005); AY852264 (Littlefield et al. 2005); EF635886, EF635889, EF635890, EF635887, EF635888, EF635891, EF635892, EF635893, EF635894 (Morin et al. 2009); AF468041, AF468040 (Weber et al. 2003); EU391656 (Sundelin et al. 2008); P. obscura J. Schröt. AF468042 (Weber et al. 2003); P. rupestris Juel EF635898 (Morin et al. 2009); P. stylidii McAlpine EF635895 (Morin et al. 2009); and P. vaginatae Juel EF635901, EF635902 (Morin et al. 2009). Both LSU and ITS sequences were aligned with MAFFT 6.611 (Katoh et al. 2002, 2005; Katoh and Toh 2008) using the L-INS-i option. Both alignments were used to count base differences between the sequences. The entire ITS alignment (length: 606 bp; variable sites: 106) was used throughout its length for phylogenetic analyses. We avoided both manipulation of the alignment by hand or manual exclusion of any positions as recommended by Giribet and Wheeler (1999) and Gatesy et al. (1993). For phylogenetic analyses, we used both neighbourjoining analysis and a Bayesian approach. For neighbourjoining, the data were first analysed with Modeltest 3.7 (Posada and Crandall 1998) to find the most appropriate model of DNA substitution. The hierarchical likelihood ratio test proposed the HKY+G DNA substitution model. Bootstrap values were calculated for 1,000 replicates. For Bayesian analysis, we used a Markov chain Monte Carlo (MCMC) technique as implemented in the computer program MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). Four incrementally heated simultaneous Markov chains were run over 2,000,000 generations using the general time-reversible model of DNA substitution with gamma-distributed substitution rates and estimation of invariant sites, random starting trees and default starting parameters of the DNA substitution model as recommended by Huelsenbeck and Rannala (2004). Trees were sampled every 100th generation resulting in an overall sampling of 20,001 trees. From these, the first 4 ,001 trees were discarded (burn-in=4,001). The trees sampled after the process had reached stationarity (16,000 trees) were used to compute a 50% majority rule consensus tree to obtain estimates for the a posteriori probabilities of groups of species. This Bayesian approach of phylogenetic analysis was repeated four times to test the independence of the results from topological priors (Huelsenbeck et al. 2002). Trees were rooted with Puccinia rupestris (EF635898). Results Morphology The width at the point of attachment of the pedicel to the distal teliospore cell was highly variable in the material studied (Table 2). The average pedicel width of the holotype of P. lagenophorae is slightly smaller than the width of P. distincta on B. perennis according to Weber et al. (1998). The average width of all specimens studied was 7.0 μm (± SE 0.05, n=750) with a minimum and maximum of 3.5 and 12 μm, respectively. Pedicels from individual specimens have significant width differences (Table 2). No pycnia were observed. Three-celled teliospores occured in variable proportions in material from Europe, Australia and North America. None were observed in any specimen from South Africa examined (not all specimens were examined 180 Table 2 Minimum, maximum and mean width (in μm) of the junction between teliospore and pedicel for teliospores of Puccinia lagenophorae a Means followed by different letters are significantly different (Ryan-Einot-Gabriel-Welsch multiple range test, alpha=0.05). Means not followed by letters were obtained from the literature and were not included in the analysis b Holotype of Puccinia lagenophorae c Sori on leaves d Sori on stems e Lectotype of Puccinia byliana Mycol Progress (2011) 10:175–187 Host plant and specimen Min.–max. Meana n SD Bellis perennis (KR 8456) Lagenophora billardieri (K(M) 162111)b Bellis perennis (KR 14476) Bellis perennis (from Weber et al. 1998) Lagenophora stipitata (VPRI 3803)c Lagenophora stipitata (VPRI 3803)d Steirodiscus tagetes (PREM 57925) Senecio sp. (PREM 57924) Senecio littoreus (PREM 46293)e Emilia spp (from Henricot and Denton 2005) Senecio vulgaris (KR 8483)d Amellus asteroides (PREM 57925) Bellis perennis (KR 8477) Senecio vulgaris (KR 8483)c Senecio vulgaris (from Weber et al. 1998) 3.5 – 8.5 4.5–10.0 4–9 5–9 4.5–8.5 5–10 5–10.5 5–9 5.5–10 5.98 6.17 6.19 6.6 6.69 7.09 7.15 7.16 7.22 7.4 7.55 7.71 7.85 8.33 8.4 100 50 100 50 50 50 50 50 50 30 50 50 100 50 50 0.97 0.97 1.1 for this character). No three-celled teliospores were observed on Amellus asteroides (L.) Druce., Arctotheca populifolia (Bergius) Norl., Dimorphotheca pluvialis (L.) Moench, Felicia tenella (L.) Nees., Steirodiscus tagetes (L.) Schlechter, Tripteris clandestina Less., and Bellis perennis L. Also, we did not observe three-celled spores in the holotype (on Lagenophora billardieri). On Senecio spp., this feature is especially variable: 0% in KR 3073a, KR 8480 (S. vulgaris L.), KR 8468 (Senecio spec.), KR 8467 (S. inaequidens); 1-5% in KR 8481, KR 8483, KR 22642 (S. vulgaris), KR 20286 (S. vulgaris ssp. denticulatus); and 10-11% in KR 8500, KR 13384 (S. vulgaris). Of the two P. saccardoi on Goodeniaceae, one had a high proportion of three-celled spores (12%, VPRI 3906) whereas the other had none at all (VPRI 3896). Ridges on teliospores were observed in many specimens, usually on less than 5% of spores but occasionally as much as on approximately 20% of spores. In the holotype of P. lagenophorae ridges were observed on one meso- and one teliospore of 50 spores. One specimen of P. saccardoi (VPRI 3906) contains an especially high proportion of teliospores with ridges. Phylogenetic analyses All analysed LSU sequences were almost identical within P. lagenophorae (ml1201 differed in position 91 of the alignment; ml1279 in position 202; ml866, ml988 (= ml871), and ml1006 in position 473; AY808061 in position 476). The sequence of Aecidium sp. on Ammobium alatum R. Br. differed in two positions, and that of the P. obscura specimens in nine positions compared with those of P. lagenophorae. ITS sequences were somewhat more variable. Within the P. lagenophorae complex, seven different ITS- 5–10 4.5–12 4.5–10.5 6–10.5 7–10 a ab ab bc cd cd cd cd de de ef f 0.9 1.1 1.29 1.13 1.05 0.98 1.62 1.04 1.08 genotypes were revealed which differed in up to 4 bp in a total of six positions of the alignment: genotype 1: AF468041, AY808060, EF212447, ml1011, ml1277; genotype 2: ml1139; genotype 3: AF468040, EU391656, ml868, ml870, ml1274; genotype 4: AY852264, EF212446, EF635886, EF635889, EF635890, ml866, ml867, ml872, ml1005, ml1006, ml1012; genotype 5: EF635887; genotype 6: EF635888, ml869; genotype 7: EF635891, EF635892, EF635893, EF635894. The genotypes are denoted in Fig. 1. Different runs of Bayesian phylogenetic analyses and the neighbour-joining analysis yielded consistent topologies. We present the consensus tree of one run of Bayesian phylogenetic analyses to illustrate the results (Fig. 1). Estimates for a posteriori probabilities are indicated on branches before the slash, bootstrap values from the neighbour-joining analysis after the slash. The analyses revealed a well-supported cluster (a posteriori probability: 97; bootstrap value: 95) of P. lagenophorae including P. distincta sensu Weber et al. (1998). Two additional species, P. saccardoi and P. stylidii, clustered within the P. lagenophorae group, the former was the only representative of genotype 5. Aecidium sp. on A. alatum clustered as a well-supported sister taxon of the P. lagenophorae complex. P. obscura and P. vaginatae are more distantly related to the specimens studied here. P. dioicae and A. brachycomes on Brachycome spathulata Gaudich. were shown to be more closely related to the P. lagenophorae complex than the previous species and “P. lagenophorae” on Senecio madagascariensis Poir. from South Africa (EF 635899, 635900). Compared to the sequences of the P. lagenophorae complex, P. stylidii differed in 10 bp, Aecidium sp. on A. alatum in 18 bp, and P. obscura in 53 bp. Mycol Progress (2011) 10:175–187 181 P. l. (EU391656: P. distincta) on Bellis perennis/Astereae (Denmark) - gt 3 P. l. (EF635894) on S. madagascariensis/Senecioneae (South Africa) - gt 7 P. l. (EF635893) on S. madagascariensis/Senecioneae (South Africa) - gt 7 P. l. (EF635892) on S. madagascariensis/Senecioneae (South Africa) - gt 7 P. l. (EF635891) on S. madagascariensis/Senecioneae (South Africa) - gt 7 P. l. (EF635888) on S. madagascariensis/Senecioneae (South Africa) - gt 6 70/- P. l. (EF635887: P. saccardoi) on G. hederacea ssp. alpestris/Asterales: Goodeniaceae (Australia) - gt 5 P. stylidii (EF635895) on Stylidium sp./Asterales: Stylidiaceae (Australia) P. l. (AF468040: P. distincta) on Bellis perennis/Astereae (UK) - gt 3 P. l. (ml869) on Dimorphotheca pluvialis/Calenduleae (South Africa) - gt 6 P. l. (ml1274) on Bellis perennis/Astereae (New Zealand) - gt 3 P. l. (ml1139) on Bellis perennis/Astereae (Germany) - gt 2 P. l. (ml868) on Bellis perennis/Astereae (Germany) - gt 3 P. l. (ml870) on Bellis perennis/Astereae (Germany) - gt 3 P. l. (EF635890) on S. madagascariensis/Senecioneae (South Africa) - gt 4 P. l. (EF635889) on S. madagascariensis/Senecioneae (South Africa) - gt 4 P. l. (EF635886) on Arctotheca populifolia/Arctotideae (Australia) - gt 4 97/95 P. l. (EF212447) on S. vulgaris/Senecioneae (Canada) - gt 1 P. l. (EF212446) on S. vulgaris/Senecioneae (Canada) - gt 4 P. l. (AY808060) on Emilia sp./Senecioneae (UK) - gt 1 P. l. (AY852264) on S. vulgaris/Senecioneae (USA) - gt 4 P. l. (AF468041) on S. vulgaris/Senecioneae (UK) - gt 1 P. l. (ml866) on Dimorphotheca sinuata/Calenduleae (South Africa) - gt 4 100/94 P. l. (ml1006) on Gazania krebsiana/Arctotideae (South Africa) - gt 4 P. l. (ml1005) on Felicia tenella/Astereae (South Africa) - gt 4 P. l. (ml1011) on Osteospermum pinnatum/Calenduleae (South Africa) - gt 1 P. l. (ml1012) on S. arenarius/Senecioneae (South Africa) - gt 4 P. l. (ml1277) on S. vulgaris/Senecioneae (New Zealand) - gt 1 100/85 P. l. (ml867) on Bellis perennis/Astereae (USA) - gt 4 P. l. (ml872) on S. vulgaris/Senecioneae (USA) - gt 4 Aecidium sp. (ml1280) on Ammobium alatum/Gnaphalieae (Australia) 79/65 P. dioicae (EF635897) on Carex bichenoviana 95/55 Aecidium brachyscomes (EF635896) on Brachyscome spathulata 100/100 P. vaginatae (EF635902) on Saussurea alpina P. vaginatae (EF635901) on Carex vaginata 55/61 100/100 P. obscura (AF468042) on Bellis perennis/Astereae (UK) P. obscura (ml1137) on Luzula sylvatica (Germany) P. obscura (ml1140) on Luzula campestris (UK) 100/99 P. l. (EF635900) on S. madagascariensis/Senecioneae (South Africa) P. l. (EF635899) on S. madagascariensis/Senecioneae (South Africa) P. rupestris (EF635898) on Carex rupestris 1 substitution/site Fig. 1 Bayesian inference of phylogenetic relationships within the sampled rust specimens: Markov chain Monte Carlo analysis of an alignment of ITS base sequences using the GTR+I+G model of DNA substitution with gamma distributed substitution rates and estimation of invariant sites, random starting trees and default starting parameters of the DNA substitution model. A 50% majority-rule consensus tree computed from 16,000 trees that were sampled after the process had reached stationarity is shown. The topology was rooted with Puccinia rupestris (EF635898). Numbers on branches before slashes are estimates for a posteriori probabilities, numbers on branches after slashes are percentage bootstrap values of 1,000 replicates. Branch lengths were averaged over the sampled trees. They are scaled in terms of expected numbers of nucleotide substitutions per site. P. Puccinia, S. Senecio, gt genotype (see the text) Taxonomy as well as on molecular data. Four taxa (P. brachycomes, P. calotidis McAlpine, P. podolepidis, and P. vittadiniae McAlpine) are listed as synonyms based only on morphological descriptions. Names listed here as synonyms for the first time are given in bold type. Puccinia lagenophorae Cooke, Grevillea 13 (65): 6 (1884) (Fig. 2) Neither molecular nor morphological data support the split of P. lagenophorae into different species. The following list of synonyms is based on morphological and biological studies of Cunningham 1931; Viennot-Bourgin 1964; Wilson et al. 1965; Scholler 1993, 1997, and other authors 182 Fig. 2 The holotype of Puccinia lagenophorae (K(M)162111) (host Lagenophora billardierei Cass., Asteraceae; holotype in K). ≡ Dicaeoma lagenophorae (Cooke) Kuntze, Revis. Gen. Pl. (Leipzig) 3: 469 (1898) = Puccinia saccardoi F. Ludw., Hedwigia 28: 362 (1889) (host Goodenia geniculata R. Br., Goodeniaceae) ≡ Dicaeoma saccardoi (F. Ludw.) Kuntze, Revis. Gen. Pl. (Leipzig) 3: 470 (1898) = Puccinia erechtitis McAlpine, Proc. Roy. Soc. Victoria 7: 216 (1894) (issued January 1895) (host Senecio quadridentatus Labill., originally as 'Erechtites quadridentata?', Asteraceae) = Puccinia hypochaeridis McAlpine (as 'hypochaeris'), Proc. Roy. Soc. Victoria 7: 217 (1894) (issued January 1895) (host Lagenophora billardierei Cass., Asteraceae, originally in error as Hypochaeris radicata), nom. illegit. Art. 53 ICBN [non Puccinia hypochaeridis Oudem. 1873], = Puccinia distincta McAlpine, Agric. Gaz. N.S.W., 6: 853 (1895) (issued 1896) (host Bellis perennis L., Asteraceae) ≡ Lindrothia distincta (McAlpine) Syd., Annal. Mycol. 20(3): 119 (1922) = Puccinia nigricaulis McAlpine, Agric. Gaz. NSW 7: 151 (1896) (hosts Goodenia glauca F. Muell. and Goodenia pinnatifida Schltdl., Goodeniaceae, a synonym of Puccinia saccardoi according to McAlpine 1906) = Puccinia macalpinei P. Syd. & Syd. (as 'macalpini'), Monographia Uredinearum I: 100, (1902) (host Lageno- Mycol Progress (2011) 10:175–187 phora billardierei Cass., Asteraceae, given originally in error as 'Hypochoeris radicata'; nom. nov. for P. hypochaeridis McAlpine q.v.). = Puccinia calendulae McAlpine, Proc. Linn. Soc. N.S.W. 28: 558 (1903) (host Calendula officinalis L., Asteraceae). Anam.: Aecidium calendulae McAlpine, Agric. Gaz. N. S.W. 7: 152 (1896) ≡ Lindrothia calendulae (McAlpine) Syd., Annal. Mycol. 20(3): 119 (1922) = Puccinia tasmanica Dietel, Ann. Mycol. 1: 535 (1903) (host Senecio vulgaris L., Asteraceae) ≡ Lindrothia tasmanica (Dietel) Syd., Ann. Mycol. 20 (3): 119 (1922) = Puccinia brachycomes McAlpine, Rusts of Australia: 150 (1906) (hosts Brachyscome ciliaris (Labill.) Less., B. diversifolia (Hook.) Fisch. & C. A. Mey, B. pachyptera Turcz., B. scapiformis DC., Asteraceae) = Puccinia calocephali McAlpine, The Rusts of Australia: 151 (1906) (hosts Calocephalus drummondii (A. Gray) Benth., C. lacteus Less., Asteraceae, a synonym of P. erechtitis according to Cunningham 1931) = Puccinia calotidis McAlpine, Rusts of Australia: 152 (1906) (hosts Calotis cuneifolia R. Br., Calotis sp., Asteraceae) ≡ Lindrothia calotidis (McAlpine) Syd., Ann. Mycol. 20 (3): 119 (1922) = Puccinia cinerariae McAlpine, The Rusts of Australia: 155 (1906) (host Cineraria sp. (cult.), Asteraceae) = Puccinia podolepidis McAlpine, Rusts of Australia: 162 (1906) (host Podolepis longipedata A. Cunn. ex DC., Asteraceae) ≡ Lindrothia podolepidis (McAlpine) Syd., Ann. Mycol. 20(3): 119 (1922) = Puccinia vittadiniae McAlpine, Rusts of Australia: 164 (1906) (host Vittadinia australis A. Rich., Asteraceae) = Puccinia cruciferae McAlpine, Rusts of Australia: 184 (1906) (host Vittadinia triloba (Gaudich.) DC., Asteraceae, originally in error as 'cruciferous plant'; see Walker 1983) = Puccinia allanii G. Cunn., Trans. Proc. N. Z. Inst. 54: 675 (1923) (host Senecio lagopus Raoul, Asteraceae). = Puccinia byliana Dippenaar, S. Afr. J. Sci. 28: 288 (1931) (as ‘P. bylianum’, name corrected in Doidge 1950). (on Senecio littoreus Thunb., S. pinnulatus Thunb, S. vulgaris L. and Senecio sp., Asteraceae; Lectotype here designated: South Africa, Western Cape, Hopefield, on Senecio littoreus Thunb., IX 1925, leg. P.A. van der Byl 2356 (PREM 46293). Anam.: Aecidium bylianum Syd., Ann. Mycol. 22: 236 (1924). = Puccinia terrieriana Mayor, Ber. schweiz. bot. Ges. 72: 266 (1962) (type host Senecio vulgaris L., Asteraceae) Mycol Progress (2011) 10:175–187 Host range A total of 41 new host records are recorded herein, 38 from South Africa, and 1 each from Germany, New Zealand and Spain (for details, see Appendix A). Discussion Nomenclature, morphology, taxonomy, phylogeny Our molecular analyses using ITS sequence data indicate that Puccinia lagenophorae forms a well-supported cluster with almost identical ITS sequences differing only in few base pairs (Fig. 1), confirming Morin et al. (2009). This cluster also includes the specimens on B. perennis which were separated by Weber et al. (1998) as P. distincta. The original sequences used by Weber et al. (2003) occur in two sub-clades, but there is very low support for this separation. Weber et al. (2003) only analysed material from western Europe, where the rust is introduced, whereas here specimens were included from throughout the geographical range, including Australia where the species originated. Because of the unique ontogeny (autopsis-form with repeating aecia, leptosporic teliospores), specific morphological features (e.g. aeciospores with hyaline plugs, formation of mesospores), and host range (Asteraceae and related Goodeniaceae) of taxa within this cluster, we consider this cluster to represent one species. Outside this cluster and not included in P. lagenophorae are two specimens of “P. lagenophorae” on S. madagascariensis (EF635899 and EF635900). Morin et al. (2009) provided evidence that these two sequences resulted from an interspecific hybridisation event, with P. lagenophorae as one of its host parents and showing P. lagenophorae morphology. This lumping of rust species contrasts with several other polyphagous “morphospecies”, for example the hostalternating Poaceae rusts P. graminis Pers. and P. coronata Corda. Both these species are genetically diverse (Abbasi et al. 2005; Szabo 2006) and probably need to be split into numerous species. In addition, we include a nonasteraceous species, P. saccardoi, in P. lagenophorae. Its host plants of the genera Goodenia, Scaevola and Velleia belong to the Goodeniaceae, a family closely related to the Asteraceae (Hansen 1997; Stevens 2001). Because of this, P. lagenophorae is considered a polyphagous species in an even broader sense than proposed by Wilson et al. (1965). Several other species from Australia may also belong to this broad concept of P. lagenophorae, including P. angustifoliae McAlpine (on Asteraceae) and P. dampierae P. Syd. & Syd. (on Goodeniaceae). Before synonymising these spe- 183 cies, however, molecular and further morphological studies are required. Puccinia stylidii, another non-asteraceous species on Stylidiaceae (also related to Asteraceae; Stevens 2001), clustered within P. lagenophorae. However, the genetic distance of P. stylidii compared the P. lagenophorae sequences is quite considerable (10 base pairs in the ITS). Its teliospores resemble P. lagenophorae, but it forms uredinia and not aecia (McAlpine 1906). This indicates that P. stylidii is a well-characterised species which likely is derived from the same or similar parental species as P. lagenophorae. In Europe, specimens on B. perennis were regarded as a distinct species from those on S. vulgaris because crossinoculations were unsuccessful and no pycnia were formed, whereas the Senecio rust sometimes formed pycnia, the teliospore pedicel width was smaller in the Bellis rust, and, finally, the Bellis rust had only two-celled and one-celled teliospores (mesopores), whereas the Senecio rust had in addition three-celled teliospores (Weber et al. 1998; Jurc and Weber 2000). Weber et al. (1998) speculated that the Bellis rust derives from the European host-alternating P. obscura (with pycnia and aecia on Bellis, and uredinia and telia on Luzula). Later, Weber et al. (2003) provided a molecular analysis, in which they regarded both the Bellis and the Senecio rust as derived from P. obscura. However, earlier authors made successful cross-inoculations from Bellis to Senecio and vice versa (Viennot-Bourgin 1964; Wilson et al. 1965). Recently, successful cross-inoculations were carried out in North America by Koike and Scholler (2001) and by Bruckart (2003); the latter author also used inoculum from P. lagenophorae on S. vulgaris from Europe (inoculum collected by C. Ellison, CABI, in March 1999, from S. vulgaris, England; W. Bruckart, personal communivation). Also, successful cross-inoculations were carried out in Germany by two of us (M.S. and G.H., unpublished data, voucher specimens in herbarium KR). Generally, we confirm the morphological description as provided by Wilson et al. (1965). We could not confirm the presence of pycnia as observed by McAlpine (1906), Cunningham (1931), and Weber et al. (1998). On the original material from New Zealand cited by Cunningham (1931) on Lagenophora pumila Cheeseman (PDD 3217, PDD 3784) and S. vulgaris (PDD 181, PDD 3543), no pycnia were observed. On one specimen (PDD 1571), pycnia were found (together with aecia and telia); however, a detailed study indicated that this specimen is likely misidentified. It differs from typical P. lagenophorae by colour of sori and teliospore cell wall, size of teliospores and by not forming mesospores. In addition, material collected by R.W.S. Weber and J. Webster (K (M) 92025, 102618) was examined and no pycnia were observed. No pycnia were observed on any other specimens examined. 184 The formation of pycnia in P. lagenophorae, if formed at all, is rare, and it is possible the records are rather of premature aecia which may have been misidentified as pycnia, or that other pycnia-forming species may have been misdentified as P. lagenophorae. Homothalism is common amongst rust fungi with reduced life cycles, and is often accompanied by the lack of pycnia (Ono 2002). The teliospore pedicel width at the point of attachment to the teliospore may depend on the host plant species and the part of the plant where sori are formed. The pedicel width of the type of P. lagenophorae is about the same as that given as characteristic for P. distincta by Weber et al. (1998) on B. perennis. It was found that this characteristic was highly variable between specimens, with one specimen on A. asteroides not only encompassing both ranges given by Weber et al. (1998) but also having greater extreme values. That P. lagenophorae on S. vulgaris consistently has a broader point of attachment than on other hosts is confirmed herein. P. distincta as defined by Weber et al. (1998) cannot be distinguished from P. lagenophorae. Although European specimens on B. perennis and S. vulgaris were significantly different in pedicel width in our analysis, other specimens were intermediate, and therefore the differences observed by Weber et al. (1998) represent two extremes of a continuum (Table 2). Threecelled teliospores are recorded as occasional in S. vulgaris (Weber et al. 1998; Littlefield et al. 2005), and also in Emilia spp. (Henricot and Denton 2005). In a specimen on S. vulgaris from New York (KR 8483), only eight threecelled teliospores were found in 4 out of 10 sori observed (400 teliospores observed in total). One of the two specimens on Goodeniaceae (VPRI 3906, formerly P. saccardoi F. Ludw.) contains a high proportion of three-celled spores whereas in the other one (VPRI 3896), not a single one was observed. Three-celled teliospores were not observed in B. perennis (Weber et al. 1998) and Senecio glaucus L. (Baka 1996), in the type on L. billardieri and on several hosts from South Africa. Ridges on the teliospore surface were illustrated by Viennot-Bourgin (1964, Fig. 2), Scholler (1993), Wilson et al. (1965, Figs. 2, 11) and Wilson and Henderson (1966). Other authors, however, such as Baka (1996) and Cunningham (1931) do not mention them. McAlpine (1906) does not mention them either although these can be seen in photographs of teliospores of P. calendulae and P. brachycomes (p. 256). Ridges were observed in the majority of specimens studied, and their occurrence was independent of the host plant and geographical origin. Therefore, we do not agree with Weber et al. (1998) who consider ridges to be an artifact in fresh spores mounted in lactic acid (the photograph in Weber et al. 1998, Fig. 8, indeed does not show ridges but shrunken spores, possibly as a consequence of insufficient heating of lactic acid microscopic mounts). According to J. Walker Mycol Progress (2011) 10:175–187 (personal communication), ridges may be the result of mutual pressure between the spores in the telia rather than true ornamentation. This is supported by the fact that the ridges were observed to mostly have formed in regions of the spore where the wall is thin. Our ontogenetical and morphological studies and those of other authors mentioned above have shown that the formation or non-formation of pycnia, three-celled teliospores and the width of the pedicel are very variable and not constant characters of P. lagenophorae. These characters may, to some degree, be influenced by the morphology of the host species, but they are not useful taxonomic characteristic as claimed by Weber et al. (1998). The results do not support the suggestion made by Weber et al. (2003) that P. obscura may be the parental species of P. lagenophorae. The molecular analyses show at least three species (A. brachycomes, Aecidium sp. and P. dioicae) being more closely related to P. lagenophorae than P. obscura, Morin et al. (2009) previously also obtained the same result. The same may hold for P. vaginatae, although support values are low. But we support Weber et al. (2003) that the parental species is likely to be, like P. obscura, a host-alternating species between Asteraceae (aecial host) and Cyperaceae/Juncaceae (telial host). Puccinia dioicae (and P. vaginatae) have such a life cycle and Aecidium sp. and A. brachycomes (both on Asteraceae) may also be part of the life cycle of a host-alternating species with a Cyperaceae/Juncaceae telial host. Host range Based on our species concept and considering the large number of host species from South Africa, the number of known hosts is increased from about 60 to 150, about 40 of which are members of the Goodeniaceae. In contrast to Wilson et al. (1965) and Scholler (1998), who listed only species of the subfamily Asterioideae, we found that members of the subfamily Cichorioideae (Arctotis, Arctotheca, Gazania, Vernonia) and of the family Goodeniaceae (Goodenia, Scaevola, Selliera, Velleia) also host P. lagenophorae. Since the host range of rust fungi is mostly restricted to groups of related plant species, P. lagenophorae may provide further information for plant systematists on relationships between the Asteraceae and related families. Annotations to geographic origin and pathways of expansion Wilson et al. (1965) were the first to name P. lagenophorae as a rust originating from Australasia (Australia, New Zealand, New Guinea) which had been introduced to Europe. Further arguments in support of this statement Mycol Progress (2011) 10:175–187 are: the first record (the holotype) is from Australia, the highest number of host plants is recorded in Australasia, there are also other related rust species with hosts on Asteraceae or related families with similar teliospore and mesospore morphology (e.g. P. brunoniae McAlpine, P. gilgiana Henn. on Goodeniaceae, and P. stylidii on Stylidaceae), and similar species are not known from elsewhere. Finally, our molecular studies indicate that there is the highest (relative) genetic diversity of P. lagenophorae in Australasia with four different ITS-genotypes in four specimens (the quotient of the number of genotypes : number of samples is 1 for Australasia, 0,43 for Europe, 0,4 for North America, and 0,33 for South Africa). Highest genetic diversity is a feature typical for the place of origin of fungi (e.g. Zhang et al. 2009). The earliest record of P. lagenophorae traced for Africa was from the Western Cape Province in South Africa collected in 1918 (see Appendix A). So far P. lagenophorae has not been recorded from elsewhere in sub-saharan Africa. However, a record from Sierra Leone from 1930 was discovered in PUR (see Appendix A). Furthermore there is a single record of an unidentified Aecidium on an ornamental Cineraria sp. from Tanzania (Wallace 1936) which may represent this species. This raises the possibility that this fungus may have reached Europe from Africa (as previously suggested by Viennot-Bourgin 1964) and not necessarily directly from Australasia. The molecular data presented herein do not provide information about the pathways of expansion of P. lagenophorae as too few isolates were included for this purpose, especially from Australasia. In each geographic region (Australasia, Europe, North America, South Africa), we found at least two different ITS genotypes (although only differing in few base pairs). It is interesting to note that the most common genotype (genotype 4) occurred in specimens from Australia, North America, and South Africa but not Europe. Also, genotype 3 was only recorded from specimens on B. perennis from Europe and New Zealand, yet the two genotypes that were most similar were only recorded from South Africa (genotypes 6 and 7). There is thus the suggestion that multiple inter-continental introductions have been made. Practical aspects Several host plants of P. lagenophorae are important ornamental plants. In Europe these include Asteriscus maritimus (L.) Less., Calendula officinalis L., Dimorphotheca pluvialis (L.) Moench and D. sinuata DC., Emilia coccinea (Sims) G. Don and E. sonchifolia (L.) DC. Senecio glaucus L. subsp. coronopifolius (Maire) Alex., and Pericallis X hybrida B. Nord. (S. cruentus DC., Pericallis cruenta (L'Hér. ) Bolle), Bellis perennis is a wild 185 plant in Europe but certain cultivars are attractive ornamental plants and rust infection causes economic losses. As a consequence, plant pathologists in various European countries developed and tested fungicides to control the daisy rust (e.g. Gullino et al. 1999; Weber and Tilston 1999; Gerlach 2000). On the other hand, numerous efforts were made to develop P. lagenophorae as a biocontrol agent against the weed S. vulgaris (e.g. Müller-Schärer and Rieger 1998; Killgore et al. 2000; Frantzen et al. 2002; Bruckart 2003; Frantzen and Müller-Schärer 2006). In this context it is noteworthy that previous authors like Viennot-Bourgin (1964), Wilson et al. (1965), Koike and Scholler (2001), and Bruckart (2003) were successful in making cross-inoculations with P. lagenophorae on Bellis and Senecio. The reason why Weber et al. (1998) failed remains unknown. Wilson et al. (1965) made successful cross-inoculations from S. vulgaris to B. perennis, Pericallis X hybrida B. Nord. and C. officinalis in the greenhouse, but did not observe this rust on B. perennis in the field. Cross-infection between Bellis and Senecio and vice versa, however, probably often occurs even in nature as suggested by floristic data. In Germany (Scholler 1994), Poland (Piatek 2003), and California (Scholler and Koike 2001; Koike and Scholler 2001), for instance, the first records on B. perennis and S. vulgaris were found in the same area outside greenhouses. Furthermore, such records were either found in the same year (in Germany in 1966, in Poland in 1998) or only one year later after direct searching (California). There are few data available to determine whether races occur within P. lagenophorae or not, and if so what are their respective host ranges. If such races do occur, their host range may be broad, as inoculation experiments have shown that inoculum derived from Senecio (Asterioideae) can infect Vernonia (Cichorioidae) (Table 1). The consequences of this finding of polyphagy in P. lagenophorae may affect the research of plant pathologists and ecologists throughout its distribution range. Our study confirms the finding by Wilson et al. (1965) that P. lagenophorae is polyphagous and therefore cognizance should be made as to whether any potential conflicts of interest occur where use is made of it as a biocontrol agent. It is recommended that Common Groundsel and other potential host plants in the vicinity of English Daisy plantings are removed as a potential control measure. Acknowledgements We thank John Walker for checking the list of synonyms, Walter Gams for reviewing an earlier version of the manuscript, Bill Bruckart for providing methodical information, Ralf Jahn for identifying Senecio rodriguezzii and the curators of Herbaria BOL, DAR, K, NBG, PDD, PUR, PREM, SAM and VPRI for providing specimens of Puccinia lagenophorae and related species. Field studies carried out by M. Menniken in South Africa were financed by a grant from BMBF, Germany (project “BIOTA”). 186 References Abbasi M, Goodwin SB, Scholler M (2005) Taxonomy, phylogeny and origin of Puccinia graminis, the black stem rust: new insights based on rDNA sequence data. Mycoscience 46:241–247 Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 Baka ZAM (1996) Occurrence and morphology of Puccinia lagenophorae on Senecio glaucus in Egypt. 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