mycological research 111 (2007) 176–185 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/mycres Polyphyly and two emerging lineages in the rust genera Puccinia and Uromyces Wolfgang MAIERa,*, Brenda D. WINGFIELDa, Mechthilde MENNICKENb, Michael J. WINGFIELDa a University of Pretoria, Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Pretoria 0002, South Africa Dolziger Str. 48, 10247 Berlin, Germany b article info abstract Article history: The phylogenetic validity of Puccinia and Uromyces, Pucciniaceae, and closely related genera Received 27 June 2006 was evaluated using nucLSU rDNA sequences. Using a wide range of rust species with dif- Received in revised form ferent life cycles and with different host specificities, Puccinia and Uromyces were shown to 6 October 2006 be highly polyphyletic and to also include representatives of the genera Aecidium, Cummin- Accepted 4 November 2006 siella, Dietelia, Endophyllum, Miyagia, and Uredo. Furthermore, the structure of the phyloge- Corresponding Editor: Gen Okada netic data did not reflect previous sub-generic delimitations based on teliospore pedicel Keywords: nia/Uromyces: Rusts with telial states on Poaceae were exclusively found in one of these Basidiomycota groupings and those with telial states on Cyperaceae resided in the other lineage. This might Molecular phylogeny suggest that the two lineages evolved in close association with these host groups in differ- Pucciniaceae ent biomes. structure, but rather suggests that at least two major lineages have evolved within Pucci- Pucciniales ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Rust fungi Uredinales Introduction Puccinia and Uromyces are by far the two largest genera of rust fungi (Pucciniales), currently including some 4000 and 600 described species, respectively (Cummins & Hiratsuka 2003). These genera have a world-wide distribution and they constitute the vast majority of the rust flora on all six continents (compare McAlpine 1906; Tranzschel 1939; Doidge 1950; Gäumann 1959; Hennen et al. 2005). Puccinia and Uromyces cannot be distinguished by the morphology of their spermogonia, aecia or uredinia or the respective spore types produced within these structures. Thus, the generic definitions were simply based on the number of the teliospore cells, which are one-celled in Uromyces and two-celled in Puccinia. This simplistic scheme is complicated by the fact, that there are species having both one- and two-celled teliospores and sometimes three- and four-celled spores. In these cases, the species have typically been relegated to Puccinia, and the one-celled teliospores have been referred to as mesospores (Gäumann 1926). For these reasons and because of the homogeneity in the morphology of the sori and spores other than teliospores, it has repeatedly been noted that Puccinia and Uromyces are not natural or monophyletic genera (Tulasne 1854; Sydow & Sydow 1904, 1910; Arthur 1934; Guyot 1938; Leppik 1959). Arthur (1906) made the first attempt at splitting the genera Puccinia and Uromyces into smaller and more manageable taxa based solely on life-cycle characteristics. This approach was * Corresponding author. E-mail address: wolfgang.maier@fabi.up.ac.za 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.11.005 Polyphyly and two emerging lineages in Puccinia and Uromyces legitimately criticised by Sydow (1921) and Dietel (1922a,b), because Dietel (1899) had previously demonstrated the phylogenetic connection between a macrocyclic (P. coronata) and a microcyclic rust (P. mesneriana) based on teliospore morphology. Details of this concept were greatly elaborated by Tranzschel (1904), and Tranzschel’s law stating that microcyclic autoecious rusts can be linked phylogenetically to macrocyclic heteroecious rusts and their telia will occur on the former aecial host thus emerged. Because of Tranzschel’s law, it became evident that a life-cycle based generic concept must lead to placement of very closely related species into different genera. Arthur (1934) consequently abandoned his life-cycle concept of genera and retained Puccinia and Uromyces based on their classical circumscription. Phylogenetically, however, he treated them as if they were one big genus. Below the genus level the robustness of the teliospore pedicel has been used to group species of both Puccinia and Uromyces (Fischer 1904; Klebahn 1914; Gäumann 1959). Arthur (1934) defined the subgenera Bullaria with fragile pedicels and consequently dehiscent teliospores and Eu-Puccinia with firm pedicels and consequently persistent teliospores, and also included Uromyces in this subgeneric definition. Besides the giant genera Puccinia and Uromyces, there are various closely related satellite genera that have been described based on peculiarities of their life-cycles or teliospore morphology. Thus, Endophyllum (Léveillé 1826) is defined by a special life-cycle or ontogeny, in which the Puccinia-type aeciospores germinate to produce basidia. The same holds true for Dietelia (Hennings 1897), which forms part of the presumably polyphyletic Pucciniosiraceae that includes only endocyclic representatives. Miyagia (Miyabe 1913) has been treated as a discrete genus because of the presence of paraphyses around the telia. Cumminsiella was split from Uropyxis, where it had originally been accommodated due to similar teliospore morphology, because of its Puccinia-like spermogonial and uredinial morphology (Arthur 1933). Contemporary evidence based on molecular phylogenetic studies using different gene regions has accumulated in support of early evidence contesting the monophyly of Puccinia and Uromyces. Thus, in an ITS-based study, it was found that Puccinia hordei, which produces both one- and two-celled teliospores, is more closely related to Uromyces scillarum than to other Puccinia species ‘‘and may be closely related to Uromyces leaf rusts on Hordeum’’ (Zambino & Szabo 1993). With the help of nucLSU rDNA data, it was shown that both Puccinia and Uromyces are polyphyletic, but also encompass Cumminsiella and Endophyllum (Maier et al. 2003). Likewise, using nucSSU rDNA data, it has been demonstrated that Miyagia and Dietelia belong to the monophyletic Puccinia-Uromyces cluster (Wingfield et al. 2004). The fact that Endophyllum is embedded in Puccinia and that Puccinia and Uromyces are polyphlyetic could also be deduced from another phylogenetic study using ITS sequences (Wood & Crous 2005). However, none of these DNA sequence-based studies have adequately considered the broader implications of their results. This is because the questions addressed in these studies were specific to particular groups of species and none of the studies included a large number of representatives of Uromyces and Puccinia. In this study, we consider the phylogenetic relationships between the genera Puccinia, Uromyces, Cumminsiella, Miyagia, 177 Dietelia and Endophyllum in considerably greater detail than has previously been attempted. The primary focus of interest is to find characters that correlate with natural groupings in the Puccinia/Uromyces complex, which eventually could help in promote a better understanding of this diverse and important group of rust fungi. Therefore, Puccinia and Uromyces species infecting a variety of plant families and displaying many different life cycle strategies were sampled. Materials and methods Sample collection and identification The European samples included in this study were mainly collected by W.M., and then identified using light microscopy. Most of the southern African specimens were collected and identified by M.M. (Mennicken & Oberwinkler 2004; Mennicken et al. 2005a,b,c). Specimens that were used in this study, with additional information on host species, life-cycle, geographic origin and GenBank accession numbers can be found in Table 1. DNA-isolation, PCR and DNA-sequencing DNA was isolated from the rust spores that were lifted from fruiting structures on infected tissue using insect pins, under a dissecting microscope. Whenever possible these spores were taken from single rust sori to avoid contamination of possible infections by multiple rust species. Spores were crushed between two microscope slides or with the help of a tissue lyser (Retsch Mixer Mill 301, Haan, Germany) by shaking the spores in an Eppendorf tube together with a steel bead 3 mm diam for 3 min at 30 Hz. The crushed spores were subsequently suspended in lysis buffer from the Qiagen Plant Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocols. PCR and direct sequencing of both strands of the 50 end of the large subunit of the ribosomal gene cluster was performed using the primer pair NL1 and NL4, LR 0R (Moncalvo et al. 1995) and LR 5 or LR 6 (Vilgalys & Hester 1990). PCR, PCR product purification and cycle sequencing settings were as described previously (Maier et al. 2003; Ritz et al. 2005). DNA sequence electrophoresis was done on automated DNA sequencers (ABI 373stretch and ABI PRISM 3100TM, Perkin-Elmer, Warrington, UK). DNA-sequencing and phylogenetic analyses Contigs of the double stranded nucleotide sequences were produced, proof-read and edited with the help of Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, MI). From the obtained sequences an alignment was produced with the help of MAFFT 5.66 (Katoh et al. 2005) using the iterative refinement method and the following settings: the NeedlemanWunsch algorithm active, 2 tree rebuilding steps, 1000 iterations and the program’s default values for gap opening and gap extension penalties. No further manual manipulation of the alignment was performed. The model of DNA substitution best fitting the data was determined with the help of the Akaike Information Criterion (Akaike 1974) implemented in 178 Table 1 – Species and specimens used in the present study Rust species I-host III-host GenBank accession no. Voucher Geographical origin Elaeagnus parvifolia Empetrum nigrum Mahonia aquifolium Mikania micrantha Euphorbia amygdaloides Sempervivum tectorum Pyrus communis Sonchus cf. oleraceus Sonchus cf. oleraceus Actaea spicata Aegopodium podagraria Dianthus barbatus Carex alba Mesembryanthemum guerichianum Aridaria noctiflora Asarum europaeum Arctium lappa Polygonum bistorta Caltha palustris Carex flacca Carex ferruginea Carex pendula Helianthus annuus Circaea lutetiana Rhamnus cathartica Carex davalliana Carex humilis Carex firma Galenia crystallina Aizoon canariense Epilobium angustifolium Triticum aestivum Hieracium murorum Adoxa moschatellina Mesembryanthemum nodiflorum Mesembryanthemum nodiflorum Bellis perennis Luzula sylvatica Alcea rosea Mentha x piperita Psilocaulon leptarthron Lampranthus otzenianus Oxyria digyna Tussilago farfara Persicaria amphibia Cirsium arvense Elaeagnaceae (Pinaceae) Berberidaceae Asteraceae Euphorbiaceae Crassulaceae (Rosaceae) – – Ranunculaceae – – Asteraceae ? ? – – Apiaceae Ranunculaceae Grossulariaceae Grossulariaceae Grossulariaceae Asteraceae – Rhamnaceae Asteraceae Asteraceae Asteraceae ? ? – Berberidaceae – Adoxaceae – – Asteraceae ? Malvaceae Lamiaceae Aizoaceae Aizoaceae ? Asteraceae Geraniaceae Asteraceae ? (Ericaceae) Berberidaceae – – – (Cupressaceae) Asteraceae Asteraceae Poaceae Apiaceae Caryophyllaceae Cyperaceae Aizoaceae Aizoaceae Aristolochiaceae Asteraceae Polygonaceae Ranunculaceae Cyperaceae Cyperaceae Cyperaceae Asteraceae Onagraceae Poaceae Cyperaceae Cyperaceae Cyperaceae Aizoaceae Aizoaceae Onagraceae Poaceae Asteraceae Balsaminaceae Aizoaceae Aizoaceae Asteraceae Juncaceae Malvaceae Lamiaceae Aizoaceae Aizoaceae Polygonaceae Poaceae Polygonaceae Asteraceae DQ917721 DQ917750 (AF426206) DQ917691 (AF426200) DQ917747 (AF426209) DQ917704 DQ917705 DQ917746 DQ917698 DQ917731 DQ917686 DQ917724 DQ917725 DQ917732 DQ917703 DQ917697 DQ917701 DQ917695 DQ917694 DQ917693 DQ917711 DQ917716 DQ917741 DQ917687 DQ917685 DQ917696 DQ917729 DQ917730 (AF426198) (L08728) DQ917688 DQ917700 DQ917727 DQ917726 DQ917692 DQ917689 (AF426208) DQ917712 DQ917728 DQ917742 DQ917735 DQ917748 DQ917702 DQ917706 WM 3523 CFB 22242 TUB 014955 IMI 393070 HeRB C-82 in ZT TUB 014957 TUB 014958 RSA 173 RSA 125 TUB 014959 TUB 014960 TUB 014961 FO 3195 RSA 153 RSA 155 TUB 014962 TUB 014963 TUB 014964 TUB 014965 TUB 014966 TUB 014967 TUB 014968 WM 3524 TUB 014969 TUB 014970 TUB 014971 TUB 014972 TUB 014973 RSA 162 RSA 148 TUB 014974 As NA CE CA CE CE CE SAf SAf CE CE CE CE SAf SAf CE CE CE CE CE CE CE SAf CE CE CE CE CE SAf SAf CE NA CE CE SAf SAf CE CE CE CE SAf SAf NE CE CE CE TUB 014975 TUB 014976 RSA 33 RSA 176 TUB 014977 TUB 014978 TUB 014979 TUB 014980 RSA 166 RSA 164 TUB 014981 TUB 014982 FO 47837 TUB 014983 W. Maier et al. Aecidium sp. Chrysomyxa empetri Cumminsiella mirabilissima Dietelia mesoamericana Endophyllum euphorbiae-sylvaticae Endophyllum sempervivi Gymnosporangium sabinae Miyagia pseudosphaeria Miyagia pseudosphaeria Puccinia actaeae-agropyri Puccinia aegopodii Puccinia arenariae Puccinia arenariicola var. caricis-montanae Puccinia aridariae Puccinia aridariae Puccinia asarina Puccinia bardanae Puccinia bistortae Puccinia calthicola Puccina caricinia var. ribesii-diversicoloris Puccinia caricina var. ribesii-ferrugineae Puccinia caricina var. ribesii-pendulae Puccinia cf. helianthi Puccinia circaeae Puccinia coronata Puccinia dioicae var. dioicae Puccinia extensicola var. linosyridi-caricis Puccinia firma Puccinia galeniae Puccinia galeniae Puccinia gigantea Puccinia graminis f. sp. tritici Puccinia hieracii Puccinia impatientis Puccinia knersvlaktensis Puccinia knersvlaktensis Puccinia lagenophorae Puccinia luzulae-maximae Puccinia malvacearum Puccinia menthae Puccinia mesembryanthemi Puccinia otzeniani Puccinia oxyriae Puccinia poarum Puccinia polygoni-amphibii Puccinia punctiformis Host species Saxifraga hieracifolia Senecio cacaliaster Senecio ovatus Taraxacum officinale agg. Carex brizoides Pennisetum glaucum Tetragonia echinata Carex acuta Carex acutiformis Carex pallescens Carex hirta Carex rostrata Solidago virgaurea Coccinia rehmannii Alchemilla vulgaris agg. Mesembryanthemum guerichianum Mesembryanthemum guerichianum Aloe arborescens Carex sempervirens Lapeirousia sp. Hesperantha sp. Ranunculus acris Ranunculus ficaria Gagea lutea Babiana tubulosa Babiana cf. sambucina Pulicaria dysenterica – – Asteraceae Asteraceae Asteraceae Solanaceae ? Urticaceae Urticaceae Urticaceae Urticaceae Urticaceae – Cucurbitaceae (Rosaceae) ? ? – Campanulaceae ? ? Ranunculaceae – – ? ? Asteraceae Saxifragaceae Asteraceae Cyperaceae Cyperaceae Cyperaceae Poaceae Aizoaceae Cyperaceae Cyperaceae Cyperaceae Cyperaceae Cyperaceae Asteraceae Cucurbitaceae (Rosaceae) Aizoaceae Aizoaceae Asphodelaceae Cyperaceae Iridaceae Iridaceae Poaceae Ranunculaceae Liliaceae Iridaceae Iridaceae Juncaceae DQ917734 DQ917699 DQ917690 DQ917707 DQ917708 DQ917743 DQ917733 DQ917719 (AF426202) DQ917717 DQ917718 DQ917720 DQ917709 DQ917710 (AF426220) DQ917722 DQ917723 DQ917740 DQ917714 DQ917737 DQ917736 DQ917745 (AF426204) (AF426208) DQ917738 DQ917739 (AF426203) TUB 014984 TUB 014985 TUB 014986 TUB 014987 TUB 014988 TUB 014989 RSA 106 TUB 014990 TUB 014991 TUB 014992 TUB 014993 TUB 014994 TUB 014995 Na 152 TUB 014996 RSA 29 RSA 153 WM 3290 GZU 10-94 RSA 211 RSA 213 TUB 014997 TUB 014998 TUB 014999 RSA 191 RSA 8 GZU 11-98 NE CE CE CE CE SAf SAf CE CE CE CE CE CE SAf CE SAf SAf SAf CE SAf SAf CE CE CE SAf SAf CE Aconitum napellus cf. Ipomoea verbascoidea Euphorbia cyparissias Ranunculus ficaria Euphorbia cyparissias Vicia pannonica – Convolvulaceae Euphorbiaceae Ranunculaceae – Fabaceae Ranunculaceae Convolvulaceae Fabaceae Poaceae Euphorbiaceae Fabaceae DQ917749 DQ917715 (AF426201) DQ917744 DQ917713 (AF426199) TUB 015000 Na 305 TUB 015001 TUB 015002 TUB 015003 TUB 015004 CE SAf CE CE CE CE Polyphyly and two emerging lineages in Puccinia and Uromyces Puccinia saxifragae Puccinia senecionis Puccinia senecionis-acutiformis Puccinia silvatica Puccinia silvatica Puccinia substriata Puccinia tetragoniae Puccina urticata var. urticae-acutae Puccinia urticata var. urticae-acutiformis Puccinia urticata var. urticae-biporulae Puccinia urticata var. urticae-hirtae Puccinia urticata var. urticae-inflatae Puccinia virgaureae Puccinia windhoekensis Trachyspora intrusa Uredo guerichiani Uredo guerichiani Uromyces aloes Uromyces caricis-sempervirentis Uromyes cf. ixiae Uromyces cf. ixiae Uromyces dactylidis Uromyces ficariae Uromyces gageae Uromyces ixiae Uromyces ixiae Uromyces junci (filed under Tuberculina sp.) Uromyces lycoctoni Uromyces otaviensis Uromyces pisi Uromyces poae Uromyces scutellatus Uromyces viciae-fabae Only the varietal epithets of the rust taxa were used in the phylogenetic trees. These varietal names also represent widely used species synonyms (see Gäumann 1959). GenBank accession numbers of sequences that had been published previously (Maier et al. 2003; Zambino & Szabo 1993) are given in parentheses. Herbarium acronyms: CFB (Northern Forestry Centre, Canadian Forest Service, Edmonton, Alberta); FO (F. Oberwinkler, private herbarium); GZU (Karl-Franzens-Universität Graz, Austria); IMI (CABI Bioscience, Egham, UK) M (Botanische Staatssammlung München, Germany); PREM (Plant Protection Research Institute, Pretoria, South Africa); TUB (Eberhards-Karls-Universität Tübingen, Germany); WM (W. Maier, private herbarium) Z þ ZT (Combined herbaria of the Universität Zürich and of the ETH Zürich). Na, RSA (collection numbers of collections made by Mennicken in Namibia and South Africa, respectively. These are housed in PREM and Z þ ZT, and PREM and M, respectively. RSA 153 displays double infections with Puccinia aridariae and Uredo guerichiani. Host relationships of the rusts at the family level are given in the columns I-host and III-host. ‘‘I’’ refers to aecial host, ‘‘III’’ to telial host. Heteroecious rusts can be identified by two different plant family names in these two columns. Autoecious macrocyclic rusts have the same family name presented twice in these two columns. ‘‘–‘‘ refers to those species lacking an aecial host and, thus, a short-cyclic rust. ‘‘?’’ refers to an unknown part of the life cycle. The host relationships of the outgroup species are cited in parentheses. Acronyms used for geographical origin: As (Asia), CA (Central America), CE (Central Europe), NA (North America), NE (Northern Europe), SAf (Southern Africa). 179 180 Modeltest 3.7 (Posada & Crandall 1998). As a result GTR þ I þ G (Tavare 1986; Rodrı́guez et al. 1990) was chosen for the following analyses. Phylogenetic estimations based on the obtained alignment were derived with the help of PAUP 4.0b10 (Swofford 2001) using Neighbour Joining (NJ (Saitou & Nei 1987) and with MrBayes 3.1.1 (Ronquist & Huelsenbeck 2003), respectively, using Metropolis Coupled Monte Carlo Markov Chains (MC3) to approximate the Bayesian posterior probability distribution. Branch support for neighbour joining was determined by 1000 bootstrap replicates. MC3 was run over one million and six million generations, respectively, starting from default (flat) values for the prior settings. Every 100th generation was sampled resulting in 10 001 and 60 001 trees. Of these the first 2001 and 40 001 trees, respectively, were discarded as burn-in and the posterior probability was estimated from the remaining 8000 and 20.000 trees, after the chains had converged to stationarity. Because MrBayes internally runs two independent analyses at once, two independent results for both, the one and six million generations were obtained. All phylogenetic trees were rooted with Trachyspora intrusa, Gymnosporangium sabinae and Chrysomyxa empetri. Results W. Maier et al. Puccinia, Uromyces, and Endophyllum were clearly polyphyletic, and Puccinia/Uromyces species with Cyperaceae and Juncaceae as telial hosts were found only in Group I. In contrast, species with Poaceae as telial hosts were found only in Group II. Species with dehiscent (‘‘Bullaria’’) as opposed to non-dehiscent (‘‘Eu-Puccinia’’) teliospores, did not correspond to either of the two large clusters accommodating the species included in this study. Species circumscription as reflected by the phylogenetic data In most cases where several collections of the same species were sequenced, the sequences were identical or differed only in one base pair in the gene region being studied (e.g. Miyagia pseudosphaeria, Puccinia aridaria, Uromyces ixiae (RSA 8, RSA 191 in the phylogenetic trees), and Puccinia silvatica. In the case of P. silvatica, sequences were obtained from both the aecial and telial hosts. In a limited number of cases, for example Uromyces cf. ixiae (RSA 211, RSA 213) compared to Uromyces ixiae (RSA 8, RSA 191) differences based on the DNA sequence data were found. It is probable that each of these represent separate taxa and, in this case, undescribed species. Data structure Discussion The phylogenetic trees obtained are based on the D1/D2 region of the nuclear large subunit of the ribosomal genes. The final alignment contained 550 characters, of which 179 were variable and 100 parsimony informative. The final alignment is deposited at TreeBASE (SN 2941; study accession number ¼ S1606, matrix accession umber ¼ M2889). In total 79 specimens representing 70 species were included in these analyses. Results of this study provide clear evidence that Cumminsiella, Dietelia, Endophyllum, Miyagia, Puccinia, Uromyces and, at least parts of the anamorph genera Aecidium and Uredo represent a highly supported monophyletic group of genera. The phylogenetic trees emerging from comparisons of DNA sequence data, however, do not support the generic boundaries of the species that we have included in the study. In this study, the two largest rust genera, Puccinia and Uromyces, emerged as polyphyletic. While this result was not unexpected, the degree of the polyphyly was surprisingly high. From the phylogenetic analyses, it is clear that the number of cells in the teliospore (one- versus two-celled), which is the basis of the separation between Puccinia and Uromyces, does not have phylogenetic significance. This supports the view of e.g. Anikster & Wahl (1979: 369) that Puccinia and Uromyces are ‘‘only artificially separated from each other’’. Thus, the transition from either one- to two-celled teliospores, or vice versa or transitions in both directions must have occurred frequently within closely related groups. The taxonomic value of this character is also flawed by species displaying intermediate forms that were included in the present study. Thus, one- and two-celled teliospores are displayed by Puccinia aridariae and Miyagia pseudosphaerica, or one, two, and threecelled by P. galeniae, or two-, three- and four-celled teliospores by P. knersvlaktensis. Phylogenetic trees showed that Cumminsiella clearly belongs to the Puccinia/Uromyces cluster, which has previously been suggested (Maier et al. 2003). Arthur (1933) recognized that Cumminsiella needed to be segregated from Uropyxis, which accommodated its representatives, because of its strikingly different spermogonial and aecial morphology. It then became clear that based on these characters Cumminsiella Phylogenetic trees The tree topologies obtained by Neighbour Joining (Fig 1) and Bayesian phylogenetic inference (Fig 2) are largely congruent for supported clades. The main difference being that in the Bayesian phylogeny, many of the non-supported groups are presented as polytomies. Two highly supported larger groupings were obvious in both phylograms (Figs 1 and 2). These are the in-group as a whole (98 % bootstrap/100 % a posteriori probability), comprising Aecidium, Cumminsiella, Dietelia, Endophyllum, Miyagia, Puccinia, and Uromyces, and the group of species designated as cluster ‘‘I’’ (91 %/100 %). Only in the Bayesian analyses a large subcluster of cluster I was also highly supported (99 %). This subcluster was lacking the taxa of Puccinia urticata and the Aecidium sp. sampled. Cluster ‘‘II’’ is then defined as all the species that do not reside in cluster I, but belong to the supported ingroup. Cluster II is however not statistically supported as a monophyletic group itself. Representatives of Aecidium, Endophyllum, Miyagia, Puccinia, and Uromyces resided in cluster I, while cluster II included representatives of Cumminsiella, Endophyllum, Puccinia, Uredo, and Uromyces. Polyphyly and two emerging lineages in Puccinia and Uromyces 181 Fig 1 – Phylogram obtained by a Neighbour Joining analysis using GTR D I D G as DNA substitution model. Bootstrap values above 60 % obtained by 1000 replicates are given above branches. Black squares behind species names refer to persistent teliospores (‘‘Eu-Puccinia’’); circles refer to dehiscent teliospores (‘‘Bullaria’’). ‘‘A’’ stands for the ‘‘African clade’’ discussed in the text. Only the variety epithets of the rust taxa were used in this tree (see the caption of Table 1). 182 W. Maier et al. Fig 2 – Majority-rule-consensus tree derived from 20000 trees sampled from the stationary phase of a Bayesian Monte Carlo Markov Chain analysis with GTR D I D G as nucleotide substitution model. A posteriori probabilities greater than 60 % are given above branches. was very similar to Puccinia, from which it differs only by having two as opposed to one germ pore per teliospore. It must, however, be noted that certain Puccinia species also display two germ pores per cell (e.g. Puccinia abutili or Puccinia cephalandrae; Mennicken et al. 2005b). All representatives of Cumminsiella are autoecious, most of them macrocyclic and they are naturally restricted to Mahonia and Berberis in the Americas (Baxter 1957; McCain & Hennen 1982). Considering these features collectively, we hypothesize that Cumminsiella is a monophyletic group within Puccinia/Uromyces, because the species included in this genus are morphologically and biologically strongly homogenous. The observation that Miyagia is part of Puccinia/Uromyces supports and enhances the findings of Wingfield et al. (2004). Polyphyly and two emerging lineages in Puccinia and Uromyces In that study, Miyagia clustered with representatives of Puccinia, Uromyces, and Dietelia with moderate support. More precisely, M. pseudosphaeria formed part of a cluster of autoecious Puccinia species parasitizing Asteraceae in the present study. This corresponds well with the fact that the genus Miyagia includes three species on Asteraceae and it ‘‘differs from Puccinia only in the peridiate [formed by palisade-like paraphyses] uredinia and telia’’ (Cummins & Hiratsuka 2003). Nevertheless, soral paraphyses represent a variable character within Puccinia species complexes (Savile 1984; Anikster et al. 2004). This fact and the phylogenetic placement of Miyagia suggest that soral paraphyses are not phylogenetically useful and it is obvious that the validity of Miyagia should be questioned. Consistent with observations regarding Miyagia, results of this study suggest that the three species of Corbulopsora, which also display uredinial and telial peridia and are parasitic on Asteraceae, will reside in the Puccinia/Uromyces cluster. Corbulopsora can be interpreted as a one-celled (‘‘Uromycestype’’) variant of Miyagia. Cummins (1940) who erected the genus treated it under Miyagia in the first edition of the ‘‘Illustrated Genera of Rust Fungi’’ (Cummins 1959) but the genus was kept separate in the second and third editions of this work (Cummins & Hiratsuka 1983, 2003). Results of this study show that both the endo-cyclic genera, Endophyllum and Dietelia, are clearly part of Puccinia/Uromyces, and that Endophyllum is polyphyletic. E. sempervivi resides in the phylogenetic Group II, whereas E. euphorbiaesylvaticae clustered with the heteroecious-macrocyclic U. pisi and the microcyclic U. scutellatus in Group I. U. pisi alternates between Euphorbia and the genera Lathyrus and Pisum (Fabaceae), while both U. scutellatus and E. euphorbiae-sylvaticae are short-cyclic on Euphorbia. This phylogenetic relationship has previously been predicted based on morphological traits and host relationships of these rust fungi by Jørstad (1952) who proposed to transfer E. euphorbiae-sylvaticae to U. euphorbiae-sylvaticae. Also from a phylogenetic point of view, Endophyllum as a whole would need to be included in Puccinia/ Uromyces, because it merely represents a special (endo) life cycle form of Puccinia/Uromyces as was clearly stated by Tranzschel (1910). The fact that Dietelia is part of the Puccinia/Uromyces cluster is consistent with previous results based on 18SrDNA sequence data (Wingfield et al. 2004). Despite the fact that Dietelia resides in Pucciniosiraceae as suggested by Cummins & Hiratsuka (2003) and not in the Pucciniaceae, this is not a surprising result, because Dietelia has the same spermogonial type as Puccinia and Uromyces and it is very similar to Endophyllum. The characters used to distinguish Dietelia from Endophyllum are subtle and include compact versus powdery aecia (aecioid telia according to the ontogentetic concept) and smooth versus ornamented aeciospores (teliospores in the ontogenetic concept) (Buriticá & Hennen 1980). Using the latter character to distinguish between the two genera was further obscured by the demonstration of verrucose aeciospores in D. codiaei (Boerema et al. 1994). Following the argument already presented for Endophyllum and based on a phylogenetic species concept, Dietelia also would need to be merged with Puccinia/Uromyces. It is no surprise that the sampled representatives of Aecidium and of Uredo belong to the Puccinia/Uromyces cluster. 183 The vast majority of species in these anamorph genera can be expected to belong here. Sub-generic classification and the host relationships of phylogenetic groupings Whether teliospores are borne on fragile versus robust pedicels correlates with phylogenetic groupings was also enquired. The relatively random distribution of this feature when plotted on a phylogenetic tree (Fig 1), however, suggests that it is a variable convergent character that can change in closely related groups. The homoplasious nature of this character had been postulated by Savile (1954, 1971), who gave a plausible ecological-evolutionary explanation for the observation that in closely related species-groups both types of teliospore pedicels can be found. Sub-generic classifications that had been based on this character, like the sub-genera Bullaria and Eu-Puccinia (Arthur 1934) do therefore not contain phylogenetic information. The two large groups that were found in the present study correlate with the biology of the rust species and thus might represent true sub-generic monophyla. Rusts parasitizing Poaceae in their uredinial and telial stages were found in various sub-clusters of species residing in cluster II, but never in cluster I. In contrast, the rusts that are parasitic on Cyperaceae or Juncaceae in their uredinial and telial stages were found only in several sub-clusters of species residing in cluster I of the phylogenetic tree. This pattern is supported by a high bootstrap support for group I, but not for group II, and a comparable pattern has also emerged in another study by van der Merwe et al. (2007) based on other gene regions and a different species sampling. Despite lacking support for the monophyly of group II, this pattern of association could be interpreted in support of the suggestion that Puccinia/Uromyces radiated mainly and independently on Poaceae and Cyperaceae and Juncaceae, respectively, as proposed by Savile (1976). While the Poaceae diversified mainly in dry grassland biomes, the Cyperaceae and Juncaceae radiated in a similar fashion predominantly in wet grassland biomes. Thus, the main-diversifications of Puccinia and Uromyces could have occurred through a mixture of radiation with Poaceae and Cyperaceae/Juncaceae, respectively, and frequent jumps to co-occurring new plant hosts in the respective biomes. Also several smaller groupings observed in the phylogenetic trees correlate with the families on which these rusts occur, while the grouping of other species indicate that host jumps are likely to have been common within Puccinia/Uromyces, as was postulated before (Savile 1971, 1990; Roy 2001). One possible example of this intricate relationship between host specificity and host jump, can be found in the group comprising Uromyces pisi, U. scutellatus, E. euphorbiae-silvaticae, U. viciaefabae and U. caricis-sempervirentis within Group I. This group is only highly supported by the MCMC analyses (99 %), nevertheless it is monophyletic in both the MCMC and NJ trees. U. caricis-sempervirentis displays a host shift between Phyteuma (Campanulaceae) and Carex (Cyperaceae), while the other species in this group alternate between Euphorbiaceae and Fabaceae (U. pisi), are macrocyclic-autoecious on Fabaceae (U. viciae-fabae) or are short-cyclic on Euphorbiaceae only (U. scutellatus, Endophyllum euphorbiae-sylvaticae). The current 184 data, however, precluded speculation as to the direction of the presumed host jump in an ancestor of this group. Geographical patterns Because the majority of species sampled in this study are of European origin, it is pertinent to briefly consider the clustering of species from other geographic origins with them. Within Group II, a cluster exclusively comprising southern African rust species can be found. Although not statistically supported in the NJ tree, a large part of this group is supported by the MCMC phylogram. All species in this group are parasitic on Aizoaceae and thus, it is not only geographic origin but also the host specificity of this group that is reflected by the phylograms. In this context, it is especially important to note that Puccinia otzeniani, which is also parasitic on Aizoaceae in southern Africa, is not part of this group. Thus, rusts on the Aizoaceae have originated from different lineages within group II and are only partly monophyletic. The majority of rusts sampled from southern Africa are part of Group II, which might reflect the fact that large parts of this area are dominated by grasslands and savannas, where Poaceae are especially frequent, and that Cyperaceae, more common in wet lands, are much less frequent. However, there was one southern African representative residing in Group I, P. windhoekensis, suggesting that its origin was from the ‘‘Cyperaceae-rust group’’. This is the first study based on a considerable taxon sampling using species from a broad range of host families and different geographic origins that has attempted to explore the phylogenetic structure of Puccinia and Uromyces and satellite genera. Intriguing phylogenetic patterns have emerged from the analyses including some that might have been expected and others that are surprising. Nonetheless, the polytomies in the Bayesian consensus tree and many statistically unsupported groupings in general show that various results of this study must be regarded as preliminary. The observed polytomies can be interpreted as reflection of the fact that less than 2 % of the 4500 or so species residing in Puccinia/Uromyces have been sampled. In addition, it is important to consider that the phylogeny is based on sequences of a single gene region, and the value of this gene region especially lies in detecting larger phylogenetic lineages within Puccinia/Uromyces. For these and for practical reasons no name changes have been attempted at this stage. However, the results should serve as a basis for further studies and for large-scale collaborations that will be necessary to address the questions raised here in more detail. Addendum Additional evidence to support this study is the research presented by van der Merwe et al. (2007). Their study came to our attention only after the experimental part of the present study had been completed. They observe the same major groupings as we report in the present study. However, van der Merwe et al. (2007) used different gene regions and a different subset of species. As the two studies reflect similar results, we have chosen to submit both studies simultaneously in order that they would be published in the same journal issue. W. Maier et al. During the review process of the present paper, a combined nuc rDNA SSU/LSU study was published dealing with higherlevel relationships of the rust fungi (Aime 2006). This study provides additional support for the view that Aecidium, Cumminsiella, Dietelia, Miyagia, Puccinia and Uromyces have a common origin. Pucciniosira and Sphenospora also formed part of that clade, and Puccinia and Uromyces were again shown to be polyphyletic. Acknowledgments We are grateful to the National Research Foundation (NRF), the NRF/DST Centre of Excellence in Tree Health Biotechnology (CTHB), the members of the Tree Protection Co-operative Programme (TPCP), the THRIP initiative of the Department of Trade and Industry (DTI) South Africa and the Deutsche Forschungsgemeinschaft (DFG) for financial support, and Sappi for a Research Fellowship to W.M. We also thank Franz Oberwinkler, Markus Göker and Reinhard Berndt for specimens as well as Matthias Lutz and Dominik Begerow for specimens and unpublished sequences, and the curators of GZU, PREM and TUB for the loan of specimens and support. Supplementary material Supplementary material associated with this article can be found, in the online version, at 10.1016/j.mycres.2006.11.005. references Aime MC, 2006. Toward resolving family-level relationships in rust fungi (Uredinales). Mycoscience 47: 112–122. Akaike H, 1974. A new look at the statistical model identification. 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