mycological research 110 (2006) 633–645
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycres
Revision of Plasmopara (Chromista, Peronosporales) parasitic on Geraniaceae Hermann VOGLMAYRa,*, Jamshid FATEHIb, Ovidiu CONSTANTINESCUc a
Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria The MASE Laboratories, P.O. Box 148, SE-751 04 Uppsala, Sweden c Museum of Evolution, Botany Section, Evolutionary Biology Centre, Uppsala University, Norbyva¨gen 16, SE-752 36 Uppsala, Sweden b
article info
abstract
Article history:
Following a phenetic and phylogenetic analysis, five species of Plasmopara are recognized
Received 12 November 2005
on Geraniaceae: P. pusilla and P. geranii-sylvatici in Eurasia, P. geranii in North America,
Received in revised form
P. praetermissa sp. nov. in Eurasia and North America, and P. wilsonii sp. nov. in North
24 January 2006
America and Far East Asia. Both the D1/D2 domains of the nuLSU-rDNA and the complete
Accepted 1 March 2006
ITS1-5.8S rDNA-ITS2 region were analysed with MP and Bayesian methods to reveal phylo-
Published online 12 June 2006
genetic relationships of the species. All species formed highly supported monophyletic
Corresponding Editor:
lineages, which is corroborated by their distinct morphology. A key for identification,
David L. Hawksworth
detailed descriptions, illustrations, and data on distribution are provided. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Keywords: Geranium Molecular phylogeny Oomycota Plant pathology
Introduction Four species of Plasmopara parasitic on Geranium spp. have been described so far, but only three (P. pusilla, P. geranii-sylvatici and P. geranii) were considered nomenclaturally valid and taxonomically sound (Constantinescu 2004). At the same time, the existence of an undescribed species, parasitic on G. sylvaticum was also revealed (Constantinescu 2004). This new species was concomitantly and independently found by H.V. (Voglmayr et al. 2004). These facts prompted us to initiate a polyphasic study of Plasmopara parasitic on Geraniaceae. During the study it turned out that the new species on G. sylvaticum may attack some other species of Geranium, and it has a wide distribution that includes various areas of Europe,
Asia and North America. At the same time, another new species was detected in North America and Eastern Asia.
Materials and methods Morphological analyses Some 1233 herbarium specimens, particularly from the exten˚ S, H, HBG, M, S, and sive collections deposited in herbaria A UPS (herbarium acronyms according to Holmgren et al. 1990) but also recently collected ones, were investigated. Due to this extensive number, only selected specimens are cited in the text: all specimens distributed as exsiccatae are listed, and for
* Corresponding author. E-mail address:
[email protected]. 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.03.005
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countries where no such exsiccatae were available one representative herbarium specimen is cited. Abbreviation of exsiccatae follows Triebel and Scholz (2001–2005). The complete list of specimens examined is deposited at herbaria UPS and WU and is available as supplementary data on Science Direct. Methods for slide preparation and examination in light microscopy have been described earlier (Constantinescu 1998; Constantinescu & Fatehi 2002). Descriptive terminology of morphological shapes is according to Systematics Association Committee for Descriptive Biological Terminology (1962) and Kirk et al. (2001:
Fig 36). Abbreviation of US state names follows Farr et al. (1989) and those of Scandinavian provinces Jonsell (2004).
Sample sources, DNA extraction, PCR and sequencing The organisms sequenced in the present publication are listed in Table 1. Besides the accessions from Geraniaceae, two species of Phytophthora (Ph. infestans and Ph. nicotianae) were used as outgroups and Plasmopara nivea as the generic type in the nuLSU rDNA dataset (Constantinescu et al. 2005). Choice of
Table 1 – Collection data and GenBank accession numbers of the taxa sequenced. The taxa are grouped alphabetically Taxon
Collection data Host
a,b
Origin/source; date, collector (voucher)
LSU GenBank accession no.
Phytophthora infestans Ph. nicotianae Plasmopara chaerophylli Pl. geranii
Solanum tuberosum
USA, Hawaii (CBS 560.95)
AF119602
Nicotiana tabacum Anthriscus sylvestris
Taiwan (CBS 305.29) Sweden, Stockholm; 12 June 1999, OC (UPS F-119992)
AY250184
Geranium maculatum
AY035520
Pl. geranii Pl. geranii
Geranium maculatum Geranium maculatum
Pl. geranii-sylvatici
Geranium palustre
Pl. geranii-sylvatici Pl. geranii-sylvatici
Geranium palustre Geranium sylvaticum
Pl. geranii-sylvatici
Geranium sylvaticum
Pl. geranii-sylvatici Pl. geranii-sylvatici Pl. nivea
Geranium sylvaticum Geranium sylvaticum Aegopodium podagraria
Pl. praetermissa
Geranium sylvaticum
Pl. praetermissa Pl. praetermissa Pl. praetermissa
Geranium sylvaticum Geranium sylvaticum Geranium sylvaticum
Pl. praetermissa Pl. praetermissa
Geranium sylvaticum Geranium richardsonii
Pl. pusilla
Geranium pratense
Pl. pusilla
Geranium sibiricum
Pl. pusilla
Geranium pratense
Pl. wilsonii Pl. wilsonii
Geranium carolinianum Geranium carolinianum
Pl. wilsonii Pl. wilsonii
Geranium molle Geranium nepalense ssp. thunbergii Geranium nepalense ssp. thunbergii
USA, Tennessee, Knoxville; 6 Apr. 2000, HV (WU), HV6.4.P.P. USA, Tennessee, Knoxville; 23 May 2003, HV (WU), PA8 USA, North Carolina, Highlands; 19 May 2003, HV (WU), PA5 Germany, Baden-Wu¨rttemberg, Tu¨bingen; 1 July 2001, HV (WU), HV942 Norway, Oslo, Skullerud; 14 Aug. 2002, HV (WU), HV2001 Germany, Baden-Wu¨rttemberg, Feldberg; 12 July 2001, HV (WU), HV946 Austria, Niedero¨sterreich, Lunz/See; 27 June 2000, HV (WU), HV411 Sweden, Uppsala; 2 July 2003, OC 03-60 (UPS F-119990) Sweden, Uppsala; 6 Aug. 2000, OC 2000 (UPS F-119991) Germany, Bayern, Oberjoch; 3 Oct. 2000, F. Oberwinkler (TUB) Norway, Hordaland, Ulvik, Finse; 10 Aug. 2002, HV (WU), HV2061 Austria, Ka¨rnten, Flattach; 8 July 2000, HV (WU) HV473 Austria, Ka¨rnten, Flattach; 10 July 2000, HV (WU) HV498 Sweden, Uppsala; 18 June 2003, OC 03-33 (UPS F-119993, holotype) Sweden, Uppsala; 18 June 2003, OC 03-40 (UPS F-119994) USA, Wyoming, 5 July 1972, W.G. Solheim & R. Solheim, 1972UPS (UPS F-119997) Austria, Steiermark, Spital/Semmering; 24 Aug. 2000, HV (WU), HV619 Austria, Wien, Botanischer Garten; 23 Apr. 1999, HV (WU), HV140 Russia, Rakitinskij floristic reserve; 4 July 1994, K.A. Pystina, 1994UPS (UPS F-119995) USA, Tennessee, Knoxville; 23 May 2003, HV (WU), PA9 USA, Washington, National Arboretum; 30 May 2003, HV (WU), PA17 USA, Maryland, Beltsville; 13 May 2003, HV (WU), PA1 Korea, Jeju; 1 Nov. 2002, H.D. Shin, HV2065 (SMK 19358 ¼ UPS F-119996) Korea, Wonju; 18 Oct. 2001, H.D. Shin, HV2066 (SMK 19276)
Pl. wilsonii
ITS GenBank accession no.
DQ131920 DQ131916
DQ148403 DQ148397 DQ148401 DQ148399 DQ148400 DQ148398 DQ131913 DQ131914 AY250162 DQ148396 AY250172 DQ148395 DQ131921 DQ131922 DQ131923 AY250168 DQ148402 DQ131915 DQ148406 DQ148407
DQ131919
DQ148405 DQ148408
DQ131918 DQ131917
DQ148404
a Collectors: HV, H. Voglmayr; OC, O. Constantinescu. b Vouchers: CBS, Centraalbureau voor Schimmelcultures, Utrecht; SMK, Korea University, Seoul; UPS-F, Uppsala University; WU, University of Vienna.
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outgroup species was based on results of previous (Go¨ker et al. 2003) and unpublished investigations showing that they are closely related to the genus Plasmopara. As it has not been possible to obtain an ITS sequence for P. nivea, the closely related P. chaerophylli was chosen instead. The DNA extraction, PCR, cycle sequencing and sequencing of the nuLSU D1/D2 region are described in Riethmu¨ller et al. (2002). For ITS, DNA was extracted directly from small pieces of infected leaf tissues from herbarium specimens. The extraction of DNA, PCR amplification of the total ITS region (including ITS1, 5.8 S rRNA gene and ITS 2), cloning of the amplified fragments and nucleotide sequencing of the ITS were performed as described in Constantinescu and Fatehi (2002). Sequencing gels were run by the Department of Animal Breeding and Genetics at Swedish University of Agricultural Sciences, Uppsala, Sweden.
probabilities that groups are monophyletic given the sequence data (posterior probabilities). To confirm that the posterior probability distribution of the MCMC processes is stationary (Huelsenbeck et al. 2002), the Bayesian analysis was repeated three times on a personal computer, always starting with random trees and default parameter values of the program. Bayesian analysis of the ITS data was performed in a similar manner as the nuLSU rDNA data, except that the best-fit selected model (by hLRT) implemented was the general time reversible substitution model with gamma-distributed substitution rates of the sites (GTR þ G; see Swofford et al. 1996). To be cautious the first 1000 saved trees were discarded, though the log likelihood reached a stable value after 4000 generations. However, inclusion of the trees from this point did not change the posterior probability values. The analysis was repeated twice.
Data analysis
Results
The nuLSU rDNA sequence alignment was produced with the aid of ClustalX (Thompson et al. 1997). The resulting alignment was checked and refined using BioEdit 5.0.9 (Hall 1999) and PAUP* version 4.0 b10 (Swofford 2002). The ITS sequences were aligned using the DNAStar computer software package (Lasergene, Madison, WI) and the alignment was subsequently visually improved. Sequence pair distances were determined between aligned sequences using the MegAlign programme in DNAStar. MP analysis of the nuLSU rDNA data was performed with PAUP* (version 4.0 b10; Swofford 2002), using 1000 replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping (MULTREES option in effect, steepest descent option not in effect), each replicate limited to 100,000 rearrangements. All molecular characters were unordered and given equal weight; gaps were treated as missing data. BS analysis with 1000 replicates was performed in the same way, but using ten rounds of random sequence addition and subsequent branch swapping during each BS replicate. MP analysis of the ITS data was performed in a similar manner, except for having 10,000 replicates of heuristic search with random addition of sequences without a limit of rearrangements. BS analysis with 1000 BS replicates was performed in the same way, but using 100 rounds of random sequence addition and subsequent TBR branch swapping during each BS replicate. For the Bayesian analysis of the nuLSU rDNA data, first the appropriate model of DNA substitution was selected by the hierarchical likelihood ratio test (hLRT) as implemented in Mr Modeltest v2.2 (Nylander 2004). The best-fit selected model (by hLRT) was that of Hasegawa et al. (1985) with gamma-distributed substitution rates of the sites (HKY þ G), which was used in subsequent Bayesian analysis. MCMC (Mau et al. 1999; Larget & Simon 1999) were performed with the computer program MrBayes (version 3.0b4; Huelsenbeck & Ronquist 2001). Four incrementally heated simultaneous Markov chains were run over 1 M generations from which every 100th tree was sampled. The trees before apparent stationary probability distribution of the cold chain were discarded (usually the first 1000 trees saved). A 50 % majority rule consensus of the remaining trees was computed to obtain estimates for the
The final alignment and the trees obtained were deposited in TreeBASE (http://www.treebase.org) and are available under study accession no. S1445. The nuLSU rDNA fragment sequenced did not differ in length between the different Plasmopara species, except for a 1 bp deletion in P. praetermissa. The final alignment used for tree calculation was 766 bp long. Considerable size variations in ITS were observed among some of the studied taxa. P. pusilla had the shortest ITS fragment (1100 bp), followed by P. geranii-sylvatici (1105 bp) and P. praetermissa (1160 bp). Significantly longer ITS sequences were obtained for P. geranii (1385 bp) and P. wilsonii (1506–1507 bp). The ITS1 region and 5.8 S rDNA were relatively conserved in length and they ranged between 209–212 bp and 160–161 bp, respectively. The great length variations of the ITS observed among some of the taxa were mostly due to the presence of large inserts in the ITS2 region of P. wilsonii and P. geranii, with lengths of 360 bp and 218 bp, respectively. In the aligned ITS sequences, the inserts of these two taxa were located at the same position (nucleotide 702–703 in P. wilsonii and nucleotide 720 in P. geranii). The sequence identity matrix of the ITS sequences is shown in Table 2. MP analysis of the partial nuLSU rDNA revealed 14,107 most parsimonious trees of score 142; one of these trees is shown in Fig 1. The topological position of the species relative to each other (tree backbone) was identical in all trees. MP analysis of the complete ITS-5.8 S rDNA-ITS2 region revealed three most parsimonious trees of score 657, which only differed in topology within the P. wilsonii clade; one of these trees is shown in Fig 2. Within both data sets, the Bayesian and the MP trees were fully compatible; the posterior probabilities were similar to but usually slightly higher than the MP BS values (Figs 1–2). All species were highly supported as monophyletic lineages. Tree topologies of the nuLSU rDNA and ITS data sets were very similar except for the position of P. praetermissa relative to P. nivea and P. chaerophylli, respectively. However, the topological position of P. praetermissa relative to the other Plasmopara species received only very low support by MP BS values and posterior probabilities in both trees (Figs 1–2). The molecular data sets confirm the morphological evidence for presence of five clearly distinct Plasmopara species on Geranium.
636
Discussion The molecular and morphological differences are clear evidence for the presence of five species on Geraniaceae. The data also indicate that they are members of the core Plasmopara clade that includes P. nivea, the lectotype of the genus Plasmopara (Constantinescu et al. 2005). Recently, the genus Plasmopara has been redefined after molecular and morphological investigations, and the new genera Plasmoverna (Constantinescu et al. 2005), Protobremia (Voglmayr et al. 2004) and Viennotia (Go¨ker et al. 2003) have been segregated from Plasmopara. Conversely, the genus Bremiella is now considered synonymous with Plasmopara (Riethmu¨ller et al. 2002; Voglmayr et al. 2004). These investigations also showed that several genetically distinct lineages representing separate species can be found on the same host family, e.g. Ranunculaceae (Constantinescu et al. 2005), Asteraceae (Voglmayr et al. 2004) and Apiaceae (Voglmayr et al. 2004). However, from the data presented here it is not possible to evaluate whether the species parasitizing Geraniaceae form a monophyletic lineage within Plasmopara. In Voglmayr et al. (2004), P. praetermissa (included as Plasmopara sp.) did not form a monophylum with the other species on Geraniaceae but was a sister taxon to all other species of the core Plasmopara clade. However, posterior probability for this position was 94 %, and MP BS support was completely absent. P. wilsonii was not included in Voglmayr et al. (2004); in a Bayesian analysis with the same nexus file and settings as in Voglmayr et al. (2004), P. wilsonii was a sister taxon to the other Plasmopara species from Geraniaceae (except P. praetermissa) with 96 % posterior probability (data not shown), which corresponds to the topologies revealed in this study (Figs 1–2). However, in the MP analysis of the same data set, such a position of P. wilsonii did not receive BS support (data not shown). Therefore, the partial nuLSU rDNA data are not adequate to evaluate monophyly versus polyphyly in Plasmopara from Geraniaceae, and additional sequence data need to be collected for the whole genus. Considering the pronounced morphological differences and its abundance in the well-studied northern latitudes of Europe, it is astonishing that P. praetermissa has not been detected earlier. The reason for this may be that the fungi
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are usually only superficially examined, the primary method of identification being just the host–parasite association. This once again demonstrates the need of thorough morphological investigations of specimens in systematic studies.
Taxonomy Plasmopara pusilla (de Bary) J. Schro¨t, in Cohn, Krypt.-Fl. Schles. 1: 237 (1886) (Fig 3). On leaves producing slight discolouration of the tissues to clearly defined epiphyllous spots; spots first pale yellowish, later becoming light to dark brown, polyangular, 1–12 mm diam, with distinct margin, sometimes coalesced, attacked tissues become necrotic. Down on the spots’ corresponding lower surface, white, lanose to felt-like. Haustoria obpyriform, 6–7 mm diam, inclusive the ca 1–1.5 mm thick sheath. Sporangiophores up to 25 in a fascicle, slender, 65–160 mm long, trunk straight, slightly curved or more or less wavy, 50–140 mm long, of more or less uniform breadth, basal end not differentiated to slightly bulbous, 4–6 (–8) mm wide above the base, 4–6 (–7) mm wide below the first branch, wall ca 0.8 mm thick; callose plugs normally absent (only exceptionally present). Branches absent, or as outgrowths, or short and simple structured. Ultimate branchlets appearing bi- or trifurcate and either sitting on the distal part of the trunk (when branches absent), or on more or less swollen ends of the very short branches; convergent or diverging mostly at ca 30–40 angle, very rarely differentiated into axial and abaxial ones, more or less cylindrical, of almost the same length, 5–22 mm long, 2–3 mm wide at the base, 1.5–2 mm just below the tip, tip blunt or round. Sporangia broadly oblong to oblong, rarely broadly ellipsoidal, (20–) 24–30 (–38) mm long, (16–) 18–22 (–27) mm wide, length:breadth ratio (1.07-) 1.25-1.43 (-1.65), n ¼ 163, greatest breadth median or supra-median, rarely sub-median, base and tip round, wall ca 0.5 mm thick, apical dehiscence apparatus 4–5 (–6) mm diam composed of a lenticular to outwardly convex 1–2 mm thick plug, pedicel either absent or visible as a scar, to slightly protruding. Resting organs in leaves; oogonia broadly globose to irregular, (33–) 40–45 (–52) mm diam, yellowish, wall folded, ca 2 mm thick, periplasm yellowish; oospores mostly plerotic, globose, 28–38
Key for the identification of Plasmopara species parasitic on Geraniaceae 1 Most sporangiophores 200 mm long and/or branching more elaborate...........................3 2(1) Trunk mostly 4–6 mm wide and callose plugs absent; ultimate branchlets two- or trifurcate, directed upwards and having both closed (round) and/or open (truncate) ends; sporangia oblong........................pusilla Trunk mostly 6–8 mm wide and callose plugs often present; ultimate branchlets facing in all directions and having closed (round) ends; sporangia broadly ellipsoidal................................geranii-sylvatici 3(1) Trunk mostly ca 6–7 mm wide and of uniform breadth, callose plug very rarely present in branches........wilsonii Trunk mostly ca 8–10 mm wide, frequently with abrupt breadth change, callose plugs frequent in branches........4 4(3) In most trunks the lower part narrower than the upper one; sporangia typically oblong, size mean values 20.5 16 mm (length:breadth ratio 1.28), oospores 23–32 mm diam...............................geranii In most trunks the lower part wider that the upper one; sporangia typically broadly oblong to subglobose, size mean values 23 19 mm (length:breadth ratio 1.21); oospores 32–48 mm diam......................praetermissa
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Table 2 – Nucleotide sequence identity matrix of the ITS1, 5.8 S rDNA and ITS2 regions between the accessions of Plasmopara studied. n not calculated as only one specimen of that species was sequenced ITS1 Pl. Pl. Pl. Pl. Pl. Pl.
geranii-sylvatici pusilla geranii wilsonii praetermissa chaerophylli
5.8S Pl. geranii-sylvatici Pl. pusilla Pl. geranii Pl. wilsonii Pl. praetermissa Pl. chaerophylli ITS2 Pl. geranii-sylvatici Pl. pusilla Pl. geranii Pl. wilsonii Pl. praetermissa Pl. chaerophylli
Pl. geranii-sylvatici
Pl. pusilla
99.6
95.5–95.7 n
99.4
98.8–99.4 n
99.7
93.7 n
Pl. geranii
Pl. wilsonii
Pl. praetermissa
Pl. chaerophylli
83.2–84.5 81–81.9 87.9–88.8 98.7–99.6
85.8–86.2 85.3 87.9 80.6–81.5 100
89.2–89.7 87.9 93.5 87.5–88.4 90.5 n
98.1–98.8 98.8 n
98.1–98.8 98.1 97.5 100
99.4–100 99.4 98.8 98.8 100
98.1–98.8 98.1 97.5 98.8 100 n
68 65.7 n
50.6–50.7 48.3–48.6 70.9–71.2 99–99.3
80.2–80.7 78–78.2 67–67.3 50.3–50.9 99.1–99.5
77.7 76.2 58.7 43.7–43.9 78.7–78.9 n
93.1–93.5 91.4 n
mm diam, with smooth, yellowish to almost colourless 2.5–3.5 mm thick wall. Frequent host: Geranium pratense. Occasional hosts: G. asphodeloides, G. dahuricum and G. sibiricum. Distribution: Europe (particularly Central Europe), and rarely west of Ural Mts. This species was reported almost indiscriminately from many places. Records from outside Europe and particularly on other hosts than G. pratense should be treated with caution. Type: on G. pratense, Fuckel, Fungi Rhen. Exs. 26, under P. pygmaea Unger (S - lectotype designated by Constantinescu 2004; M-57224, W - isolectotypes designated here). Additional specimens examined: on G. asphodeloides (1): Poland: S´la˛skie, Wroc1aw, May 1880, J. Schro¨ter (WRSL). On G. dahuricum (1): Russia: Tomsk, 20 Jul. 1924, Rozhdestvenskij (LEP). On G. pratense (204): Austria (11): Niedero¨sterreich, Hollabrunn distr., Hardegg, Riegersburg, 17 May 2005, H. Voglmayr HV2168 (WU); Belgium (1): Roumegue`re, Fungi Sel. Exs. 4320 (UPS); Czech Republic (52): Kerner von Marilaun, Fl. Exs. Austro-Hung. 778 (BUC, C, GZU, H, M-57235, W, WU); Petrak, Crypt. Exs. 3501 (C, GZU, H, M-57241, UPS, W); Petrak, Fl. Bohem. Morav. Exs. Pilze 449 (HBG, M-57242, PRM 732422, W); Petrak, Fungi Eichleriani 34 (HBG, S, W); Svrcˇek, Fungi sel. exs. 1 (C, GZU, OULU, UPS); Denmark (1): Fyn, 12 July 1880, E. Rostrup (S); Finland (6): Liro, Mycoth. Fenn. 309 (GZU, H, TUR, UPS); France (1): Jura, Chapois, 3 July 1967, G. Chevassut (MPA); Germany (89): Thu¨men, Mycoth. Univ. 422 (BUC, GZU, H, HBG, M-57249, UPS, W, WRSL, WU); Allescher & Schnabl, Fungi Bav. 555 (BUC, GZU, H, HBG, M-57189, S); Krieger, Fungi Sax. Exs. 196a (GZU, H, HBG, M-57220, S, W); Rabenhorst, Fungi Eur. Exs. 1371 (H, HBG, M57218, UPS, W, WU); Sydow, Mycoth. Germ. 2903 (C, H, HBG, M-57213, S, UPS, W, two spec., WU); Sydow, Phycomyc. Protomyc. 78 (HBG, M-57257, UPS, W, WU); Sydow, Mycoth. March. 326 (HBG, UPS, W); Great Britain (3): Llanfrynach, Brecon, 22 Jul. 1927, P.G.H. Rhodes (M-57240, S); Italy (4): Su¨dtirol, 7 Sep. 1904, A. Heimerl rvete, Uplejas (HBG); Latvia (2): Dobele, Te farm, 27 July 1961, E. Vimba 3086 (RIG); Poland (9): Raciborski, Fungi Parasit. Polon. Exs. 55 (HBG, S); Kochman, Mycoth. Polonica 3 (H, UPS, W); Schroeter, Pilze Schles. 101 (HBG, WRSL); Schroeter, Pilze Schles. 4 (HBG,
vulescu, WRSL); Romania (10): Borza, Fl. Roman. Exs. 2331 (C, W); Sa Herb. Mycol. Roman. 118 (BUCM, M-57233, W, WU); Russia (8): Triebel, Microf. Exs. 179 (GZU, H, MA, UPS); Bucholtz & Bontarzev, Fungi Ross. Exs. Ser. B 604 (CUP, WSP 14184); Slovakia (7): Vysoke´ Tatry, Poprad, June 1926, K. Keissler (W). On G. sibiricum (3): Austria (3): Wien, Botanical Garden of the University, 23 April 1999, H. Voglmayr HV140 (WU).
Comments: The distal end of the ultimate branchlets in P. pusilla either remains open after sporangium secession, a feature encountered also in Plasmoverna (Plasmopara) pygmaea (Constantinescu et al. 2005), or a new wall is formed and the tip appears round (Fig 3). Nevertheless, in contrast to P. pygmaea, the plug of the dehiscence apparatus is firmly attached to the sporangium wall. P. pusilla differs from P. geranii-sylvatici by the ultimate branchlets mostly directed upwards and the oblong sporangia, and from P. praetermissa by its simple structured sporangiophores with narrow trunk and absence of callose plugs. This fungus is mostly restricted to G. pratense. The records on G. sylvaticum refer to either P. geranii-sylvatici (often) or to P. praetermissa (more rarely). Several records and specimens labelled P. pusilla on Geranium spp., like the ones cited in Hruby (1932) and preserved in S and HBG contain Ramularia. Similarly, the record by Vanev et al. (1993) on G. reflexum L. is based on a mixture of Geranium harbouring Ramularia geranii, and Anemone sp. parasitized by Plasmoverna (Plasmopara) pygmaea (Constantinescu & Denchev 2004). Like in other downy mildews, several herbarium specimens labelled P. pusilla turned out to contain Ramularia, anamorphs of Erysiphaceae, or even Ceratobasidium. vul. & O. Sa vul., Bul. S Plasmopara geranii-sylvatici Sa x ti. Acad. Republ. Pop. Rom. Sectx. S x ti. Biol. Geol. Geogr. 3: 352 (1951) (Fig 4). On leaves producing slight discolouration of the tissues to clearly defined epiphyllous spots. Spots first pale yellowish,
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Pl. geranii-sylvatici G. palustre HV942 99 100
Pl. geranii-sylvatici G. palustre HV2001 Pl. geranii-sylvatici G. sylvaticum HV946
100
Pl. geranii-sylvatici G. sylvaticum HV411
100 100 75
100
85
Pl. pusilla G. sibiricum HV140 Pl. pusilla G. pratense HV619
Pl. geranii G. maculatum PA5 91 98 87 100
Pl. geranii G. maculatum HV6.4.P.P. Pl. geranii G. maculatum PA8 Pl. wilsonii G. nepalense ssp. thunbergii HV2066 Pl. wilsonii G. carolinianum PA9 100
58 54
100
Pl. wilsonii G. molle PA1 Pl. wilsonii G. carolinianum PA17 Pl. wilsonii G. nepalense ssp. thunbergii HV2065
Pl. praetermissa G. sylvaticum HV498
100 100
100 100
Pl. praetermissa G. sylvaticum HV2061 Pl. praetermissa G. sylvaticum HV473
Pl. nivea AY250162 Ph. infestans AF119602 Ph. nicotianae AF250184 5 substitutions Fig 1 – One of the 14,107 most parsimonious trees inferred from the partial nuLSU rDNA data. Branch lengths correspond to the number of substitutions, numbers above branches show MP BS values (>50 %), below branches posterior probabilities obtained from a Bayesian analysis of 1 M generations.
later becoming light brown, to reddish, to violet brown, irregular to polyangular, from ca 2-12 mm diam to covering large areas or even the whole leaf surface, attacked tissues becoming necrotic. Down hypophyllous, whitish, consisting of scattered patches of conidiophores to almost felt-like. Haustoria vesicular, 5-7 mm diam including the sheath. Sporangiophores stout to slender, (75-) 100-160 (-190) mm long, n ¼ 39; trunk straight, rarely slightly curved, (65-) 80-135 (-160) mm long, from more or less uniform breadth to gradually broadening or tapering upwards, basal end not differentiated to slightly bulbous and then up to 10 mm wide, (5-) 6-8 (-9) mm wide above the base, 6-8 (-10) mm wide below the first branch, wall ca 0.6-0.8 mm thick, often thinner in the lower part; callose plugs often present, located mostly in the median part or slightly lower. Branches (0-) 1-2 (-3) on each sporangiophore, mostly
reduced to short outgrowths, rarely more differentiated and then up to 30 mm long, branching monopodial, in one order, very rarely two orders; callose plugs absent. Ultimate branchlets either single or in groups of 2-4 arising from a slightly inflated base, mostly not differentiated into axial and abaxial ones, more or less finger-like, 4-15 mm long, 2-3 mm wide at the base, ca 1.5 mm wide just below the tip, tip round, sometimes very slightly inflated. Sporangia broadly ellipsoidal, occasionally almost oblong, (20-) 23.5-29 (-35) mm long, (16-) 19.5-23.5 (-27) mm wide, length/breadth ratio (1.07-) 1.14-1.3 (-1.45), n ¼ 129, greatest breadth median or supra-median, very rarely sub-median, base and tip round, wall ca 0.5-0.6 mm thick; apical dehiscence apparatus 3.5-5 mm diam, composed of an outwardly convex to lenticular ca 1.5 mm thick plug; pedicel absent to slightly protruding. Resting organs in leaves, easily
Revision of Plasmopara (Chromista, Peronosporales) parasitic on Geraniaceae
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Pl. geranii-sylvatici G. sylvaticum OC03-60 Pl. geranii-sylvatici G. sylvaticum OC2000 Pl. pusilla G. pratense 1994UPS Pl. geranii G. maculatum HV6.4.P.P.
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Pl. wilsonii G. nepalense ssp. thunbergii SMK19358 100 100
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Pl. wilsonii G. molle PA1 Pl. wilsonii G. carolinianum PA17
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Pl. chaerophylli Anthriscus sylvestris
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Pl. praetermissa G. sylvaticum OC03-33 Pl. praetermissa G. sylvaticum OC03-40 Pl. praetermissa G. richardsonii 1972UPS
Ph. infestans AF266779 Ph. nicotianae AF266776 50 substitutions Fig 2 – One of three most parsimonious trees resulting from an analysis of the ITS sequence data including ITS1, ITS2 and 5.8 S rRNA gene. Branch lengths correspond to the number of substitutions, numbers above branches represent MP BS values (>50 %) and numbers below indicate posterior probability values obtained from a Bayesian analysis of 1 M generations.
visible on the upper leaf surface as dark or black spots; oogonia broadly globose to irregular by mutual pressure, (35-) 40-45 (-55) mm diam, wall yellowish to brownish to reddish brown, more or less wrinkled, (1-) 1.5-2 mm thick; periplasm yellowish; oospores plerotic, rarely aplerotic, globose, 30-42 mm diam, with colourless to yellowish, ca 3 mm thick wall that may reach 7 mm in warmed up slides. Frequent hosts: Geranium sylvaticum and G. palustre. Occasional hosts: G. collinum, G. erianthum, G. nodosum, and G. sylvaticum ssp. pseudosibiricum. Distribution: Eurasia. Because this species is sympatric with P. praetermissa, particularly in high altitude and latitude areas, the occurrence records are unreliable and should be checked against the specimens. Type: on Geranium sylvaticum, Weese, Eumyc. Sel. Exs. 457 (BUCM 3202 - lectotype designated by Constantinescu 2004; UPS F-120166 - isolectotype designated by Constantinescu 2004; M-57229, W - isolectotypes designated here). Additional specimens examined: on G. collinum (1): Ukraine: Kiev, around Smely, 29 Jul. 1912, Sgarbowsky (LEP). On G. erianthum (3): Russia (3): Kamchatka, near the river Paratunki, Jun. 1908, coll. V.L. Komarov, det. W.A. Tranzschel (LE, W); on G. nodosum (7): Italy (7): Saccardo, Mycoth. Ital. 724 (H, HBG, PRM 732430); Briosi & Cavara, Fung. Paras. Piante Colt. Utili Ess. 278 (M-57204, S, UPS). On G. palustre (118): Austria (3): Vorarlberg, near Frastanz, 12 Aug. 1909, P. Magnus (HBG); Czech Republic (5): Cechy, Teplice, 22 Aug. 1889, P. Magnus (HBG, M-57210); Germany (76): Allescher & Schnabl,
Fungi Bav. 353 (GZU, H, HBG, M-57259, S); Allescher & Schnabl, Fungi Bav. 560 (H); Krieger, Fungi Sax. Exs. 196b (H, HBG, M-57221, S, WRSL), Krieger, Fungi Sax. Exs. 197 (BUC, H, GZU, HBG, K, M57222, S, W 1901-1291, WRSL); Sydow, Pycomyc. Protomyc. 33 (HBG, M-57216, S, UPS, WU, W); Triebel, Microf. Exs. 101 (GZU, H, MA, UPS); Latvia (13): Smarods, Fungi Lat. Exs. 551 (H, M-57205, PRM 732428, RIG, UPS, W); Norway (1): Oslo, Skullerud, 200 m E Lake Skrapenrudtjern, 14 Aug. 2002, H. Voglmayr HV2001 (WU); Poland (1): Schneider, Herb. Schles. Pilze 5 (WRSL); Romania (10): Negrean, Herb. Mycol. Roman. 3096 (BUCM 72000, H, I, IBIR, UPS, W); Russia (7): Jaczewski, Komarov & Tranzschel, Fungi Ross. Exs. 1 (H, HBG, S, UPS, WU); Ukraine (2): Stryj, Stryalkow, 29 Jun. 1917, F. Petrak (M-57245). On G. sylvaticum (418): Austria (30): Petrak, Crypt. Exs. 4416 (BUCM 3204, C, GZU, H, M-57206, M-57208, PC, S, UPS, W); Czech Republic (9): Thu¨men, Fungi Austr. Exs. 422 (BUC, ˚ rhus, 11 Jun. 1968, GZU, UPS, W, WU); Denmark (1): Jutland, A coll. L. Jensen et al., det. G. Negrean (BUCM 70498); Finland (93): Liro, Mycoth. Fenn. 308 (BUCM, GZU, H, TUR, UPS); France (7): Alpes Maritimes, Tende, Fontanalba, 28 June 1961, E. Mu¨ller (M-57211); Germany (39): Sydow, Phycomyc. Protomyc. 79 (HBG, M-57247, S, UPS, W, WU); Krieger, Fungi Sax. Exs. 340 (BUC, H, HBG, M-57231, M-57232, S, W, W, WRSL); Italy (1): Valle d’Aosta, Morgex, 12 June 1962, J. Poelt (GZU); Latvia (1): Madona, Vidzemeˇ, Vestiena, 21 June 1936, K. Starcs 3654 (RIG); Norway (88): Tr, Nordreisa parish, Reisa Valley, Molliusfoss, 3 Aug. 1961, J.A. Nannfeldt 16980 (UPS); Poland (14): Sydow, Mycoth. Germ. 674 (C, HBG, M-57230, S, UPS, W, WU); Schneider, Herb. Schles. Pilze 4 (K); Russia (5): Karelia, Kirjavalahti, 1 Aug. 1929, V. Heikinheimo & J.I. Liro (H); Slowakia (8): Belianske´ Tatry Mts, Dolina Javorova´ valley, 1110 m s.m., 10 Aug.
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Figs 3–5 – Plasmopara on Geranium. (a) Sporangiophores. (b) Trunk base. (c) Resting organs. (d) Haustoria. (e) Tip of branches and ultimate branchlets. (f) Sporangia. Bars [ 20 mm. Fig 3. P. pusilla (lectotype, S). Fig 4. P. geranii-sylvatici (lectotype, BUCM 3202). Fig 5. P. wilsonii (holotype, UPS).
Revision of Plasmopara (Chromista, Peronosporales) parasitic on Geraniaceae
1998, W. Mułenko (LBL); Sweden (101): Eriksson, Fungi Paras. Scand. Exs. 44 (BUC, C, H, HBG, S, UPS); Lundell & Nannfeldt, Fungi. Exs. Suec. 897 (C, S, UPS, W); ditto 1697 (C, S, UPS, W); Sydow, Phycomyc. Protomyc. 80 (HBG, M-57214, S, UPS, W); Switzerland (22): Wallis, Ta¨sch, 17 Jul. 1929, leg. H. Poeverlein (HBG, W). On G. sylvaticum ssp. pseudosibiricum (1): Russia: Irkutsk, Kirensk, 25 Jul. 1909, S. Ganeskhin (LE).
Comments: The repeatedly twisted trunk, resulting in a screw-like appearance in both fresh and herbarium preserved specimens is characteristic for this species. Geranium sylvaticum is also parasitized by P. praetermissa (q.v.), and plants harbouring either P. geranii-sylvatici or P. praetermissa can be found very close to each other. Rarely, both fungi may be present on the same plant or even the same leaf. Plasmopara wilsonii Voglmayr, Fatehi & Constant., sp. nov. (Fig 5) Etym.: In the memory of Guy West Wilson (1877–1956) who made significant contributions to the knowledge of Peronosporales. Haustoria obpyriformia, 4–8 mm diam. Sporangiophora 160–480 mm longa; truncus 80–390 mm longus, aequicrassus; ramuli terminales acutati vel cylindracei. Sporangia fere late ellipsoidea vel ellipsoidea, 21–27 16.5–21 mm. Oogonia globosa vel irregularia, 40–55 mm diam, pariete fere luteolo vel brunneolo, laevi vel plicato. Oosporae globosae, 30–40 mm diam., pariete incolorato vel luteolo.
Typus: USA: IL: Union Co., near Anna, on Geranium carolinianum 28 Apr. 1882, A.B. Seymour in Rabenh.-Winter, Fungi europ. 3176 (UPS F-120167 - holotype; H, HBG, M, S, WU isotypes). On leaves producing slight discolouration of the tissues to clearly defined epiphyllous spots; spots first pale yellowish, later becoming reddish-brown to violaceous-brown, polyangular, from 0.5–22 mm when coalescent, but often covering the whole leaf surface, with diffuse margin to vein limited; attacked tissues becoming necrotic. Down hypophyllous, white to greyish, dense, felt-like. Haustoria obpyriform, 4–8 mm diam, including the sheath. Sporangiophores slender, 160–480 mm long, trunk and the main axis straight or slightly curved, 80–390 mm long, of more or less uniform breadth, rarely changing in diameter, basal end not differentiated to slightly bulbous, 4–7 (–9) mm above the base, 4–8 (–9) mm wide below the first branch, wall ca 0.8 mm thick, callose plugs often present, either one, located slightly above the middle of the trunk, or sometimes two along the trunk. Branches 2–4 (–5) on each sporangiophore, more or less simple structured, branching monopodial, in one to two orders, first order branches alternate, very rarely opposite, arising at ca 45–65 angle to the main axis, 15–85 mm long, callose plugs occasionally present. Ultimate branchlets arranged in groups, formed laterally and at the end of the branches, common base not or only rarely inflated, rarely differentiated into an axial (usually longer) and abaxial ones, acute to almost cylindric, of variable length, 12–20 mm when axial, 6–16 when abaxial, 2–5 mm at the base, 1–1.5 mm just below the tip, tip round (cup-like when collapsed), but mostly swollen up to 3 mm diam. Sporangia broadly ellipsoidal to ellipsoidal, rarely ovoidal or almost oblong, (17–) 21–27.5 (–33) mm long, (12–) 16.5–21 (–28) mm wide, length:breadth ratio (1–) 1–1.5 (–2), n ¼ 209, greatest breadth median or sub-median, rarely supra-median, base and tip round, wall ca 0.5 mm thick, apical dehiscence
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apparatus (3.5) 4–5 (–5.5) mm diam, composed of a lenticular ca 1.5 mm thick plug, pedicel not visible to slightly protruding. Some giant sporangia 42–50 26–33 mm present in several collections. Resting organs in leaves, visible on the upper leaf surface, often in necrotic tissues as black dots; oogonia broadly globose to irregular by mutual pressure, 40–55 mm diam, wall yellowish to brownish, smooth, more or less wrinkled, uniformly 2.5–4 mm thick; periplasm yellowish to brownish; oospores plerotic or aplerotic, globose to irregular by mutual pressure, 30–40 mm diam, with smooth, colourless to yellowish wall; wall ca 2 mm thick but easily swollen up to 4 mm. Frequent host: G. carolinianum. Occasional hosts: Erodium cicutarium, G. bicknellii, G. koreanum, G. molle, and G. nepalense ssp. thunbergii. Additional selected specimens examined: on Erodium cicutarium (1): USA: TX, Jacksonville, 18 Mar. 1953, coll. P.A. Young, det. J.A. Stevenson (BPI 187672). On G. bicknellii (1): USA: WI: Spooner, 1 Sep. 1924, J.J. Davis (WU). On G. carolinianum (65): UK (2): Devonshire: Bermuda Isl.:, 7 Dec. 1921, coll. H.H. Whetzel, det. G.W. Wilson (CUP); USA (63): Bartholomew, Fungi Columb. 2974 (H); ditto 3275 (H, S, WSP 10499); Ellis, N. Amer. Fungi 1404 (BUC, M-57183, UPS); Ellis & Everhart, Fungi Columb. 405 (HBG two spec.); Seymour & Earle, Econ. Fungi, Suppl. A 3 (BUCM 47410, ILL 2473, K). On G. koreanum (6): Korea (6): Kangnung: 13 Oct. 1991, H.-D. Shin (SMK 11308). On G. molle (1): USA: MD: Beltsville, USDA Building, 13 May 2003, H. Voglmayr HV-PA1 (WU). On G. nepalense ssp. thunbergii (5): Korea (5): Jeju, 1 Nov. 2002, H.-D. Shin (SMK 19358, UPS F-119996).
Comments: Differs from other Plasmopara on Geranium by its long and narrow trunk, swollen tip of the ultimate branchlets, and variable size of sporangia. Distribution: NE USA, South and Far East Asia (Korea). Plasmopara geranii (Peck) Berl. & De Toni, in Saccardo, Syll. Fung. (Padova) 7: 242 (1888) (Fig 6). Commonly present on leaves, producing slight discolouration of the tissues to clearly defined epiphyllous spots. Spots first pale yellowish, later becoming reddish-brown, polyangular, margin diffuse to vein limited, 2–20 mm diam, covering larger areas by coalescence; attacked tissues becoming necrotic. Down hypophyllous, white to yellowish, consisting of scattered patches of sporangiophores, very rarely of a more dense structure. Haustoria vesicular, 4–8 mm diam (including the callose sheath). Sporangiophores slender to stout, 135–490 mm long, trunk and the main axis straight to slightly curved; trunk 75–300 mm long, the basal end not differentiated to slightly bulbous, (5–) 8–11 (–16) mm wide above the base, (6–) 8–10 mm wide below the first branch, wall ca 1 mm thick; callose plugs 1 (–3), commonly present; in most specimens the trunk breadth often noticeably modified at the place of callose plug that is located predominantly below the first branch. Branches (2–) 3 (–4) on each sporangiophore, more or less simple structured to showing an elaborate, arborescent structure, some terminating in a subulate tip; branching monopodial, in one to two orders; first-order branches alternate (rarely opposite), arising at ca 60–70 (–80) angle to the main axis, (15–) 30–100 mm long, more or less uniform in breadth, at the base usually not constricted, callose plugs often present. Ultimate branchlets arranged in 2–3 clusters, formed laterally and at the end of the branches, arising from a common, slightly to definitely inflated base, sometimes differentiated into an axial (usually longer and wavy) and abaxial ones, long-conical to
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Figs 6–7 – Plasmopara on Geranium. (a) Sporangiophores. (b) Trunk base. (c) Resting organs. (d) Haustoria. (e) Tip of branches and ultimate branchlets. (f) Sporangia. (g) Callose plugs and trunk change in diameter. Bars [ 20 mm. Fig 6. P. geranii (lectotype, NYS 1336a). Fig 7. P. praetermissa (holotype, UPS).
Revision of Plasmopara (Chromista, Peronosporales) parasitic on Geraniaceae
almost cylindric, of almost the same length, often flexuous, 20–27 mm when axial, 7–23 mm when abaxial, 2–6 mm wide at the base, 1–1.5 mm wide just below the tip, tip round to slightly inflated (but appearing cup-like when collapsed), 2 mm diam. Sporangia oblong, (14–) 18–23 (–28) mm long, (13–) 14.5–17.5 (–20) mm wide, length:breadth ratio (1.07–) 1.19–1.36 (–1.5), n ¼ 160, greatest breadth median (rarely sub- or supra-median), base and tip round; apical dehiscence apparatus 3–4 (–6) mm diam, composed of an outwardly convex, ca 1–1.5 mm thick plug; pedicel slightly protruding to short-conical. Resting organs in leaves; oogonia broadly globose to irregular, (30–) 35– 40 (–50) mm diam, wall yellowish, of uniform thickness, more or less wrinkled, 1.5–3.5 mm thick; oospores plerotic to aplerotic, globose to irregular by mutual pressure, 23–32 mm diam, with smooth, colourless to yellowish, 3–4.5 mm thick wall. Frequent host: Geranium maculatum. Occasional hosts: G. oreganum, G. sibiricum, G. texanum, and G. vulcanicola. Distribution: E Canada, NE USA, Mexico. Type: USA: NY: North Greenbush, Jun. 1874, C.H. Peck (NYS 1336a - lectotype designated by Constantinescu 2004) Additional specimens examined: on G. maculatum (72): Canada (21): Bartholomew, Fungi Columb. 2048 (CUP, H, HBG, S, UPS); ditto 2476 (K, H, RMS 0665, S, UPS, WSP 10501); Sydow, Phycomyc. Protomyc. 286 (C, H, HBG, M-57182, S, UPS, W, WU); USA (50): Bartholomew, Fungi Columb. 4173 (N, WSP 10498); Ellis, N. Amer. Fungi 218 (BUC, L 910.234-1319, M-57186, UPS). On G. oreganum (1): USA (1): OR: Marion Co., Salem, 26 Jun. 1920, coll. J.C. Nelson 3174, det. C.G. Shaw (WSP 36725). On G. sibiricum (3): USA (4): WI: Dane Co., near Dane, 17 Aug. 1954, H.C. Greene 1750 (BPI 187826, RMS 30213, TUR, WSP 39860). On G. texanum (2): USA (2): LA: Baton Rouge, 2 Feb. 1921, C.W. Edgerton 2365 (BPI 187829). On G. vulcanicola (1): Mexico: 2 Oct. 1896, collector unknown, det. C.G. Shaw & M. Gaines (WSP 311213).
Comments: Up to three types of sporangiophores are often present in the same collection: (1) In the most characteristic the trunk consists of a lower part, 5–6 mm wide, with ca 0.5 thick wall, and a wider, 7–10 mm upper part, extending into the branched portion, and having a thin, ca 0.5 mm wall. The two parts are separated by a callose plug. Rarely the lower portion is wider and the upper one narrower. (2) Less frequent, in which the whole trunk is ca 8–10 mm wide, reminiscent of the sporangiophores in P. praetermissa. (3) Rarely encountered, with trunk uniformly ca 5–6 mm wide, rather similar to the one present in P. wilsonii. In numerous specimens the ultimate branchlets are sitting on a slightly swollen base, and the axial one is longer. Sporangia characteristic for this species are oblong and most are ca 20–22 mm long, but in some collections the length can reach 24–26 mm. Peck (1876) did not mention the presence of resting organs, but they were described by Berlese and De Toni (1888) when the fungus was transferred to Plasmopara. Later, Berlese (1898, 1901) provided an accurate description and illustration. In fact, the resting organs are rather common but overlooked probably because they are situated on the upper leaf surface. Plasmopara praetermissa Voglmayr, Fatehi & Constant., sp. nov. (Fig 7) Etym.: praetermissus ¼ overlooked, in reminiscence of the fact that although much collected, this fungus was only recently distinguished from P. geranii-sylvatici. Haustoria obpyriformia vel globosa, 5–7 mm diam. Sporangiophora 80–340 mm longa; truncus 60–240 mm longus, ex duo
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partibus latitudibus inaequalibus constans, partibus obturamentum callosum separatis; ramuli terminales digitiformes. Sporangia fere globosa vel ellipsoidea, 15–34 14.5–26 mm. Oogonia globosa vel irregularia, brunneola, 40–55 mm diam, pariete brunneolo, laevi vel plicato. Oosporae globosae, 32–48 mm diam., pariete luteolo.
Typus: Sweden: Uppsala: Uppland, Va˚rdsa¨tra sa¨tesga˚rd, shadow places, 59.48N 17.36E, on Geranium sylvaticum 18 Jun. 2003, O. Constantinescu 03-33 (UPS F-119993, holotype). On leaves producing slight discolouration of the tissues to clearly defined epiphyllous spots; spots first yellowish, later becoming reddish to reddish-brown, irregular to polyangular, 2–12 mm diam, vein limited, covering larger areas by coalescence; attacked tissues become necrotic. Down hypophyllous, white to greyish, consisting of scattered to a relatively dense mat of sporangiophores. Haustoria obpyriform to vesicular, 5–7 mm wide including the sheath. Sporangiophores slender to stout, (80–) 100–220 (–340) mm long, straight to slightly curved; trunk (60–) 90–170 (–240) mm long, gradually tapering upwards, basal end not differentiated or slightly bulbous and then up to 15 mm wide, (8–) 9–12 mm wide above the base, (6–) 8–10 mm wide below the first branch; trunk often differentiated into a lower, broader part with a ca 0.5 mm thick wall, and an upper, narrower part having a ca 0.5 mm thick wall, the two parts separated by a callose plug located in the median part or below the first branch; more rarely the upper part being wider. Branches 1–3 on each sporangiophore, alternate, rarely opposite, most reduced to a 10–15 mm long, inflated outgrowth; sometimes the lower branch up to 60 mm long and then showing second order branching; callose plugs often present; branching monopodial. Ultimate branchlets arranged in groups of 2–4 (–5), arising from a slightly to definitely inflated base at the end of the branches, only rarely differentiated into axial and abaxial ones, mostly finger-like, often flexuous, up to 20 mm long when axial, 6–10 mm long when abaxial, 2–4 mm wide at the base, 1–1.5 mm wide just below the tip, tip round, rarely slightly inflated. Sporangia broadly ellipsoidal to oblong, rarely subglobose or ellipsoidal, (15–) 19–27 (–34) mm long, (14.5–) 16.5–21.5 (–26) mm wide, length:breadth ratio (1.03–) 1.13–1.25 (–1.42), n ¼ 156, greatest breadth median, more rarely supra-median or sub-median, base and tip round, wall ca 0.5 mm thick; apical dehiscence apparatus (2.5–) 3–4 (–5) mm diam, composed of an outwardly convex, 1–1.5 mm thick plug; pedicel from a scar to slightly protruding, rarely short-conical and then 1-1.5 mm long and 1.5-2 mm wide at the base. Resting organs in leaves, more visible as brown to blackish dots on the upper leaf surface; oogonia broadly globose or irregular by mutual pressure, 40–55 (–66) mm diam., wall yellowish to brownish, smooth to more or less wrinkled, uniformly (2–) 3–-3.5 mm thick; periplasm yellowish; oospore mostly plerotic, globose, 32–48 mm diam., with smooth, colourless to yellowish, 3–3.5 mm thick wall that may reach 5–6 mm in warmed-up slides. Frequent hosts: Geranium sylvaticum and G. richardsonii. Occasional hosts: G. albiflorum, G. palustre, G. viscossisimum, and G. viscossisimum var. nervosum. Additional specimens examined: on G. albiflorum (1): Russia: Polarnyj Ural, 3 Aug. 1924, K. Benua (LE). On G. palustre (6): Latvia (5): Madona, Vestiena, 24 June 1932, K. Starcs 221 (H, M-57244, S, TUR, W); Russia (1): Karelia, 20 June 1898, J.I. Liro (H). On G. richardsonii (24): USA (25): Bartholomew, Fungi Columb. 4683 (H, WSP), Solheim, Mycofl. Saximont. Exs., Cent. 12, 1113 (B,
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CUP, RMS 36082 & 45913, WSP 49964 & 62865, W); ditto Cent. 1417, 1521 (CUP, RMS 45913, UPS F-119997, WSP 62865, W). On G. sylvaticum (279): Austria (4): Ka¨rnten, Mo¨lltal, ca 2.8 km WSW Innerfragant, Schoberto¨rl, 1860 m s. m., map grid 9044/1, margin of a subalpine Rododendron ferrugineum thicket, 10 Jul. 2000, H. Voglmayr HV498 (WU); Denmark (1): Bornholm, Nexø, Rytterga˚rd, 14 Jun. 1977, H. Roivainen (H); Faroes (2): Klaksvig, 12 Aug. 1938, coll. F.H. Møller, det. N.F. Buchwald (C); Finland (129): EnL, ESomashja¨rvi, 30 Jul. 1960, L. & H. Roivainen (C, UPS); France (1): Savoie, Val d’Ise`re, alt. 1918 m, Aug. 1967, M. Ponchet (BUCM 117079); Georgia (1): Chewi, Great Caucasus, NE-exposed slope ca 1 km WNW above Gerget’i (WNW opposite of village Kazbegi), Betula litwinowii forest, 2100 m s. m., 44 380 E, 42 390 3000 N, 20 Jul. 1997, P. Scho¨nswetter & A. Tribsch (WU); Italy (5): Su¨dtirol, Vahrn, Steinwend, Kinigadner-Wiese, 28 Jun. 1905, A. Heimerl (HBG, W); Norway (29): Sydow, Phycomyc. Protomyc. 34 (HBG, M nis 57217, S, UPS, W, WU); Romania (1): Suceava, Obcina Mesteca x Mt., Lucina horse farm, 13 Jul. 1986, 47 390 1100 N, 25 100 2200 E, alt. 1234 m, G. Negrean (BUCM 99290); Russia (3): Moskwa, Podolsk distr., Mikhailovskoje, 21 May 1896, F. Bucholtz (WU); Slowakia (3): Za´padne´ Tatry Mts, Ticha´ dolina valley, Zadna Ticha dolina valley, 1520 m s.m., 28 Jul. 2001, W. Mułenko (LBL); Sweden (91): Lundell & Nannfeldt, Fungi Exs. Suec. 897 (S, UPS, W); Eriksson, Fungi Paras. Scand. Exs. 44 (BUC, C, HBG, S, UPS); Switzerland (9): Graubu¨nden, Pontresina, Aug. 1888, G. Lagerheim (S). On G. viscossisimum (1): USA: WY: Shoshone National Forest, 25 mi. N. of Dubois, 12 Aug. 1950, C.G. Shaw & Eats (WSP 27141). On G. viscossisimum var. nervosum (2): USA (2): WY: Shoshone National Forest, 25 mi. N. of Dubois, 12 Aug. 1950, C.G. Shaw & Eats (WSP 27137).
Distribution: Northern Europe, Northern European Russia and Arctic Urals (commonly north of 56 ), Caucasus Mts., alpine areas of Europe (Alps and Carpathians above 1400 m), and USA (above 2000 m). Because in many areas the distribution area of this species is sympatric with P. geranii-sylvatici, it is almost impossible to find out to which of the species the published records refer to. Comments: P. praetermissa can easily be distinguished from P. geranii-sylvatici by its longer, wider, mostly collapsed trunk of the sporangiophore, and the frequently present callose plugs in both trunk and branches. In addition, the trunk often consists of two parts of unequal breadth and, although may show one or two twirls, is never regularly, spirally twisted as in P. geranii-sylvatici. Plasmopara on Geranium sylvaticum was intensively collected in Finland, Norway and Sweden, some 490 collections were examined during this study. Of these, 283 (53 %) harboured P. geranii-sylvatici, and 249 (47 %) P. praetermissa. The sites of collections are scattered between 56–70 370 N. Although these two species have a sympatric distribution in this territory, both the southern and northern limits of the distribution area of P. praetermissa are about 0.5–2 further north. For several years the presence of P. geranii-sylvatici and P. praetermissa was monitored in few locations around Uppsala. In some cases the plants harbouring these two fungi were separated by a distance of ca 2 m. Only exceptionally the presence of both fungi on the same plant specimen was detected. In two cases, on the same leaf, surfaces covered by one fungus were in the close vicinity with surfaces covered by the second fungus. These two fungi cannot be differentiated according to the symptoms produced, and the characters of the down under dissecting microscope may prove deceptive. Consequently, the only reliable method is to make slides from every spot, which is a very time consuming procedure. It seems that
H. Voglmayr et al.
the concomitant presence of these two Plasmopara on the same plant specimen contradicts the ‘two genera rule’ (Constantinescu 1991: 476; 2002: 299). Nevertheless, if the taxonomic discrepancy between these two fungi is at the species or genus level has to be further investigated.
Acknowledgements The Directors and Curators of the following herbaria are thanked for allowing access to specimens in their keeping: ˚ S, B, BPI, BUC, BUCM, C, FH, GZU, H, HBG, I, IBIR, ILL, L, LBL, A LE, LEP, M, MA, MPA, NYS, OULU, PRM, RIG, RMS, S, SMK, TUR, UPS, VLA, W, WRSL, WSP, and WU. The work was partially sponsored by grants form the Swedish Natural Science Council (NFR) to O.C., Royal Swedish Academy of Agriculture and Forestry (KSLA) to J.F. and O.C., and the Strategic Foundation for Environmental Research (MISTRA) to J.F. O.C. thanks the owners of Va˚rdesa¨tra sa¨tesga˚rd for permission to collect specimens on their property.
Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mycres.2006.03.005.
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