Diversity and species boundaries in floricolous downy mildews ...

Mycol Progress (2013) 12:321–329 DOI 10.1007/s11557-012-0837-7

ORIGINAL ARTICLE

Diversity and species boundaries in floricolous downy mildews Marco Thines & Volker Kummer

Received: 30 December 2011 / Revised: 6 June 2012 / Accepted: 12 June 2012 / Published online: 27 July 2012 # German Mycological Society and Springer 2012

Abstract Floricolous downy mildews are a monophyletic group of members of the genus Peronospora (Oomycota, Peronosporales). These downy mildews can be found on a variety of families of the Asteridae, including Asteraceae, Campanulaceae, Dipsacaceae, Lamiaceae, and Oroban- chaceae. With the exception of Peronospora radii, which can also cause economically relevant losses, sporulation usually takes place only on floral parts of their hosts. However, only very few specimens of these mostly inconspicuous downy mildews have so far been included in molecular phylogenies. Focusing on Lamiaceae, we have investigated multiple specimens of floricolous downy mildews for elucidating species boundaries and host specificity in this group. Based on both mitochondrial and nuclear loci, it became apparent that phylogenetic lineages in the Lamiaceae seem to be host genus specific and significant sequence diversity could be found between lineages. Based on distinctiveness in both phylogenetic reconstructions and morphology, the downy mildew on flowers of M. Thines (*) Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, 60325 Frankfurt am Main, Germany e-mail: [email protected] M. Thines Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt am Main, Siesmayerstr. 70, 60323 Frankfurt am Main, Germany M. Thines Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, 60325 Frankfurt am Main, Germany V. Kummer Institut für Biochemie und Biologie, University of Potsdam, Maulbeerallee 1, 14469 Potsdam, Germany

Stachys palustris is introduced as a new species, Peronospora jagei sp. nov., which can be morphologically distinguished from Peronospora stigmaticola by broader and shorter conidiospores. The diversity of the floricolous down mildews might be higher than previously assumed, although specimens from a much broader set of samples will be needed to confirm this view. Keywords cox2 . Internal transcribed spacer . Peronosporaceae . Phylogeny

Introduction Several oomycetes causing downy mildew are able to infect floral tissues, although most of these are causing symptoms systemically or predominantly on non-floral parts of their hosts. These include, e.g. P. rumicis Corda (Gustavsson 1959a), P. sparsa Berk. (Tate 1981), P. statices Lobik (Skrzypczak and Marasek 1998), P. destructor (Berk.) Casp. ex Berk. (Cook 1930), P. linariae Fuckel (Magnus 1891), and the graminicolous downy mildews (Kenneth 1981). But there are a few species, termed floricolous downy mildews, which are mostly inconspicuous and cause symptoms exclusively on flowers, with the exception of Peronospora radii de Bary, which has also been observed on leaves (Ben-Ze’ev et al. 1987; Constantinescu 1989; Koike et al. 2004). However, there is good indication that these downy mildews are in fact systemic (Horáková and Skalický 1989), but with sporulation being triggered in flowers. This is also corroborated by the results of Stearn (1929), who observed conidiosporophores inside distorted buds of Campanula cochleariifolia Lam. (syn. C. pusilla Haenke). Conidiosporophores in this group are mostly stout, and conidiospores are borne on short ultimate branchlets, which do not discharge the conidiospores upon desiccation

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as an adaptation to dispersal by pollinators (Raunkiær 1893; Gustavsson 1959a). This constitutes an important synapomorphy of this group, which is apparently monophyletic (Voglmayr 2003). Peronospora radii and P. violacea Berk., which are relatively easy to spot, have been reported from a wide range of Asteraceae (Constantinescu 1989) and Dipsacaceae (Gäumann 1923), respectively. Members of the floricolous downy mildews affecting other families are often less easy to observe, as sporulation is profound only within the flower, especially in the Lamiaceae. In this family, several members of the genus Mentha are known to be hosts of a floricolous downy mildew (Teppner 1978), which is sporulating mostly on the stigma of their host, a fact that is reflected by its scientific name, P. stigmaticola Raunk. However, sporulation can also be present on stamens and petals (Greene 1957). The inconspicuous nature of the pathogen is also reflected by the fact that it had not been reported for Austria prior to 1978 (Teppner 1978). It has been demonstrated that species of Peronospora mostly have very restricted host ranges and are often host species specific (Gäumann 1923). Whether this also applies to the floricolous downy mildews has so far not been evaluated, as only a few specimens of these mostly rare and easily overlooked species were included in previous phylogenetic investigations. Nuclear ribosomal

and mitochondrial sequences have proven to be useful for species delimitation and phylogenetic reconstructions in oomycetes, and also in Peronospora species on Lamiaceae these loci have been used successfully (Voglmayr 2003; Thines et al. 2009; Choi et al. 2009). It was the aim of this study to investigate the diversity and species boundaries of floricolous downy mildews, with emphasis on two genera of Lamiaceae, using molecular phylogenetics and morphological investigations.

Materials and methods The oomycete specimens used in this study are listed in Table 1. Morphological investigations were done by removing small amounts of sporulating tissue from the specimens and transferring this material to a droplet of 3 % aqueous KOH solution on a slide to restore turgidity. Measurements were performed using a Zeiss Axioscope light microscope (Zeiss, Germany) using a magnification of ×200 and ×1,000. LM micrographs were taken with a Coolpix 4500 camera (Nikon, Japan). DNA was extracted using the innuprep plant DNA kit (Analyticjena, Germany) according to the instructions of the manufacturer. PCR for cox2 amplification was done according to Hudspeth et al. (2000), and ITS amplifications

Table 1 Oomycete material used and GenBank accession numbers for sequences obtained in this study Pathogen

Host

Origin

FR herbarium number

V. Kummer herbarium number

cox2 accession number

ITS accession number

P. jagei TYPE

Stachys palustris

Germany, BB, Gatow, leg. V. Kummer

FR0046042

1800-4

JQ031172

JQ031186

P. jagei

Stachys palustris


n.a.

1800-5

n.a.

n.a.

P. jagei

Stachys palustris


FR0046043

1800-6

JQ031173

JQ031187

P. radii

Tripleurospermum perforatum

Germany, BB, Potsdam, leg. V. Kummer

FR0046024

1986-2

JQ031163

n.a.

P. radii


Germany, MV, Göhren-Lebbin, leg. V. Kummer

FR0046025

1986-3

n.a.

JQ031183

P. radii


Germany, TH; Berka, leg. V. Kummer

FR0046026

1986-4

JQ031162

JQ031180

P. radii


Germany, BB, Potsdam, leg. V. Kummer

FR0046027

1986-5

JQ031167

n.a.

P. radii


Germany, BB, Finowfurth, leg. V. Kummer

FR0046028

1986-7

JQ031165

JQ031182

P. radii


Germany, TH, Berka, leg. V. Kummer

FR0046029

1986-15

JQ031166

JQ031184

P. radii


Germany, BB, Uetz, leg. V. Kummer

FR0046030

1986-16

JQ031164

JQ031185

P. radii


Germany, BB, Casekow, leg. V. Kummer

FR0046031

1986-17

JQ031160

n.a.

P. radii

Matricaria recutita

Germany, BB, Fahrland, leg. V. Kummer

FR0046032

1984-2

JQ031161

JQ031181

P. stigmaticola

Mentha aquatica

Germany, BB, Guteborn, leg. V. Kummer

FR0046041

1832-3

JQ031170

JQ031188

P. stigmaticola

Mentha arvensis

Germany, BB, Wittenberge, leg. H. Jage

FR0046036

1834-2

JQ031169

n.a.

P. stigmaticola

Mentha arvensis

Germany, BB, Himmelpfort, leg. V. Kummer

FR0046038

1834-4

JQ031171

JQ031190

P. stigmaticola

Mentha arvensis

Germany, BB, Rheinsberg, leg. V. Kummer

FR0046039

1834-6

JQ031168

JQ031189

P. stigmaticola

Mentha x verticillata

Germany, MV, Untergöhren, leg. V. Kummer

FR0046040

1833-5

n.a.

n.a.

P. violacea

Knautia arvensis

Germany, BB, Klosterfelde, leg. V. Kummer

FR0046033

1519-6

JQ031174

JQ031177

P. violacea

Knautia arvensis

Germany, BB, Finowfurth, leg. V. Kummer

FR0046034

1519-7

JQ031176

JQ031179

P. violacea

Knautia arvensis

Germany, BB, Geesow, leg. V. Kummer

FR0046035

1519-13

JQ031175

JQ031178

n.a. not available


were done as reported previously (Thines et al. 2009). Sequencing was done at the BiK-F Laboratory Centre (BiK-F, Germany), with the primers used for PCR. Sequences were edited using Genious (v.5.15) and aligned using mafft (Katoh et al. 2002) with the Q-INS-I algorithm (Katoh et al. 2005). Alignments were trimmed for leading and trailing gaps. No manual edits were done to the alignment for not introducing any bias. Phylogenetic reconstruction for Maximum Likelihood inference was done using RAxML (Stamatakis 2006), as implemented on the RAxML webservers (Stamatakis et al. 2008). Minimum Evolution inference was done using MEGA 5.0 (Tamura et al. 2011) with default values, except for choosing the Tamura-Nei substitution model and allowing heterogeneous substitution rates between lineages. Bayesian Inference was done using MrBayes (Huelsenbeck and Ronquist 2001), version 3.12 as described previously (Runge et al. 2011), using 4,000,000 generations and discarding the first 50 % of the trees and using the remaining trees for calculating posterior probabilities. All sequences have been submitted to GenBank (Table 1). Alignments and the tree shown in Fig. 1 have been deposited in TreeBase (www.treebase.org) under the accession number S12114.

Results Molecular phylogeny Phylogenetic analysis based on cox2 and ITS sequences (Fig. 1) did not reveal any conflicting support, except for the placement of the foliar pathogen Peronospora knautiae Fuckel, which was placed basal to the floricolous downy mildews in the ITS and within the outgroup in the cox2based tree. It should, however, be noted that the sequences were derived from two distinct hosts. The phylogenetic reconstructions based on cox2 showed a significantly higher resolution on the backbone of the phylogenetic trees, while the terminal branches were equally well resolved. With the exception of the closely related genera Anthemis, Matricaria, and Tripleurospermum, specimens from a single host genus always clustered together with strong to maximum support in Maximum Likelihood and Bayesian Analysis, which was also the case for the Minimum Evolution analysis based on cox2. Only in the Minimum Evolution Analysis of ITS did the clustering of the sequences representing P. stigmaticola receive only 87 % bootstrap support. All other terminal groupings received high to maximum support. In the ITS-based phylogenetic reconstruction, the floricolous downy mildews from Lamiales, euasterids I (lamiids) sensu APG III (APG III 2009; Chase and Reveal 2009) were grouped together with moderate bootstrap support in ME (82 %), but received strong (96 %) to maximum support in

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Maximum Likelihood and Bayesian analyses, respectively, and contained three distinct lineages representing the floricolous downy mildews of Melampyrum, Mentha, and Stachys. The specimens from the Lamiaceae, Mentha and Stachys, were grouped together with moderate bootstrap support (80 %) in the Minimum Evolution analysis. In the cox2-based phylogenetic reconstruction, the pathogens from Lamiaceae were also grouped together with moderate support in Maximum Likelihood (bootstrap support of 82 %) and Bayesian analysis (posterior probability of 0.85), but with stronger support in the Minimum Evolution analysis (bootstrap support of 92 %). In the cox2-based inference, the floricolous downy mildews of euasterids II (campanulids) sensu APG III (APG III 2009; Chase and Reveal 2009) fall into two different groups, with one group containing sequences of P. violacea, while the other group consists of P. radii from various Asteraceae. The latter show a strongly supported sister-group relationship with the floricolous downy mildews of the Lamiaceae of the euasterids I sensu APG III (APG III 2009; Chase and Reveal 2009). The same, yet only moderately supported, topology was observed in the Bayesian analysis of ITS, but a conflicting weakly supported topology, grouping the floricolous downy mildews of euasterids II sensu APG III (APG III 2009; Chase and Reveal 2009), was observed in the Minimum Evolution analysis of the ITS sequence data. The monophyly of the floricolous downy mildews with respect to the closely related species in the outgroup, which were chosen according to the phylogenetic tree presented in Voglmayr (2003), received strong support in the ITS-based phylogenetic analyses and maximum support in the cox2-based analyses. Morphology The results of detailed measurements of some morphological characters on the two phylogenetically distinct species on Lamiaceae, Peronospora stigmaticola (Fig. 2a, b) and the undescribed phylogenetic lineage on Stachys palustris L. (Fig. 2c, d) are summarised in Table 2. The overall conidiosporophore morphology is similar in both species, with straight short ultimate branchlets and a similar branching pattern. However, the height of the conidiosporophores of the new species on Stachys palustris (mean 464 μm) seems to be greater than for P. stigmaticola on Mentha arvensis L. (mean 373 μm) or Mentha × verticillata L. (mean 180 μm). The ratio of the length of the conidiosporophore to the length of the trunk is 1.29, 1.34, and 1.54 for these accessions, in the same order as above. While the gross morphology of the conidiosporophore shows a high degree of variation for the three accessions, the measurements for the length of the ultimate branches and the ultimate branchlets revealed a high degree of congruence between the specimens from the two Mentha species investigated in this study (see Table 2). Means for the

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Mycol Progress (2013) 12:321–329 Peronospora radii ex Tripleurospermum perforatum FR0046025

ITS

Peronospora radii ex Tripleurospermum perforatum FR0046029 Peronospora radii ex Tripleurospermum perforatum FR0046028 100 / 99 / 1.0

Peronospora radii ex Matricaria recutita FR0046032 Peronospora radii ex Tripleurospermum perforatum FR0046026 Peronospora radii ex Anthemis austriaca AY198296

54 / - / *

Peronospora radii ex Tripleurospermum perforatum FR0046030 Peronospora violacea ex Knautia arvensis AY198297 -/*/100 / 99 / 1.0

Peronospora violacea ex Knautia arvensis FR0046034 Peronospora violacea ex Knautia arvensis FR0046035 Peronospora violacea ex Knautia arvensis FR0046033

92 / 89 / 1.0

Peronospora stigmaticola ex Mentha arvensis FR0046038 87 / 97 / 1.0

Peronospora stigmaticola ex Mentha arvensis FR0046039 Peronospora stigmaticola ex Mentha aquatica FR0046041

80 / - / -

Peronospora stigmaticola ex Mentha longifolia AY198295 Peronospora jagei ex Stachys palustris FR0046042

99 / 100 / 1.0 82 / 96 / 1.0

Peronospora jagei ex Stachys palustris FR0046043 Peronospora tranzscheliana ex Melampyrum pratense AY198294 Peronospora knautiae ex Scabiosa columbaria AY198302

Peronospora sherardiae ex Sherardia arvensis AY198301

61 / - / 99 / 99 / 1.0

Peronospora aparines ex Galium aparine AY198300 Peronospora calotheca ex Galium odoratum AY198298

0.005 subsitutions / site

Peronospora stigmaticola ex Mentha arvensis FR0046038

cox2

97 / 98 / 0.99

Peronospora stigmaticola ex Mentha aquatica FR0046041 Peronospora stigmaticola ex Mentha arvensis FR0046036

92 / 82 / 0.85

Peronospora stigmaticola ex Mentha arvensis FR0046039 99 / 100 / 1.0

Peronospora jagei ex Stachys palustris FR0046043 Peronospora jagei ex Stachys palustris FR0046042

92 / 96 / 0.99

Peronospora radii ex Tripleurospermum perforatum FR0046027 Peronospora radii ex Tripleurospermum perforatum FR0046031 Peronospora radii ex Matricaria recutita FR0046032 100 / 100 / 1.0

Peronospora radii ex Tripleurospermum perforatum FR0046026 Peronospora radii ex Tripleurospermum perforatum FR0046024

100 / 100 / 1.0

Peronospora radii ex Tripleurospermum perforatum FR0046030 Peronospora radii ex Tripleurospermum perforatum FR0046029 Peronospora radii ex Tripleurospermum perforatum FR0046028 75 / 82 / 0.70 100 / 99 / 0.96

Peronospora violacea ex Knautia arvensis FR0046035 Peronospora violacea ex Knautia arvensis FR0046033

Peronospora violacea ex Knautia arvensis FR0046034 99 / 62 / 0.86

Peronospora calotheca ex Galium odoratum DQ365721 Peronospora knautiae ex Knautia sylvatica EU826096 Peronospora aparines ex Galium aparine DQ365717

0.01 subsitutions / site


325

R Fig. 1

Phylogenetic reconstruction of the floricolous downy mildews based on ITS and cox2. The phylograms shown are based on Minimum Evolution Analysis, numbers at the branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order

measurements of branches and branchlets were 11.2 μm and 5.2 μm, respectively, for Mentha arvensis, and 11.4 μm and 5.2 μm, respectively, for Mentha × verticillata. Ultimate branches and branchlets of the new species on Stachys palustris were slightly longer, with means of 13.1 μm and 6.2 μm, respectively. The ratio of the length of these two morphological features (mean) was similar for the Mentha and Stachys accessions (Table 2), with 2.20 for the floricolous downy mildew of Stachys palustris, 2.17 for P. stigmaticola on Mentha arvensis, and 2.26 for the same species on Mentha × verticillata. Conidiospores of P. stigmaticola and the new species are differing in various aspects. They are shorter and wider in the new species on Stachys palustris in comparison with those of P. stigmaticola (see means and ratio of conidiospores in

Table 2 and Fig. 2). The mean length to width ratio for P. stigmaticola was 2.80 and 2.60 on Mentha arvensis and Mentha × verticillata, respectively, while this ratio was only 1.61 in the new species on Stachys palustris. Furthermore, the conidiospores of P. stigmaticola were tapering towards both base and apex, resulting in an elongate fusiform shape (elongate limoniform in some smaller conidiospores). In contrast those of the floricolous downy mildew on Stachys palustris were tapering only towards the base, resulting in broad ellipsoidal, tear-shaped conidiospores. Only a few oospores could be observed in the specimens from Mentha × verticillata and Stachys palustris and no oospores were found in the stigmata of Mentha arvensis. Oospores were slightly larger in P. stigmaticola (mean of 29.2 μm) compared to the new species on Stachys palustris (mean of 27.5 μm). Furthermore, the conidiosporophores of the new Peronospora species on Stachys palustris were usually found on the corolla and very rarely on the filaments or stylus of the flowers, whereas the conidiosporophores of P. stigmaticola were normally found on the stigmata or stylus of the Mentha flowers.

a

Taxonomy

b

c

d

Based on differences in cox2 and ITS sequence data, as well as morphological characteristics, the new phylogenetic lineage of Peronospora on flowers of Stachys palustris is described as a new species here. Peronospora jagei Thines & Kummer sp. nov., MycoBank MB 800593, Fig 2c, d Diagnosis – Differs from Peronospora stigmaticola in having broad ellipsoidal to tear-shaped conidiospores generally less than two times longer than wide, tapering only towards the base, while in Peronospora stigmaticola, conidiospores are tapering towards both base and apex and are mostly more than two times longer than wide, and differs from Peronospora scutellariae in having less elongate conidiospores. Etymology – Dedicated to Dr. Horst Jage for his longsta ndin g c on tributio ns to the kno wledg e o f th e Peronosporales and his outstanding dedication to recording and collecting of phytoparasitic fungi and oomycetes in Central Europe. Habitat – Live flowers of Stachys palustris L. Known distribution – Germany. Type – On Stachys palustris, Germany, Brandenburg, Gatow, leg. Volker Kummer, June 2007, specimen FR0046042.

Discussion Fig. 2 Morphological characteristics of Peronospora stigmaticola (a, b) and Peronospora jagei (c, d). Scale bars (a, b) 50 μm, (c, d) 20 μm

Floricolous downy mildews are a poorly known group of obligate plant parasitic Peronosporaceae and have been

d

c

b

a

Ratio conidiospore length to width (2.12–)2.50–2.80–3.09(–3.42), n085 (1.80–)2.28–2.60–2.91(–3.64), n049

(4.0–)4.8–6.2–7.7(–10.0), n054d Width of conidiospores (10.0–)11.2–13.2–15.2(–21.0), n085 (11.0–)12.1–13.3–14.5(–15.5), n049

(1.38–)1.59–2.20–2.81(–4.00), n054

(2.0–)3.6–5.2–6.8(–9), n092 (1.0–)4.0–5.2–6.4(–9.5), n048

(4–)5–5–6(–7), n028

(4–)5–6–7(–8), n027 (4–)4–5–6(–6), n016

Number of ramifications

Oospore size n.a. (26.0–)26.0–29.2–32.4 (–35.0), n010 (23.0–)26.5–28.9–31.3(–37.5), n0108 (14.0–)16.5–18.0–19.6(–23.5), n0108 (1.38–)1.48–1.61–1.73(–2.08), n0108 (24.0–)25.2–27.5–29.8 (–32.0), n017

Length of conidiospores (27.0–)32.0–36.6–40.3(–48.0), n085 (27.0–)30.5–34.5–38.5(–41.0), n049

Specimens FR0046042 and 1800/5, Random selection from the terminal ramnification

Specimen FR0046039, Specimen FR0046040

pathogen and host binomials Peronospora stigmaticola ex Mentha arvensisa Peronospora stigmaticola ex Mentha × verticillatab Peronospora jagei ex Stachys palustrisc

Peronospora stigmaticola ex Mentha arvensisa (7.5–)9.0–11.2–13.3(–16.0), n046 (7.5–)7.8–11.4–14.9(–23.0), n024 Peronospora stigmaticola ex Mentha × verticillatab (9.0–)10.6–13.1–15.6(–18.0), n054 Peronospora jagei ex Stachys palustrisc

Length of ultimate branch

pathogen and host binomials

(1.11–)1.15–1.29–1.45(–1.71), n028

Ratio total length to trunk length (1.24–)1.25–1.34–1.43(–1.60), n027 (1.39–)1.41–1.54–1.67(–1.90), n016

Ratio length ultimate branch to ultimate branchlets (1.57–)1.89–2.17–2.45(–2.78), n046 (1.67–)1.64–2.26–2.88(–4.00), n024

(170–)241–369–497(–560), n028

(260–)327–464–601(–700), n028 Length of ultimate branchlets

Length of conidiosporophore trunk (120–)214–281–349(–380), n027 (75–)92–117–142(–155), n016

Total length of conidiosporophores (165–)290–373–456(–515), n027 (115–)139–180–221(–240), n016

Pathogen and host binomials Peronospora stigmaticola ex Mentha arvensisa Peronospora stigmaticola ex Mentha × verticillatab Peronospora jagei ex Stachys palustrisc

Table 2 Morphological analysis of Peronospora stigmaticola and P. jagei, two floricolous downy mildew pathogens of Lamiaceae

326 Mycol Progress (2013) 12:321–329


reported only from very few hosts in Asteraceae, Campanulaceae, Lamiaceae, and Orobanchaceae (Schröter 1874; Kochman and Majewski 1970; Constantinescu and Negrean 1983; Teppner 1978; Vanev et al. 1993; Brandenburger and Hagedorn 2006), all of which are members of the euasterids sensu APG III (APG III 2009; Chase and Reveal 2009). Most of the species have been reported from Europe and North America, where they are present from Mediterranean climates (Pantidou 1973, Ben Ze’ev et al. 1987, García-Blásquez et al. 2007) to the regions with sub-Mediterranean and temperate climates, and cool temperate regions (Savile 1951; Gustavson 1959b; Novotelnova and Pystina 1985). However, the less frequent reports from central Asia might be a result of sampling bias, as Europe is the continent with the most detailed record for obligate biotrophic oomycetes, and the floricolous downy mildews are mostly rather inconspicuous. Interestingly, the floricolous downy mildews are a monophyletic assemblage within the Peronosporaceae (Voglmayr 2003; this study), whereas the hosts affected have more distant phylogenetic relationships. The phylogenetic origins of this group of Peronospora species are possibly in foliar pathogens of the Dipsacaceae (Voglmayr 2003), but the pathogens of Rubiaceae also seem to be closely related to this group. Following the establishment of the floricolous habit, the floricolous downy mildews radiated on several hosts in the Asteridae by host jumps to different phylogenetic groups. Subsequently, they underwent further differentiation on Lamiaceae, again potentially by a host jump, given the close phylogenetic relationships of the floricolous pathogens and the more distant relationships of the hosts affected. These host jumps might have been enabled by similarities in the physiology of the infected organs and facilitated by the fact that all hosts of the floricolous downy mildews are members of the euasterids. Also, Runge and Thines (2011) have discussed a potential contribution of the host physiology to the facilitation of host jumps, on the example of the host jump by Pseudoperonospora from vining Cannabaceae to vining Cucurbitaceae. It has been revealed that floricolous downy mildews grow systemically in the host plant (Horáková and Skalický 1989), but with few exceptions (Ben-Ze’ev et al. 1987; Constantinescu 1989; Koike et al. 2004) usually sporulate only on floral tissues, or even show a strong preference for certain floral organs. The hosts, therefore, look superficially healthy, although it has been reported that infections can lead to sterilisation (Molliard 1893, 1901; Prell 1943). This is similar to infections by several members of the Microbotryales or Ustilaginales. The floricolous downy mildews show several adaptations to the floral regime, including rather rigid conidiosporophores with short and strait ultimate branchlets that do not discharge the conidiospores upon desiccation, which is likely a result of adaptation to

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insect vectors. It seems likely that, also on the interaction level, an adaptation to the colonisation of floral tissues and the sporulation on these took place, given the strong limitation of symptoms to flowers in most floricolous downy mildews (Thines and Kamoun 2010). Our morphological investigations revealed a strong divergence in the height of the conidiosporophores in Peronospora stigmaticola on two different hosts, Mentha arvensis and Mentha × verticillata, and also in the ratio of the length of the total conidiosporophore to the length of the trunk (Table 2). The sequences in both cox2 and ITS were identical, however, providing evidence for the conspecificity of the downy mildew pathogens on these hosts. This is also corroborated by the characteristics of the ultimate branches and the conidiospores, which showed only minor variation on the two hosts. The divergence in the gross morphology of the conidiosporophores could be due to the different hosts tissues affected. While P. stigmaticola on Mentha arvensis was found mostly on the stigma of the host, this species affected other floral parts, including stylus, filament, and corolla on Mentha × verticillata. Previous studies have shown that the organ affected can have an influence on conidiosporophore morphology (e.g. Delanoё 1972), but also the different host matrix is a plausible explanation for the morphological divergence, as has recently been demonstrated for Pseudoperonospora cubensis (Berk. & M.A. Curtis) Rostovzev by Runge and Thines (2011). Therefore, conidiosporophore morphology might not be useful for species delimitation in downy mildews on different hosts. Which morphological features are influenced the least by the organ and host species affected is currently not ascertained, but the results of this study indicate that especially the ultimate branchlets and the conidiospores might be revealed to be useful in this respect. Our measurements of these characters for P. stigmaticola are in agreement with previous measurements (Raunkiær 1893; Gäumann 1923; Kochman and Majewski 1970; Novotelnova and Pystina 1985; Vanev et al. 1993), although no standard deviation is given in any of these studies. The new species described in this study, P. jagei, differs markedly from P. stigmaticola, but also from P. scutellariae Bejlin, in which conidiospores are, according to Novotelnova and Pystina (1985), also more elongate (ratio of length to width of 1.9) than in P. jagei. In this character, P. scutellariae seems to be intermediate between P. stigmaticola and P. jagei, although the conidiosporophore length of P. scutellariae (150–300 μm; Kochman and Majewski 1970; Novotelnova and Pystina 1985) is much shorter than in both P. stigmaticola (115–515 μm, this study; 400–500 μm, Kochman and Majewski 1970; Novotelnova and Pystina 1985) and P. jagei (260–700 μm). Future studies are needed to infer which of these species is more closely related to P. scutellariae. Given the inconspicuous nature of the floricolous

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downy mildews, it seems possible that additional specialised species, or species adapted to certain climatic or ecological niches (Ploch et al. 2010; Voglmayr and Göker 2011), might be present in this group and still await their discovery. Acknowledgments Svenja Kürten is gratefully acknowledged for technical assistance in performing molecular biology work. Cordial thanks to Sebastian Ploch for GenBank and TreeBase submissions. This study was supported by the research funding programme “LOEWE – Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz" of the Ministry of Higher Education, Research, and the Arts of Hesse. Author contributions: M.T. designed the study, V.K. collected downy mildew specimens, V.K. performed measurements, M.T. and V.K. performed the analyses of the morphological data, M.T. edited and analysed the sequence data, M.T. performed the phylogenetic analyses, M.T. and V.K. performed taxonomic investigations, M.T. and V.K. wrote the manuscript. This manuscript is dedicated to Ovidiu Constantinescu (1933-2012) for his important and critical contributions to the taxonomy of the biotrophic oomycetes.

References APG III (2009) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Bot J Linn Soc 161:105–121 Ben-Ze’ev IS, Kenneth RG, Bonde MR (1987) Peronospora radii de By., a causal agent of downy mildew of Anthemideae: complementary description and new hosts recorded in Israel. Phytoparasitica 15:51–67 Brandenburger W, Hagedorn G (2006) Zur Verbreitung von Peronosporales (inkl. Albugo, ohne Phytophthora) in Deutschland. Mitt Biol Bundesanst Land- Forstwirtsch Berlin-Dahlem 405:1–174 Chase MW, Reveal JL (2009) A phylogenetic classification of the land plants to accompany APG III. Bot J Linn Soc 161:122–127 Choi Y-J, Shin H-D, Thines M (2009) Two novel Peronospora species are associated with recent reports of downy mildew on sages. Mycol Res 113:1340–1350 Constantinescu O (1989) Peronospora complex on Compositae. Sydowia 41:79–107 Constantinescu O, Negrean G (1983) Check-list of Romanian Peronosporales. Mycotaxon 16:537–556 Cook HT (1930) The presence of mycelium of Peronospora schleideni in the flowers of Allium cepa. Phytopathology 20:139–140 Delanoë D (1972) Biologie et épidémiologie du mildiou du tournesol (Plasmopara helianthi Novot.). CETIOM Inf Tech 29:1–49 García-Blásques G, Constantinescu O, Tellería MT, Martín MP (2007) Preliminary check list of Albuginales and Peronosporales (Chromista) reported from the Iberian Peninsula and Balearic Islands. Mycotaxon 98:185–188 Gäumann E (1923) Beiträge zu einer Monographie der Gattung Peronospora Corda. Beitr Kryptogamenflora Schweiz 8(4) Greene HC (1957) Notes on Wisconsin parasitic fungi. XXIII. Trans Wis Acad Sci 46:140–158 Gustavsson A (1959a) Studies on Nordic Peronosporales I. Taxonomic revision. Opera Bot 3:1–271 Gustavsson A (1959b) Studies on Nordic Peronosporales II. General account. Opera Bot 3(2):1–61 Horáková J, Skalický V (1989) Contribution to the ecology of Peronospora violacea Berk. Česka Mykol 43:13–29 Hudspeth DSS, Nadler SA, Hudspeth MES (2000) A cox2 molecular phylogeny of the Peronosporomycetes. Mycologia 92:674–684

Mycol Progress (2013) 12:321–329 Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755 Katoh K, Misawa K, Kuma K-I, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066 Katoh K, Kuma K-I, Toh H, Miyata T (2005) MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res 33:511–518 Kenneth RG (1981) Downy mildews of graminaceous crops. In: Spencer DM (ed) The downy mildews. Academic, London, pp 367–394 Kochman J, Majewski T (1970) Grzyby (Mycota) Tom IV Glonowce (Phycomycetes) Wroślikowe (Peronosporales). Warzawa Koike ST, Fogle D, Tjosvold SA, King AI (2004) Downy mildew caused by Peronospora radii on marguerite daisy (Argyranthemum frutescens) in California. Plant Dis 88:1163 Magnus P (1891) Über den Einfluss, den die Vegetation einiger parasitischer Pilze in der Blüte der Wirtspflanze auf die Ausbildung der Blütenteile ausübt. Verh Bot Ver Prov Brandenburg 33:VI– VIII Molliard M (1893) Sur deux cases de castration parasitaire observés chez Knautia arvensis Coulter. Compt Rend Hebd Séances Acad Sci 116:1306–1308 Molliard M (1901) Fleures doubles et parasitisme. Compt Rend Hebd Séances Acad Sci 133:548–551 Novotelnova NS, Pystina KA (1985) Flora Plantarum Cryptogamarum URSS. Vol. XI Fungi (3) Ordo Peronosporales (Fam. Pythiaceae, Phytophthoraceae, Peronosporaceae, Cystopaceae). Leningrad Pantidou ME (1973) Fungus-host index for Greece. Athens Ploch S, Choi Y-J, Rost C, Shin H-D, Schilling E, Thines M (2010) Evolution of diversity in Albugo is driven by high host specificity and multiple speciation events on closely related Brassicaceae. Mol Phylogen Evol 57:812–820 Prell HH (1943) Aantasting van Knautia arvensis Coulter door Peronospora violaceae Berkeley. Europ J Plant Pathol 4:124–125 Raunkiær C (1893) Et par nye Snyltesvampe. Botan Tidskr 18:108– 111 Runge F, Thines M (2011) Host matrix has major impact on the morphology of Pseudoperonospora cubensis. Europ J Plant Pathol 129:147–156 Runge F, Choi Y-J, Thines M (2011) Phylogenetic investigations in the genus Pseudoperonospora reveal overlooked species and cryptic diversity in the P. cubensis species cluster. Europ J Plant Pathol 129:135–146 Savile DBO (1951) Peronospora stigmaticola in Canada. Mycologia 43:113–114 Schröter J (1874) Über Peronospora violacea Berk. und einige verwandte Arten. Hedwigia 13:177–184 Skrzypczak C, Marasek A (1998) Statice downy mildew, host symptoms and disease development. Meded Fac Landbouwkd Biol 63:899–900 Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690 Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web-servers. Syst Biol 57:758–771 Stearn WT (1929) A new disease of Campanula pusilla (Peronospora corollae). Gardening illustrated 51:565 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Tate KG (1981) Aetiology of dryberry disease of boysenberry in New Zealand. New Zealand J Exp Agric 9:371–376 Teppner H (1978) Der Falsche Mehltau Peronospora stigmaticola – neu für Österreich. Mitt Naturwiss Ver Steiermark 108:177– 178

Mycol Progress (2013) 12:321–329 Thines M, Kamoun S (2010) Oomycete-plant coevolution: recent advances and future prospects. Curr Opin Plant Biol 13:427–433 Thines M, Telle S, Ploch S, Runge F (2009) Identity of the downy mildew pathogens of basil, coleus, and sage with implications for quarantine measures. Mycol Res 113:532–540 Vanev SG, Dimitrova EG, Ilieva EI (1993) Fungi Bulgaricae 2 Tomus Ordo Peronosporales. Sofia

329 Voglmayr H (2003) Phylogenetic relationships of Peronospora and related genera based on nuclear ribosomal ITS sequences. Mycol Res 107:1132–1142 Voglmayr H, Göker M (2011) Morphology and phylogeny of Hyaloperonospora erophilae and H. praecox sp. nov., two downy mildew species co-occurring on Draba verna sensu lato. Mycol Progr 10:283–292