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Phylogenetic relationships of Albugo species (white blister

rusts) based on LSU rDNA sequence and oospore data

Hermann VOGLMAYRa,*, Alexandra RIETHMULLERb

a
Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, A-1030 Wien, Austria
b
Fachbereich 18 Naturwissenschaften, Fachgebiet Okologie, Universitat Kassel, Heinrich-Plett-Strabe 40, D-34132 Kassel, Germany

article info
Phylogenetic maximum parsimony and Bayesian analyses of 60 collections belonging to 12
Article history: species of Albugo (Peronosporales) and two species of Pythium (Pythiales) were performed us-
Received 18 May 2005 ing nuclear large subunit ribosomal DNA sequences containing the D1 and D2 regions.
Received in revised form These data were supplemented with detailed light and scanning electron microscopical
18 September 2005 analyses of oospore morphology, and the morphological data of insufficiently studied
Accepted 24 September 2005 taxa (e.g. A. caryophyllacearum, A. gomphrenae) are revised. Molecular data revealed two
Corresponding Editor: main clades: one containing the collections from hosts belonging to the Caryophyllales
Susumu Takamatsu and Asteraceae, and the other containing the collections from hosts belonging to the Bras-
sicaceae and Convolvulaceae. Separation into these two clades was also corroborated by oo-
Keywords: spore morphology. Whereas the Albugo collections from Caryophyllales did not form
Host specificity a monophyletic lineage, the collections originating from Brassicaceae, Convolvulaceae and
Oomycetes Asteraceae each formed highly supported monophyletic clades. According to DNA sequence
Peronosporales data and oospore morphology, the host genus Amaranthus harbors two distinct species, Al-
Plant pathology bugo amaranthi and Albugo bliti. The DNA sequence data further indicate that Albugo candida
Straminipila and Albugo tragopogonis each may consist of several distinct lineages, but additional data
need to be collected before further taxonomic conclusions can be made.
2005 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Until recently, the phylogenetic position of the genus

The genus Albugo (white rust or white blister) is a widely dis-
tributed genus of obligatory plant parasites, containing more
than 40 species (Dick 2002a) described from various dicotyle-
donous host plant families. Several species are known as
crop pathogens (e.g. A. candida on various Brassicaceae, A. trag-
opogonis on sunflower, A. ipomoeae-panduratae on sweet potato,
A. occidentalis on spinach). Despite its prominent role as a plant
pathogen, a detailed modern monographic study of the genus
Albugo is still lacking; the keys of Biga (1955) and of Choi &
Priest (1995) being merely compilations of the published
data, and a critical re-examination of the genus Albugo using
modern techniques is needed.
Albugo was unclear to some extent. Traditionally, a monotypic
family Albuginaceae has been classified within the Peronospor-
ales, together with the Peronosporaceae (downy mildews) (Kirk
et al. 2001; Dick 2002a, b), and, sometimes, Pythiaceae (Water-
house 1973). Morphologically, the genus Albugo is markedly
distinct from all other Oomycetes in its catenate sporangial
chains produced by tightly packed, short, erect sporangio-
phores which are located in subepidermal pustules, the spo-
rangia being liberated after rupture of the epidermis. Its
isolated phylogenetic position is evident from morphology
and was generally accepted. However, the morphological dis-
tinctiveness precluded detailed hypotheses on phylogenetic
relationships to the other biotrophic genera of Peronosporales.

* Corresponding author.
E-mail address: hermann.voglmayr@univie.ac.at
0953-7562/$ see front matter 2005 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2005.09.013
76
Therefore, it is understandable that the proposed phylogenetic
relationships based on morphology were little more than
speculative (e.g. Shaw 1981; Dick 2002b).
Molecular phylogenetic analyses based on LSU rDNA
(Petersen & Rosendahl 2000; Riethmuller et al. 2002), and
cox2 (Hudspeth et al. 2003) shed some light on the phylogenetic
placement of Albugo, and new evolutionary hypotheses were
established. The isolated position of Albugo was confirmed.
In all analyses, a basal position within the Peronosporomycetidae
was evident, and a closer relationship to the downy mildews
(Peronosporaceae) could be ruled out. In addition, the LSU
rDNA data showed a high number of sequence substitutions
compared to the other taxa (Riethmuller et al. 2002), which is
in line with the hypothesis of an ancient origin and diversifica-
tion of the Albugo lineage (Dick 1988, 2002b). The data also
showed that the obligatory biotrophism of downy mildews
and Albugo are likely to have evolved independently. This
isolated phylogenetic position led Thines & Spring (2005) to
propose a new subclass, Albuginomycetidae.
The previous molecular phylogenetic analyses concentrated
on phylogenetic relationships of Oomycetes in general, and
quite few species of Albugo were included. Therefore, detailed
molecular systematic investigations within the genus Albugo
were still lacking, and the aim of the present study is to add
additional data towards a molecular phylogeny of Albugo, and
to enhance our understanding of the evolutionary radiation
of the genus Albugo on its hosts. In addition, the sequence
data were combined with data on oospore morphology, which
were obtained by thorough light microscopical and SEM inves-
tigations. Therefore, the present publication should be a fur-
ther step towards a modern systematic classification of Albugo.
Materials and methods
Sample sources, DNA-extraction, PCR and sequencing
The organisms studied in the present publication are listed in
Table 1. Due to the lack of a modern classification of the genus
Albugo, the species were determined and classified mainly
according to Biga (1955) and Choi & Priest (1995). The DNA ex-
traction, PCR, cycle sequencing and sequencing of the nuclear
large subunit D1/D2 region are described in Riethmuller et al.
(2002).
Data analysis
Initially, the sequence alignment was produced with the aid of
the MEGALIGN Module of the LASERGENE System (DNASTAR,
Inc.) and visually checked and refined with Se-Al version 2.0
(Rambaut 1996). Due to alignment problems in part of the
alignment, it was realigned with DIALIGN 2 (Morgenstern
1999; http://bioweb.pasteur.fr/seqanal/interfaces/dialign2-
simple.html) which compares whole segments of the sequen-
ces without applications of gap penalties. This approach is
especially efficient where sequences are not globally related
but share only local similarities (Morgenstern 1999). The
resulting alignments were checked and refined using BioEdit
5.0.9 (Hall 1999) and PAUP* version 4b10 (Swofford 2002). Due
to the alignment problems, phylogenetic analyses were
H. Voglmayr, A. Riethmuller
performed with both the complete dataset and a dataset
only containing the well-conserved first 374 base pairs.
Maximum parsimony (MP) bootstrap analyses were per-
formed with PAUP* version 4b10. 1000 bootstrap replicates
were done using heuristic search with random addition of
sequences and subsequent TBR branch swapping (MULTREES
option in effect, steepest descent option not in effect). Each
bootstrap replicate consisted of 10 rounds of random se-
quence addition and subsequent branch swapping, each
round limited to 10 000 rearrangements. Of the resulting trees,
50 % bootstrap consensus trees were calculated.
Metropolis-coupled Markov chain Monte Carlo analyses
(MCMC; Larget & Simon 1999; Mau et al. 1999) were performed
with the computer program MrBayes (version 3.0b4; Huelsen-
beck & Ronquist 2001) based on the general time-reversible
model of DNA substitution, additionally assuming a proportion
of invariant sites with gamma-distributed substitution rates of
the remaining sites (GTRIG; Swofford et al. 1996). Four incre-
mentally heated simultaneous Markov chains were run over
one million generations from which every 100th tree was sam-
pled. The trees before apparent stationary probability distribu-
tion of the cold chain were discarded (usually the first 1000
saved trees). A 50 % majority rule consensus of the remaining
trees was computed to obtain estimates for the probabilities
that groups are monophyletic given the sequence data (poste-
rior probabilities). Branch lengths were computed as mean
values over the sampled trees. 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 ran-
dom trees and default parameter values of the program. One of
these analyses was run over five million generations.
Morphological analyses
If possible, the same specimens as used for sequencing were
also used for morphological investigations. In the few cases
when oospore features could not be observed in these speci-
mens, the investigations were supplemented by dried refer-
ence specimens in WU, W, and BPI.
Scanning electron microscopy (SEM). For the investigation
of sporangia, dry leaf or stem fragments containing sporangial
pustules were glued on Cambridge stubs. Small parts of host
tissue containing oospores were soaked in a drop of distilled
water on a coverslip, picked to pieces using preparation nee-
dles and washed several times with distilled water. Then,
the host tissue and the oogonial wall was digested with a
4 % driselase (Sigma, St Louis, mo, USA) solution in 0.1 M sodi-
um citrate buffer (pH 5.0) for 6-24 h at 37
C. After the oogonial
wall had disintegrated, the oospores were washed several
times with distilled water, transferred to a clean coverslip,
air dried and the coverslip glued on Cambridge stubs. The
specimens were sputter coated with gold and examined in
a Jeol T 300 scanning electron microscope at 10 kV.
Light microscopy (LM). Host tissue containing oospores
was soaked in water, picked to pieces using preparation nee-
dles and carefully squashed with a coverslip. In parallel, also
oospores digested for SEM study (see above) were investigated
with the light microscope to check whether oospore ornamen-
tation is altered by enzyme digestion. Photographs were made
Phylogeny of Albugo species 77

Table 1 Collection data and GenBank accession number of the taxa sequenced
Taxon Collection dataa-b GenBank
accession no.
Isolated from/host Origin/source (voucher)

Albugo achyranthis Achyranthes aspera Namibia, near Wilhelmstal, MM (PREM), Na148, AR383 DQ007507
A. achyranthis A. sicula Namibia, Windhoek, leg. MM (PREM), Na160, AR384 DQ007508
A. amaranthi A. hybridus Germany, Baden-Wurttemberg, near Uberlingen, DQ007509
RBa (TUB), AR290
A. amaranthi A. powellii Austria, Upper Austria, St Willibald, HV (WU), HV441 AY035543
A. bliti A. blitum Taiwan, Chia Yi, Chung Pu, RK (TUB), AR291 DQ007503
A. bliti A. blitum ssp. Austria, Lower Austria, Marchegg, JW (WU), HV2137 DQ007504
emarginatus
A. candida Arabidopsis thaliana Austria, Upper Austria, St Willibald, HV (WU), HV783 DQ007481
A. candida Arabis alpina 1 Germany, Bavaria, Oberjoch, MP (TUB), AR156 AY035539
A. candida A. alpina 2 Germany, Bavaria, near Oberjoch, MP (TUB), AR224 DQ007465
A. candida A. stellulata Austria, Karnten, Villach, ML (WU), AR338 DQ007470
A. candida Armoracia rusticana 1 Germany, Baden-Wurttemberg, DQ007474
Tubingen-Unterjesingen, MM (TUB), AR330
A. candida A. rusticana 2 Austria, Upper Austria, St Willibald, HV (WU), HV954 DQ007475
A. candida Aurinia saxatilis Germany, Baden-Wurttemberg, Tubingen-Hirschau, DQ007464
AR (TUB), AR337
A. candida Berteroa incana 1 Germany, Berlin, Teufelsberg, MM (TUB), AR300 DQ007462
A. candida B. incana 2 Germany, Berlin-Marzahn, MM (TUB), AR304 DQ007463
A. candida Capsella bursa-pastoris 1 Germany, Mecklenburg-Vorpommern, Greifswald, AY035538
MG (TUB), MG15-7
A. candida C. bursa-pastoris 2 AF235938c
A. candida Cardamine amara Germany, Bavaria, Gruntensee near Wertach, DQ007478
MG (TUB), MG1834
A. candida C. diphylla USA, Tennessee, Knoxville, HV (WU), HV5.4.A.C AY035540
A. candida Cleome gynandra Namibia, Erichsfelde, MM & RB (PREM), Na31, AR257 DQ007477
A. candida Coronopus squamatus Germany, Baden-Wurttemberg, Tubingen, HV, AR283 DQ007482
A. candida Diplotaxis erucoides France, Dept. Aude, E Narbonne, HV & AR (TUB), F35 DQ007467
A. candida D. tenuifolia Germany, Berlin Alt Stralau, MM (TUB), AR295 DQ007468
A. candida Erysimum cheiranthoides Germany, Berlin Prenzlauer Berg, MM (TUB), AR296 DQ007479
A. candida Eutrema japonica Taiwan, Wasabi, RK (TUB), AR174 DQ007466
A. candida Lunaria annua 1 France, Dept. Drome, S Lyon, W Romans s/Isere, DQ007471
HV & AR (TUB), F20
A. candida L. annua 2 Germany, Baden-Wurttemberg, Tubingen-Hagelloch, DQ007473
FO (TUB), AR253
A. candida L. annua 3 Germany, Baden-Wurttemberg, Tubingen-Hagelloch, DQ007472
KH (TUB), AR155
A. candida Raphanus raphanistrum Reunion, Plaine des Cafres, MG (TUB), MG1874 DQ007476
A. candida R. sativus Germany, Berlin-Lubars, MM (TUB), AR359 DQ007461
A. candida Sinapis alba 1 Germany, Baden-Wurttemberg, Tubingen-Waldhausen, DQ007460
MG (TUB), MG1866
A. candida S. alba 2 Germany, Baden-Wurttemberg, Tubingen, DQ007459
AR (TUB), AR339
A. candida Sisymbrium irio Bolivia, Dpto. La Paz, Prov. Manco, Kapak, MP et al. DQ007469
2590 (LPB, TUB)
A. candida S. loeselii Germany, Berlin-Friedrichshain, MM (TUB), HV918 DQ007480
A. caryophyllacearum Spergularia salina Austria, Burgenland, Illmitz, Neusiedler See, DQ007499
HV (WU), HV2131
A. evolvuli Evolvulus alsinoides 1 Namibia, Erichsfelde, MM (PREM), Na133, AR376 DQ007487
A. evolvuli E. alsinoides 2 Namibia, Sonop, MM (PREM), Na121, AR377 DQ007489
A. evolvuli E. alsinoides 3 Namibia, Omatako, MM (PREM), Na139, AR378 DQ007488
A. gomphrenae Gomphrena martiana Argentinia, Prov. Catamarca, Chumbicha, DQ007501
WT (WU), HV2139
A. aff. gomphrenaed Iresine diffusa Costa Rica, Prov. Limon, Bribri, MP (TUB), AR166 AY035545
A. ipomoeae-panduratae Ipomoea plebeia Namibia, Miles 46, MM & RB (PREM), Na26, AR255 DQ007486
A. ipomoeae-panduratae Ipomoea sinensis Namibia, near Okahandja, MM (PREM), Na145, AR382 DQ007484
ssp. blepharosepala 1
A. ipomoeae-panduratae I. sinensis Namibia, Ombeameiata housecamp, MM (PREM), DQ007485
ssp. blepharosepala 2 Na186, AR385
A. ipomoeae-panduratae Ipomoea sp. Namibia, Erichsfelde, MM (PREM), Na132, AR381 DQ007483
A. occidentalis Spinacia oleracea L. USA, Texas, MCB, AR362e DQ007500
A. platensis Boerhavia deserticola Namibia, Windhoek, MM (PREM), Na151, AR375 DQ007502
(continued on next page)
78 H. Voglmayr, A. Riethmuller

Table 1 (continued)
Taxon Collection dataa-b GenBank
accession no.
Isolated from/host Origin/source (voucher)

A. portulacae Portulaca oleracea 1 Austria, Vienna, HV (WU), HV374 AY035544

A. portulacae P. oleracea 2 Namibia, Steinhausen, MM (PREM), Na301, AR374 DQ007506
A. portulacae Portulaca sp. Reunion, St Leu, MH, HMH3226, AR 305 DQ007505
A. tragopogonis Ambrosia Austria, Burgenland, Apetlon, HV (WU), HV1024 DQ007492
artemisifolia
A. tragopogonis Centaurea scabiosa Switzerland, Appenzell, Schwendenbachtal, RB & FB (TUB), AR217 AY035542
A. tragopogonis Cirsium arvense Germany, Baden-Wurttemberg, Tubingen, MM (TUB), AR298 DQ007490
A. tragopogonis C. oleraceum Austria, Styria, Mariazell, WM (TUB), MG9-4 AY035541
A. tragopogonis Crepis sancta France, Dept. Drome, S Lyon, W Romans s/Isere, HV & AR (WU), F18 DQ007496
A. tragopogonis Scorzonera laciniata France, Dept. Herault, SW Montpellier, near Montbazin, DQ007497
HV & AR (TUB), F40
A. tragopogonis Senecio vulgaris France, Dept. Drome, S Lyon, between Tain-lHermitage DQ007498
and Romans s/Isere, HV & AR (TUB), F12
A. tragopogonis Tanacetum parthenium Germany, Baden-Wuttemberg, Tubingen, HV (WU), HV920, AR271 DQ007493
A. tragopogonis Tragopogon orientalis Germany, Baden-Wurttemberg, Tubingen, HV (TUB), HV904, AR268 DQ007495
A. tragopogonis T. pratensis Germany, Brandenburg, between Lubars and Schildow, MM (TUB), AR302 DQ007494
A. tragopogonis Urospermum France, Dept. Herault, SW Montpellier, near Montbazin, DQ007497
dalechampii HV & AR (TUB), F41
Pythium middletonii Soil CBS 528.74, AR116 AF119608
P. undulatum Water Germany, Baden-Wurttemberg, Kanzach, AR, AR 55 AF119603

a Collectors: AR, A. Riethmuller; FB, V. Faust-Berndt; FO, F. Oberwinkler; HV, H. Voglmayr; JW, J. Walter; KH, L. Kisimova-Horovitz; MCB, M. C.
Black; MG, M. Goker; MH, M. Hendrichs; ML, M. Lutz; MM, M. Mennicken; MP, M. Piepenbring; RB, R. Berndt; RBa, R. Bauer; RK, R. Kirschner; WM,
W. Maier; and WT, W. Till.
b Vouchers: PREM, Plant Protection Research Institute, Pretoria; TUB, University of Tubingen; WU, University of Vienna; and CBS, Centraalbur-
eau voor Schimmelcultures, Utrecht.
c Specimen sequenced by Petersen & Rosendahl (2000).
d Erroneously identified and published as Albugo achyranthis in Riethmuller et al. (2002).
e Supplied as DNA extract by Deborah and Michael Hudspeth.

using a Olympus BX50 light microscope equipped with a Nikon In the molecular analyses, two main lineages are present:
D70 digital camera and a Olympus SIS Color View3 digital a clade containing accessions from Brassicaceae and Convolvu-
camera system, respectively. laceae; and a clade containing accessions from Caryophyllales
and Asteraceae. Within the two main lineages, highly sup-
ported subgroups are present which correspond to the host
Results
systematics: accessions from Brassicaceae, Convolvulaceae,
Asteraceae and the majority of Caryophyllales each form groups
Molecular phylogenetic analyses
highly supported at least in the MP analyses (Figs 1-2). The
only exception is the clade containing A. occidentalis (from Che-
The final alignment, 786 base pairs long, and the trees
nopodiaceae, North America) and A. caryophyllacearum (from
obtained were deposited in TreeBASE (http://www.treebase.
Caryophyllaceae), which is basal to the Asteraceae-core Caryo-
org) and are available under study accession no. S1407.
phyllales clade in the MP and MCMC analyses (Figs 1-2). How-
Whereas all sequences were well aligned for the first 374
ever, this sister group relationship is only moderately
base pairs, base pairs 375-786 contained regions which were
supported in MP and MCMC analyses. In addition, whereas
well aligned only within the accessions from the same host
highly supported (94 % posterior probability) for the short
families and orders, respectively, but poorly aligned between
dataset (Fig 1), support for the clade containing accessions
the accessions from the different host groups. However, also
from Brassicaceae was decreased to 54 % for the large dataset
within this region, some conserved motifs were present.
in the MC analysis with 5 M generations (Fig 2).
These conserved motifs could be identified with DIALIGN 2,
Within the core of Albugo species from Caryophyllales, several
and they served as anchor points for an improved alignment.
distinct lineages are evident. On Amaranthus, two distinct
To test the effects of the poorly alignable regions, in a first
species are present, A. bliti (on Amaranthus blitum), and A. amar-
analysis only the first 374 base pairs were used (Fig 1). The
anthi (on Amaranthus hybridus, A. powellii and related species).
comparison with analyses of the complete dataset (Fig 2)
revealed no significant differences concerning the topology,
but the resolution within the accessions from the same host SEM and LM studies
group was much improved. On the other hand, the internal
support for many nodes remained similar, but in some cases SEM of the sporangia revealed a verrucose to reticulate orna-
was altered considerably (Figs 1-2). mentation of the sporangial wall, depending on the species
Phylogeny of Albugo species 79

A. candida (ex Sinapis alba 1)

A. candida (ex Sinapis alba 2)
A. candida (ex Raphanus sativus)
A. candida (ex Berteroa incana 1)
A. candida (ex Berteroa incana 2)
A. candida (ex Aurinia saxatilis)
A. candida (ex Arabis alpina 1)
A. candida (ex Arabis alpina 2)
62 A. candida (ex Eutrema japonica)
75
93 A. candida (ex Raphanus raphanistrum)
A. candida (ex Diplotaxis erucoides)
A. candida (ex Diplotaxis tenuifolia)

Brassicaceae
A. candida (ex Sisymbrium irio)
A. candida (ex Arabis stellulata)
A. candida (ex Lunaria annua 1)
A. candida (ex Lunaria annua 2)
A. candida (ex Lunaria annua 3)
98
A. candida (ex Capsella bursa-pastoris 1)
94
A. candida (ex Capsella bursa-pastoris 2) a
A. candida (ex Amoracia rusticana 1)
A. candida (ex Amoracia rusticana 2)
A. candida (ex Cleome gynandra)
A. candida (ex Cardamine amara)
80
A. candida (ex Erysimum cheiranthoides)
88 A. candida (ex Sisymbrium loeselii)
98
100 A. candida (ex Cardamine diphylla)
A. candida (ex Arabidopsis thaliana)
A. candida (ex Coronopus squamatus)
A. ipomoeae-panduratae (ex Ipomoea. sp.)

Convolvulaceae
73
A. ipomoeae-panduratae (ex Ipomoea sinensis 1)
100 75
A. ipomoeae-panduratae (ex Ipomoea sinensis 2)
100
99 A. ipomoeae-panduratae (ex Ipomoea plebeia)
98 A. evolvuli (ex Evolvulus alsinoides 1)
100
A. evolvuli (ex Evolvulus alsinoides 2)
100
A. evolvuli (ex Evolvulus alsinoides 3)
b
55 A. tragopogonis (ex Cirsium arvense)
52
70 A. tragopogonis (ex Cirsium oleraceum)
54 61 A. tragopogonis (ex Centaurea scabiosa)
100 83 77 96 A. tragopogonis (ex Crepis sancta)
Asteraceae

100 100 100 A. tragopogonis (ex Urospermum dalechampii)

68 A. tragopogonis (ex Scorzonera laciniata)
* A. tragopogonis (ex Ambrosia artemisifolia)
83
A. tragopogonis (ex Tanacetum parthenium)
*
100 72 A. tragopogonis (ex Tragopogon pratensis)
100 91 A. tragopogonis (ex Tragopogon orientalis) c
A. tragopogonis (ex Senecio vulgaris)
72 100 A. aff. gomphrenae (ex Iresine diffusa)
99 100 A. gomphrenae (ex Gomphrena martiana)
A. platensis (ex Boerhavia deserticola)
76 A. bliti (ex Amaranthus blitum)
100
95 A. bliti (ex Amaranthus blitum ssp. emarginatus)
Caryophyllales

100 99
73 A. portulacae (ex Portulaca sp.)
97
69 76 A. portulacae (ex Portulaca oleracea 2)
96 99 A. portulacae (ex Portulaca oleracea 1)
100
99 A. achyranthis (ex Achyranthes aspera)
100 A. achyranthis (ex Achyranthes sicula)
98 A. amaranthi (ex Amaranthus cruentus)
100 A. amaranthi (ex Amaranthus powellii) d
95 A. caryophyllacearum (ex Spergularia salina) 2 m
96 A. occidentalis (ex Spinacia oleracea)
Pythium undulatum
Pythium middletonii

Fig 1 50 % maximum parsimony bootstrap consensus tree based on the first 374 bp of the D1/D2 alignment (LSU rDNA),
calculated from the trees saved in an analysis with 1000 bootstrap replicates, using 10 rounds of random sequence addition
and subsequent TBR branch swapping (MULTREES option in effect, steepest descent option not in effect), each round limited to
10 000 rearrangements. Numbers above the branches are maximum parsimony bootstrap values, below the branches the
posterior probabilities of a one-million MCMC analysis, discarding the first 600 trees. Asterisks (*) denote nodes in conflict
with the MCMC consensus tree, (a-d) sporangial ornamentation characteristic for the major lineages: (a) A. candida (ex Sinapis
alba HV729, WU); (b) A. ipomoeae-panduratae (ex Ipomoea sinensis Na145); (c) A. tragopogonis (ex Tragopogon orientalis HV1042,
WU); and (d) A. amaranthi (ex Amaranthus powellii HV441, WU).
 a 64/91
b
A. candida (ex Sinapis alba 1)
1 A. candida (ex Sinapis alba 2)
A. candida (ex Raphanus sativus)
61/98
A. candida (ex Berteroa incana 1)
A. candida (ex Berteroa incana 2)

Brassicaceae
A. candida (ex Aurinia saxatilis)
100/ 1 A. candida (ex Arabis alpina 2)
54 A. candida (ex Arabis alpina 1)
A. candida (ex Eutrema japonica)
87/ 100/65 A. candida (ex Diplotaxis erucoides)
100 A. candida (ex Diplotaxis tenuifolia)
A. candida (ex Sisymbrium irio)

Convolvulaceae
100/ A. candida (ex Arabis stellulata)
100 A. candida (ex Lunaria annua 1)
2 A. candida (ex Lunaria annua 2)
100 A. candida (ex Lunaria annua 3)
A. candida (ex Capsella bursa-pastoris 2)

Asteraceae
100 100/
100/ 54 A. candida (ex Capsella bursa-pastoris 1)
100
3 A. candida (ex Amoracia rusticana 1)
59/ 67/97 A. candida (ex Amoracia rusticana 2)
59 A. candida (ex Raphanus raphanistrum)
100/
A. candida (ex Cleome gynandra)
Caryophyllales

100
92/ A. candida (ex Cardamine amara)
100 69/58
4 A. candida (ex Erysimum cheiranthoides)
95/91 A. candida (ex Sisymbrium loeselii)
A. candida (ex Cardamine diphylla)
5 A. candida (ex Arabidopsis thaliana)
Outgroup (Pythium) 99/64
A. candida (ex Coronopus squamatus)
0.05 substitutions/site
100/90 A. ipomoeae-panduratae (ex Ipomoea. sp.)
A. ipomoeae-panduratae (ex Ipomoea sinensis 1)
100/ A. ipomoeae-panduratae (ex Ipomoea sinensis 2)
100 100/100 A. ipomoeae-panduratae (ex Ipomoea plebeia)
A. evolvuli (ex Evolvulus alsinoides 1)
A. evolvuli (ex Evolvulus alsinoides 2)
2 100/100 A. evolvuli (ex Evolvulus alsinoides 3)

82/91
A. tragopogonis (ex Cirsium arvense)
80/98
A. tragopogonis (ex Cirsium oleraceum)
75/100
A. tragopogonis (ex Centaurea scabiosa)
70/75
A. tragopogonis (ex Scorzonera laciniata)
3 81/88 100/100 A. tragopogonis (ex Crepis sancta)
100/75 A. tragopogonis (ex Urospermum dalechampii)
A. tragopogonis (ex Ambrosia artemisifolia)
100/100 A. tragopogonis (ex Tanacetum parthenium)
A. tragopogonis (ex Tragopogon pratensis)
96/95
98/100 A. tragopogonis (ex Tragopogon orientalis)
A. tragopogonis (ex Senecio vulgaris)

4 100/100 A. aff. gomphrenae (ex Iresine diffusa)

A. gomphrenae (ex Gomphrena martiana)
76/97
99/100 A. portulacae (ex Portulaca sp.)
100/ A. portulacae (ex Portulaca oleracea 1)
100 /53
A. portulacae (ex Portulaca oleracea 2)
72/85 A. achyranthis (ex Achyranthes aspera)
100/100 A. achyranthis (ex Achyranthes sicula)
87/94
A. bliti (ex Amaranthus blitum)
100/100
100/100 A. bliti (ex Amaranthus blitum ssp. emarginatus)
A. platensis (ex Boerhavia deserticola)
A. amaranthi (ex Amaranthus cruentus)
100/100 A. amaranthi (ex Amaranthus powellii)

5
100/99 A. caryophyllacearum (ex Spergularia salina)
0.05 substitutions/site A. occidentalis (ex Spinacia oleracea)

Fig 2 Bayesian phylogenetic analysis using Markov chain Monte Carlo (MCMC) of 60 specimens of Albugo based on the
complete D1/D2 alignment (LSU rDNA). 50% majority rule consensus tree from an MCMC analysis over five million genera-
tions, in which every 100th tree was sampled, discarding the first 600 trees; branch lengths are averaged over the sampled
trees. First numbers on the branches are maximum parsimony bootstrap values (for methodological details see Fig 1), second
numbers the estimates for the posterior probabilities of the Bayesian analysis for monophyly of the respective clades. (a)
Simplified complete tree, showing the branch lengths and MP bootstrap/MCMC posterior probability values of the tree
backbone. Clade numbers correspond to the clades shown in detail in Fig 2b. (b) Terminal groups of the tree in detail, rep-
resenting the accessions from Brassicaceae (1), Convolvulaceae (2), Asteraceae (3) and Caryophyllales (4, 5).
Phylogeny of Albugo species
(Fig 1). Ornamentation of Albugo candida and Albugo from Con-
volvulaceae was verrucose (Fig 1a, b), that of A. tragopogonis ir-
regularly verrucose-reticulate (Fig 1c), and Albugo from
Caryophyllales irregularly verrucose, the verrucae often being
striate and confluent (Fig 1d).
Enzyme digestion of the host tissue and oogonial wall with
driselase worked well in most cases; however, in Albugo candida
and A. ipomoeae-panduratae the oogonial wall was resistant to
driselase digestion in all accessions tested. Comparison of
non-digested and digested oospores with both LM and SEM
showed that the oogonial wall was not altered markedly during
driselase treatment.
LM and SEM of the oospores showed various types of orna-
mentation (Figs 3-26). Oospore ornamentation was observed
to be species-specific. Oospores of the species investigated
were either smooth (A. ipomoeae-panduratae; Fig 25, but see
note below), coarsely-bluntly verrucose (A. candida; Figs 14,
26), irregularly reticulate (A. bliti; Figs 4, 15), or more regularly
reticulate (A. achyranthis, A. amaranthi, A. gomphrenae, A. occi-
dentalis, A. platensis, A. portulacae; Figs 3, 5-8, 16-22) with a small
to wide reticulum depending on the species. SEM shows that
some of these distinctly reticulate ornaments have tubercles
on them (A. achyranthis, A. amaranthi, A. occidentalis, A. portula-
cae; Figs 3, 6-7, 10). Within A. caryophyllacearum, ornamenta-
tion can vary between irregularly verrucose with confluent
verrucae to incompletely reticulate (Figs 7, 21-22), often even
within the same oospore. A. tragopogonis usually shows a com-
bination of a very fine reticulum with fine tubercles in LM, but
in SEM the reticulum is often not apparent and ornamentation
appears to be distinctly finely verrucose (Figs 9-10, 24). The re-
ticulate oospores of A. achyranthis are described for the first
time, they measure 50-65 mm, and the irregularly penta- to
heptagonal meshes measure 4.5-7 mm diam.
SEM investigations of oospores of A. candida and A. ipo-
moeae-panduratae proved to be difficult due to the pronounced
resistance of the oogonium wall to enzyme digestion. However,
in A. candida it was possible to mechanically remove the oogo-
nium wall without severe damage in a few cases (Fig. 14). In
A. ipomoeae-panduratae this was not successful as the oospore
wall appears to be firmly connected to the inner oogonium
wall. In contrast to the other species investigated, the persis-
tent oogonium wall of A. ipomoeae-panduratae was distinctly
verrucose (Fig 13, 25).
Discussion
The phylogenetic relationships inferred from nLSU data cor-
roborates the results of Riethmuller et al. (2002) that collec-
tions originating from the same host families and orders,
respectively, usually form highly supported, distinct lineages.
The only exception is Albugo from Caryophyllales, which is par-
aphyletic (Figs. 1-2). The two species with the widest host
ranges, A. candida (Brassicaceae) and A. tragopogonis (Asteraceae)
do not form homogeneous lineages, but indicate the presence
of genetically distinct entities which may represent distinct
species. Critical re-examination of morphology showed that
morphological features are fully in line with the results of
the phylogenetic analyses.
81
Recently, sporangium wall ornamentation revealed by SEM
has been proposed to be of diagnostic value for different line-
ages of the genus Albugo (Spring 2004; Spring & Thines 2004;
Thines & Spring 2005), and two new segregate genera, Pustula
and Wilsoniana, were described (Thines & Spring 2005). In our
SEM observations, sporangium wall ornamentation of the ge-
nus Albugo varied from verrucose (A. candida, Albugo spp. from
Convolvulaceae), verrucose-striate (Albugo spp. from Caryophyl-
lales) to irregularly reticulate (A. tragopogonis) (Fig 1a-d). How-
ever, ornamentation can vary greatly even within the same
collection; for example, in A. tragopogonis the reticulum is
not distinctive in all conidia, which then appear to be densely
verrucose. Although diagnostic for the major lineages, it is
therefore unlikely that sporangium wall ornamentation can
be used for the distinction of closely related species.
Although oospore features have been considered as impor-
tant diagnostic features for species delimitation (Biga 1955;
Choi & Priest 1995), they were rarely used for hypotheses on
phylogenetic relationships within the genus (e.g. Wilson
1907). The results of the molecular analyses correspond with
oospore morphology. The lineage containing A. candida,
A. evolvuli, and A. ipomoeae-pandurata, has a persistent oogonium
wall which is resistant to enzyme digestion (Figs 13-14, 25-26);
in addition, the outer oospore wall is partly connected with the
oogonium wall, so that the oospores could not be separated
mechanically from the oogonial wall without damage
(Fig 14). The oogonial walls of this lineage are either smooth
(A. candida) or distinctly verrucose (A. ipomoeae-panduratae),
whereas the outer oospore walls are either coarsely verrucose
(A. candida) or smooth (A. ipomoeae-panduratae) (Figs 13-14, 25-
26). Within this lineage, oospore walls are comparatively light
brown in the light microscope, and they are usually smaller
than 50 mm (Choi & Priest 1995).
Conversely, the lineage containing A. tragopogonis s. lat.
and Albugo spp. from Caryophyllales is characterised by a non-
persistent, smooth oogonium wall which is digested easily
with cellulolytic enzymes, and the outer oospore wall is either
reticulate, reticulate-verrucose or finely verrucose (Figs 3-10,
15-24). In addition, oospore walls of mature oogonia are usually
dark to blackish brown, and the oospores are usually larger
than 50 mm (Choi & Priest 1995). Within that lineage, oospore
ornamentation is much more diverse than in the former.
Albugo from Caryophyllales
The molecular data do not reveal the Albugo species from
Caryophyllales as a monophyletic group (Figs 1-2). However,
the basal position of the A. occidentalis/caryophyllacearum group
is only weakly to moderately supported (Figs 1-2). Oospore
morphology does not conflict with the phylogenetic analyses,
as the oospore ornamentation of A. occidentalis is similar to
that of A. tragopogonis; both species show a fine reticulum
with verrucae on the ridges where they fuse (Figs 10, 23).
This type of oospore ornamentation may therefore be plesio-
morphic for the whole lineage. The core Albugo clade from Car-
yophyllales is highly supported (Figs 1-2), and although closely
related, it is evident that the collections from the different
hosts are distinct species. This is also corroborated by oospore
morphology, which is, although basically reticulate, diagnos-
tic for the different species (Figs 3-10, 15-23).
Figs 3-14 SEM pictures of oospores of Albugo spp. Fig 3. A. amaranthi (HV441, WU); Fig 4. A. bliti (HV2137, WU); Fig 5. A. cf
gomphrenae (ex Gomphrena sp., BPI 184163); Fig 6. A. portulacae (Na301); Fig 7. A. achyranthis (ex Achyranthes bidentata, Japan,
Morioka, Iwate, K. Togashi, W); Fig 8. A. platensis (ex Boerhaavia sp., USA, Arizona, Tucson, W. G. Solheim [Mycoflora Saximon-
tanensis Exsiccata no. 401] W); Fig 9. A. caryophyllacearum (HV2131); Fig 10. A. occidentalis (ex Spinacia oleracea, USA, Texas,
Winter Haven, Ivanoff, W); Fig 11. A. tragopogonis (F41); Fig 12. A. tragopogonis (ex Tragopogon pratensis, Hungary, near Budapest
Magocsy-Dietz, WU); Fig 13. A. ipomoeae-panduratae (ex Ipomoea leptophylla, USA, Kansas, Stokton, W. Petrak [Mycotheca Generalis
no. 601], W); Fig 14. A. candida (ex Capsella bursa-pastoris, Prencov, 1. Sep. 1887, A. Kmet [Fungi Schemnitzenses], WU).
Arrow denotes position where outer oospore wall and inner oogonium wall were fused. Bar 20 mm.
Phylogeny of Albugo species 83

Figs 15-26 Light microscopy of oospores. Fig 15. A. bliti (HV2137, WU); Fig 16. A. portulacae (Na301); Fig 17. A. achyranthis
(ex Achyranthes bidentata, Japan, Morioka, Iwate, K. Togashi, W); Fig 18. A. platensis (ex Boerhaavia sp., USA, Arizona, Tucson,
W. G. Solheim [Mycoflora Saximontanensis Exsiccata no. 401], W); Fig 19. A. amaranthi (HV441, WU); Fig 20. A. gomphrenae
(ex Gomphrena perennis, Argentinia, San Juan, Mar. 1904 Spegazzini, LPS 28054-type); Figs 21-22. A. caryophyllacearum
(HV2131); Fig 23. A. occidentalis (ex Spinacia oleracea, USA, Texas, Winter Haven, Ivanoff, W); Fig 24. A. tragopogonis (F41); Fig 25.
A. ipomoeae-panduratae (ex Ipomoea leptophylla, USA, Kansas, Stokton, W. Petrak [Mycotheca Generalis no. 601], W); Fig 26. A.
candida (ex Armoracia rusticana, Bohemia, Bodenbach, de Thumen [Fungi Austriaci no. 430], WU). Arrows denote oogonium
walls. Bar 20 mm.
84
In this context, it has to be mentioned that morphological data
in the literature should be treated with caution. Careful re-ex-
amination of oospore morphology showed that such data are
sometimes incomplete or even incorrect. Oospore ornamenta-
tion of A. caryophyllacearum, given as verrucose (Wilson 1907;
Biga 1955), could be shown to vary between irregularly verru-
cose with confluent verrucae to incompletely reticulate
(Figs 21-22) often even within the same oospore. Although
originally described as smooth (Spegazzini 1909), the oospore
wall of A. gomphrenae is distinctly reticulate in the type speci-
men and all other specimens examined (Figs 5, 20).
Interestingly, the molecular phylogenetic analyses revealed
two distinct Albugo lineages within the host genus Amaranthus,
which was also confirmed by oospore morphology. Investiga-
tion of numerous specimens showed significant differences
between oospores from A. blitum and those from the other
Amaranthus species. Whereas the collections from A. blitum
had irregular sinuous ridges forming, if at all, only a very irreg-
ular reticulum (Figs 4, 15), those from other species of Amaran-
thus showed a regular reticulum (Figs 3, 19). These
morphological differences were already noticed by Zalewski
(1883), who recognised two species: Cystopus bliti (confined to
A. blitum) and C. amaranthacearum (all other Amaranthaceae).
However, subsequent authors (Fischer 1892: 423; Wilson
1907) did not accept Zalewskis (1883) split, and his data were
ignored. As Schweinitz (1831: 292) already described Caeoma
subgen. Uredo amaranthi (now Albugo amaranthi) from American
Amaranthus paniculatus (a synonym of A. cruentus, which is host
for the species with reticulate oospores), this name has priority
over the species described by Zalewski (1883). As Albugo amar-
anthi is a very common parasite apparently confined to Amer-
ican Amaranthus species, it is likely that it has quite recently
been introduced to the Old World together with its hosts. Con-
versely, Albugo bliti, parasitic on Amaranthus blitum (and proba-
bly some other close relatives from the Old World), is
considered native to the Old World.
Albugo from Asteraceae
This group is highly supported, and differs significantly in its
LSU sequence from the other species. According to Whipps &
Cooke (1978), Albugo has been recorded from more than 300
host genera of Asteraceae. Besides A. tragopogonis, several addi-
tional species and varieties have been described from astera-
ceous hosts (Biga 1955). Choi & Priest (1995) listed A. solivae
and A. chardiniae as separate species, which should differ in oo-
spore ornamentation from A. tragopogonis. Whereas the molec-
ular data confirm the presence of genetically distinct lineages
which may represent different species, we consider the de-
scription of numerous new species premature before extensive
molecular investigations of a more representative sample on
a world-wide basis are carried out. However, according to our
preliminary results, it may be difficult to find morphological
characteristics for unequivocal species delimitation.
Albugo from Brassicaceae
Traditionally within Brassicaceae, a single species (Albugo can-
dida) has been recognized, but high host specificity has been
observed (Hiura 1930) and several host specific forms
H. Voglmayr, A. Riethmuller
(Sa vulescu & Rayss 1930), varieties (Biga 1955), or races (Pound
& Williams 1963; Hill et al. 1988) have been distinguished.
However, clear-cut morphological distinction of different en-
tities could not be always verified (Makinen & Hietajarvi
1965). From the closely related Capparidaceae, now also includ-
ed in Brassicaceae (Angiosperm Phylogeny Group 2003), an ad-
ditional species (A. capparidis) has been described. The Albugo
collections from Brassicaceae are split into two main groups in
the molecular phylogenetic analyses (Figs 1-2). In his mono-
graph of Albugo, Biga (1955) accepted two varieties within
Albugo candida, var. candida and var. macrospora, the latter hav-
ing slightly larger sporangia. However, morphological investi-
gations of the sporangia showed that the tree topologies
obtained with molecular data were neither congruent with
such a differentiation, nor with the host list for the two varie-
ties given in Biga (1955). No features were found which could
be used for morphological differentiation of the two lineages
obtained with molecular data. Therefore, we presently refrain
from proposing species rank for the separate lineages.
Synonymy of A. capparidis and A. candida is supported by
the position of the collection on Cleome gynandra, which is
placed within A. candida (Figs 1-2). This is in line with the ab-
sence of clearly distinct morphological features.
Albugo from Convolvulaceae
Several species have been described from Convolvulaceae, of
which Choi & Priest (1995) accepted six species in their key.
Species were suggested to differ in sporangial wall thickening
and oospore ornamentation (Biga 1955; Choi & Priest 1995).
According to Biga (1955), Albugo ipomoeae-panduratae has the
widest host range and an almost cosmopolitan distribution;
following that concept, our accessions from South Africa are
placed in this taxon. However, it should be noted that a taxo-
nomic revision of the Albugo species from Convolvulaceae is
badly needed; the oospore morphology especially, which rep-
resents a prime character for the distinction of the species of
this group, should be re-investigated. This is particularly diffi-
cult as the oospores are not produced in the leaves but in dis-
torted stems, which are only rarely collected. In the present
study, no oospores were found in the South African specimens
of A. ipomoeae-panduratae and A. evolvuli investigated, and the
data were obtained from North American material of A. ipo-
moeae-panduratae on Ipomoea pandurata (the type host) and
I. leptophylla. Whereas the oospore wall of A. ipomoeae-pandur-
atae has been described as verrucose by Wilson (1907),
re-examination of the same collections examined by him (BPI
185737, BPI 796200) showed that the oospore wall was smooth
(Fig. 25). Wilson (1907) probably mistook the persistent, wrin-
kled-verrucose oogonium wall (Figs 13, 25) for the oospore
wall, which was already assumed by Choi & Priest (1995).
Acknowledgements
Cordial thanks are due to Viola Faust-Berndt, Markus Goker,
Matthias Hendrichs, Deborah and Michael Hudspeth, Roland
Kirschner, Liuba Kisimova-Horovitz, Matthias Lutz, Mechthild
Mennicken, Franz Oberwinkler and Meike Piepenbring for
Phylogeny of Albugo species
collecting and/or providing specimens for analysis, and to
Johannes Walter for determining and providing infected
Amaranthus specimens. HV wishes to thank Irmgard Krisai-
Greilhuber and Ron H. Petersen for their support in organising
collecting trips and the latter for sending literature. Thanks
are also due to Michael Hesse for permission to use the SEM
facilities, and to Alfred Glaser and Heidi Halbritter for techni-
cal support. The curators of the collections at BPI, LPS, TUB
and W are gratefully acknowledged for sending herbarium
specimens, and Walter Till (WU) for handling the loan
requests. The present publication is part of the GLOPP (Global
Information System for the Biodiversity of Plant Pathogenic
Fungi) project financed by the Bundesministerium fur Bildung
und Forschung (BMBF), which is gratefully acknowledged.
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