Mycologia, 100(5), 2008, pp. 716–726. DOI: 10.3852/08-030 # 2008 by The Mycological Society of America, Lawrence, KS 66044-8897
Molecular data do not support a southern hemisphere base of Nothofagus powdery mildews Seiko Niinomi Susumu Takamatsu1
INTRODUCTION
The Erysiphaceae is a group of obligately biotrophic fungi that cause powdery mildew disease on about 10 000 angiosperm species (Amano 1986) and consists of 16 genera and ca. 650 species (Braun and Takamatsu 2000, Braun et al 2002, Takamatsu et al 2005a, b, Liberato et al 2006). The host range of this fungal group is confined strictly to angiosperms, and the fungi never have been reported to infect ferns or gymnosperms (Amano 1986). Molecular phylogenetic analyses demonstrated that the Erysiphaceae form a distinct monophyletic group (Mori et al 2000b, Lutzoni et al 2004, Takamatsu 2004, Wang et al 2007). Thus the Erysiphaceae are derived from a single ancestral taxon that might have acquired the ability to parasitize plants only once. The question as to when and where powdery mildew fungi originated is an interesting subject to consider with regard to evolution of the fungi. Heluta (1994) hypothesized that powdery mildew fungi originated in southeastern China based on paleogeography and paleobotany data. Hirata (1972) reported that host species are abundant in temperate regions of the northern hemisphere compared with tropical regions or the southern hemisphere. These reports suggest that the northern hemisphere is the geographic origin of powdery mildews. In contrast no reports suggest that powdery mildews originated in the southern hemisphere. However angiosperms now are considered to have originated potentially during the late Jurassic–early Cretaceous in western Gondwana (southern hemisphere; Anderson et al 1999, Hill et al 1999). If the evolution of powdery mildews is closely related to the evolution of angiosperms the possibility that they originated in the southern hemisphere cannot be excluded. Molecular phylogenetic analyses suggest that the powdery mildew genus Golovinomyces evolved in affinity with their hosts, the Asteraceae (Matsuda and Takamatsu 2003). Because the Asteraceae originated in South America it could be assumed that the geographic origin of Golovinomyces was the southern hemisphere if the affinity of Golovinomyces and the Asteraceae began in South America. To evaluate this possibility Takamatsu et al (2006) conducted molecular phylogenetic analyses of South American Golovinomyces species. However the results indicated that the Golovinomyces species of South America were imported from the northern hemisphere after the
Graduate School of Bioresources, Mie University, 1577 Kurima-Machiya, Tsu 514-8507, Japan
Maria Havrylenko Department of Botany, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, San Carlos de Bariloche, Rio Negro, Argentina
Abstract: Three powdery mildew species present on Nothofagus (viz. Erysiphe magellanica, E. nothofagi and E. patagoniaca) are endemic to South America and have unique ascomatal appendages that are not found in powdery mildews of the northern hemisphere. We determined the nucleotide sequences of the rDNA internal transcribed spacer regions and D1/D2 domains of the 28S rDNA of these three powdery mildew species to reveal their phylogenetic relationships with powdery mildews of the northern hemisphere. Although the molecular phylogenetic analyses indicated that the three Nothofagus powdery mildews are closely related to each other they did not group into one clade in either the ITS or 28S trees. Kishino-Hasegawa, Shimodaira-Hasegawa and Templeton tests could not significantly reject the constrained trees that were constructed based on the assumption that the Nothofagus powdery mildews would form a single clade. Based on this result and the evidence that all Nothofagus powdery mildews are endemic to South America and have similar morphological characteristics, it is likely that these three species diverged from a single ancestor present on Nothofagus. Calibration of evolutionary events with molecular clocks suggested that the Nothofagus powdery mildews split from the northern hemisphere relatives 22–16 million y ago (Ma) in the middle Miocene, and divergence among the Nothofagus powdery mildews occurred 17–13 Ma. These results do not support a southern hemisphere base of the Nothofagus powdery mildews. Key words: Erysiphales, Erysiphe, internal transcribed spacer, molecular clock, molecular phylogeny, ribosomal DNA
Accepted for publication 10 June 2008. 1 Corresponding author. E-mail:
[email protected]
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Asteraceae expanded their distribution into the northern hemisphere. Genus Nothofagus, the southern beech, is one of the most interesting taxa among the Gondwana floristic elements. About 37 extant species of Nothofagus are distributed only in the southern hemisphere, including South America, Australia, New Zealand, New Guinea and New Caledonia (Setoguchi et al 1997, Swenson et al 2001). Nothofagus species traditionally have been classified into the Fagoideae of Fagaceae. However recent molecular phylogenetic analyses revealed the distant relationship of Nothofagus and the Fagaceae. Three powdery mildew species (viz. Erysiphe magellanica, E. nothofagi and E. patagoniaca) have been reported on Nothofagus in Argentina and Chile (FIGS. 1–3; Thaxter 1910, Havrylenko 1997, Havrylenko and Takamatsu 2003). All three of these Erysiphe species are endemic to South America and belong to section Uncinula based on the presence of ascomatal appendages with uncinate to circinate tips. Appendages of E. nothofagi and E. patagoniaca are coiled helically many times at the upper half of or throughout appendages, which is a unique characteristic of these fungi. Appendages of Uncinula with uncinate to circinate tips are known to be the most ancestral characteristic among powdery mildews (Mori et al 2000a). Why do these Erysiphe species with ancestral characters occur on Nothofagus, which are the most famous Gondwanan plants? To address this question we determined the nucleotide sequences of the rDNA internal transcribed spacer (ITS) regions and D1/D2 domains of the 28S rDNA for these three powdery mildew species of Nothofagus to reveal their phylogenetic relationships with powdery mildews of the northern hemisphere. In addition we considered the possibility of a southern hemisphere base of these Nothofagus powdery mildews. MATERIALS AND METHODS
DNA extraction and amplification.—Sources of the powdery mildew specimens used for molecular analyses and the database accession numbers of their DNA sequences are provided (TABLE I). Whole-cell DNA was isolated from chasmothecia or mycelia with the chelex method (Walsh et al 1991, Hirata and Takamatsu 1996). The ITS region including 5.8S rDNA and the 59 end of 28S rDNA, which includes the variable domains D1 and D2, were amplified separately by two sequential PCR reactions with partially nested primer sets. PCR reactions were conducted with TaKaRa Taq DNA polymerase (TaKaRa, Tokyo, Japan) in a TP-400 thermal cycler (TaKaRa) under these thermal cycling conditions: an initial denaturation step for 2 min at 95 C, 30 cycles of 30 s at 95 C, followed by 30 s at 52 C for annealing, and 30 s at 72 C for extension, and a final
FIGS. 1–3. Ascomata of the powdery mildew fungi of Nothofagus. 1. Erysiphe magellanica (MUMH 2493). 2. Erysiphe nothofagi (MUMH 2486). 3. Erysiphe patagoniaca (MUMH 2508). Bars 5 100 mm. extension for 7 min at 72 C. A negative control that lacked template DNA was included in each set of reactions. PCR products were subjected to electrophoresis in a 1.5% agarose gel in TAE buffer, excised from the ethidium bromide-stained gel, and purified with the JETSORB Kit (Genomed, Oeynhausen, Germany) according to the
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FIG. 4. Phylogenetic analysis of the divergent domains D1 and D2 sequences of the 28S rDNA for 58 sequences from genus Erysiphe including Nothofagus powdery mildews. The tree is a phylogram of the tree with the highest likelihood score among the 158 most parsimonious trees with 326 steps and was obtained by a heuristic search employing the random stepwise addition option of PAUP*. Gaps were treated as missing data. Horizontal branch lengths are proportional to the number of
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TABLE I. Sources of powdery mildew fungi on Nothofagus used for molecular analyses and DNA database accession numbers Fungal species
Host
Location
Date
Erysiphe
Nothofagus
magellanica magellanica magellanica magellanica magellanica magellanica nothofagi nothofagi
antarctica antarctica antarctica antarctica antarctica antarctica alpina antarctica
Lago Lacar, Neuquen, Argentina Lago Curruhue, Neuquen, Argentina Villa La Angostura, Rio Negro, Argentina Villa La Angostura, Rio Negro, Argentina Lago Mascardi, Rio Negro, Argentina Pampa Linda, Rio Negro, Argentina Lago Lacar, Neuquen, Argentina Isla Fuego, Argentina
25 APR 2001 26 APR 2001 3 MAR 2004 3 MAR 2004 6 MAR 2004 6 MAR 2004 25 APR 2001 24 MAR 1996
nothofagi nothofagi patagoniaca
obliqua pumilio 3 antarctica
Lago Lacar, Neuquen, Argentina Devil Throut, Rio Negro, Argentina Espejo Lago Chico, Neuquen, Argentina
2 MAR 2004 6 MAR 2004 27 APR 2001
patagoniaca patagoniaca
pumilio pumilio
Devil Throut, Rio Negro, Argentina Devil Throut, Rio Negro, Argentina
6 MAR 2004 6 MAR 2004
Voucher No.a Accession No.b
MUMH 1472 MUMH 1473 MUMH 2493 MUMH 2494 MUMH 2496 MUMH 2516 MUMH 1471 BCRU 3868 MUMH 1475 MUMH 2486 MUMH 2511 BCRU 4337 MUMH 1474 MUMH 2508 MUMH 2511
AB378738 AB378739 AB378742 AB378743 AB378744 AB378748 AB378737 AB378736 AB378741 AB378746 AB378740 AB378745 AB378747
a
Sources: BCRU, Institutional Herbarium of Centro Regional Universitario Bariloche, San Carlos de Bariloche, Argentina; MUMH, Mie University, Mycological Herbarium, Japan. b DDBJ, EMBL, and GenBank database accession number of the nucleotide sequence data. manufacturer’s protocol. Nucleotide sequences of the PCR products were obtained for both strands with direct sequencing in a DNA sequencer CEQ2000XL (Beckman Coulter, Fullerton, California). The sequence reactions were conducted with the CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter) according to the manufacturer’s instructions. For amplification of the ITS region primers ITS5 (White et al 1990) and P3 (Kusaba and Tsuge 1995) were used for the first amplification. One microliter of the first reaction mixture was used for the second amplification along with the partially nested primer sets ITS5 and ITS4 (White et al 1990). The ITS5/ITS4 fragment was subjected to cyclesequencing with primers ITS1, ITS4, T3 and T4 (Hirata and Takamatsu 1996). For amplification of 28S rDNA primers PM3 (Takamatsu and Kano 2001), and TW14 (Mori et al 2000a), and NL1 (Mori et al 2000a) and TW14 were used respectively for the first and second amplifications. Primers NL1, NL2, NL3 (Mori et al 2000a) and NLP2 were used for cycle sequencing. Phylogenetic analysis.—Sequences were aligned initially with the Clustal 3 package (Thompson et al 1997). The alignment was visually refined with a word processing program using color-coded nucleotides. The alignments were deposited in TreeBASE (http://www.treebase.org/) under accession number S2123. Phylogenetic trees were obtained from the data with maximum parsimony (MP) in PAUP* 4.0 (Swofford 2001) and Bayesian analysis in
MrBayes 3.1.1 (Ronquist and Huelsenbeck 2003). MP analyses were performed with the heuristic search option using the tree bisection reconstruction (TBR) algorithm with 100 random sequence additions to find the global optimum tree. All sites were treated as unordered and unweighted, with gaps treated as missing data. The strength of the internal branches of the resulting trees was tested with bootstrap (BS) analyses using 1000 replications with the stepwise addition option set as simple (Felsenstein 1985). BS values higher than 70% were shown. For Bayesian phylogenetic analyses, the best-fit evolutionary model was determined for each dataset by comparing different evolutionary models via the Akaike information criterion (AIC) using PAUP* and MrModeltest 2.2 (Nylander 2004). MrBayes was launched with random starting trees for 1000 000 generations and Markov chains were sampled every 100 generations, resulting in 10 000 sampled trees. To ensure that the Markov chain did not become trapped in local optima, we used the MCMCMC algorithm, performing the estimation with four incrementally heated Markov chains. Of the resulting 10 000 trees the first 2000 (burn-in) were discarded. The remaining 8000 trees were summarized in a majority rule consensus tree, yielding the probability of each clade being monophyletic. Bayesian posterior probability (PP) values higher than 0.9 were shown. The Kishino-Hasegawa (KH) (Kishino and Hasegawa 1989) and Shimodaira-Hasegawa (SH) (Shimodaira and Hasegawa 1999) likelihood tests and the Templeton test
r nucleotide substitutions that were inferred to have occurred along a particular branch of the tree. Percentage bootstrap support (1000 replications; $ 70%) and the Bayesian posterior probability value ($ 0.90) are shown on and under the branches, respectively.
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TABLE II.
Kishino-Hasegawa, Shimodaira-Hasegawa and Templeton tests
Data
Constrained tree
Parsimony tree length (step)
28S
Best unconstrained tree (FIG. 4) Nothofagus PM monophyletic Best unconstrained tree (FIG. 5) Nothofagus PM monophyletic
326 327 671 674
ITS a b
Difference 2Ln L
2Ln La
3208.1373 3210.2068 4147.5086 4161.4407
(best) 2.0695 (best) 13.9321
P KH-test/SH-test Templeton test
Significantly worse?b
0.414/0.414
0.706
no
0.190/0.190
0.664
no
Difference in log-likelihood compared to that of the best tree. The constrained tree is considered to be significantly worse if the P-value is lower than 0.05.
(Templeton 1983) were conducted with PAUP* to compare the best tree topology obtained using the nucleotide sequence data with a constrained tree. Constrained tree topology was constructed based on the hypothesis that the three Erysiphe species group into a clade with MacClade ver. 4.05 (Maddison and Maddison 2002). Then the most parsimonious tree consistent with the constrained tree was found with the heuristic search in PAUP*. We then calculated the likelihood of obtaining our data given the most parsimonious tree or the constrained tree using PAUP*. The test distribution option was set as RELL, and one-tailed tests were conducted. The HKY85 model (Hasegawa et al 1985) was used as the substitution model for the calculation. A likelihood ratio test was performed to assess whether the molecular clock hypothesis was applicable to the 28S rDNA and ITS datasets. RESULTS
28S phylogeny.—A total of 58 sequences of the 28S rDNA, including 11 sequences from Nothofagus powdery mildews, were used to construct the phylogenetic tree of genus Erysiphe (including Brasiliomyces trina and Typhulochaeta japonica). Erysiphe australiana was used as outgroup taxon based on Mori et al (2000a). The dataset consisted of 820 characters, of which 142 were variable and 98 were phylogenetically informative for parsimony analysis. A total of 158 equally MP trees with 326 steps (CI 5 0.5276, RI 5 0.8236, RC 5 0.4345) were constructed by MP analysis. The tree with the highest likelihood score among the 158 MP trees is provided (FIG. 2). Most internal branches are supported in the strict consensus of the 158 trees. Bayesian analysis generated a similar tree topology. Five sequences from E. magellanica formed a clade with strong supports (BS 5 100%, PP 5 1.0). Each of the three sequences from E. nothofagi and E. patagoniaca grouped together into a clade (BS 5 98%, PP 5 1.0). The two species formed respective separate subclades, although they were not supported by BS and PP values. The three Erysiphe species of Nothofagus were related closely but did not form a clade. However KH, SH and Templeton tests could not reject a constrained tree
constructed based on the assumption that the Nothofagus powdery mildews would form a single clade (TABLE II). Erysiphe gracilis, E. mori, E. simulans, Typhulochaeta japonica and Brasiliomyces trina formed a clade with the Nothofagus powdery mildews with strong supports (BS 5 74%, PP 5 1.0). ITS phylogeny.—A total of 44 sequences of the ITS region, including 13 sequences from Nothofagus powdery mildews, were used to construct the phylogenetic tree of genus Erysiphe, including Brasiliomyces trina and Typhulochaeta japonica. Erysiphe australiana was used as outgroup taxon based on Mori et al (2000a). The dataset consisted of 712 characters, of which 212 were removed from the analysis due to ambiguous alignment. Of the remaining 500 characters, 231 were variable and 170 were phylogenetically informative for parsimony analysis. A total of 324 equally MP trees with 671 steps (CI 5 0.5410, RI 5 0.7753, RC 5 0.4194) were constructed by MP analysis. The tree with the highest likelihood score among the 324 MP trees is provided (FIG. 5). Most internal branches are supported in the strict consensus of the 324 trees. Bayesian analysis generated a similar tree topology. The ITS tree was almost consistent with the topology of the 28S tree. Six sequences from E. magellanica formed a clade with strong supports (BS 5 100%, PP 5 1.0). Seven sequences from E. nothofagi and E. patagoniaca grouped together into a clade (BS 5 99%, PP 5 1.0). Erysiphe patagoniaca formed a subclade (BS 5 70%, PP 5 0.84), and E. nothofagi was paraphyletic to E. patagoniaca. The three Erysiphe species of Nothofagus were closely related but did not form a clade. However KH, SH and Templeton tests could not reject a constrained tree based on the assumption that the Nothofagus powdery mildews would form a single clade (TABLE II). Erysiphe flexuosa, E. gracilis, E. mori, E. prunastri, E. simulans, E. wadae, Typhulochaeta japonica and Brasiliomyces trina formed a clade with the Nothofagus powdery mildews with strong supports (BS 5 94%, PP 5 1.0). Dating of evolutionary events.—We used a molecular
NIINOMI ET AL: NOTHOFAGUS POWDERY MILDEWS clock to estimate the timing of divergence of the Nothofagus powdery mildews. At first the 28S rDNA dataset was used for a likelihood ratio test to check whether a molecular clock hypothesis was applicable. Because the likelihood ratio test rejected the molecular clock hypothesis for the dataset 31 sequences were removed from the dataset. This reduced dataset comprising 27 28S rDNA sequences was not rejected by the likelihood ratio test and was used to construct a tree by the unweighted pair-group method with arithmetic averages (UPGMA) using PAUP*. Kimura’s two-parameter model (Kimura 1980) was applied for calculation of pairwise genetic distances. The molecular clock (6.5 3 10210 substitutions per site year21) reported by Takamatsu and Matsuda (2004) was used for the calculation. In the UPGMA tree the three Erysiphe species of Nothofagus together formed a clade (FIG. 6). They split from the sister taxa of the northern hemisphere 16.5 Ma in the middle Miocene, and the first split within the Nothofagus powdery mildews occurred 13.5 Ma. Because the ITS dataset was too variable to align unambiguously we constructed a reduced dataset consisting of 22 closely related sequences. The reduced dataset was not rejected by the likelihood ratio test and was used to construct a UPGMA tree (FIG. 7). The molecular clock (2.52 3 1029 substitutions per site year21) reported by Takamatsu and Matsuda (2004) was used for the calculation. Together the three Erysiphe species of Nothofagus again formed a clade in the ITS tree. They split from the sister taxa of the northern hemisphere 22.2 Ma, and the first split within the Nothofagus powdery mildews occurred 16.6 Ma. DISCUSSION
The present molecular phylogenetic analyses indicated that the three Erysiphe species of Nothofagus each represent a different group delineated by morphological characteristics. Of the three species E. nothogfagi and E. patagoniaca, which share appendages with spiral coils (Thaxter 1910, Havrylenko 1997, Havrylenko and Takamatsu 2003), also are related closely in phylogeny and together formed a clade with strong supports (BS 5 98–99%, PP 5 1.0) in both the ITS and 28S rDNA trees. Erysiphe magellanica, with simply uncinulate appendages, formed a separate clade. Thus morphological similarity is consistent with phylogenetic relationships among the three powdery mildew species of Nothofagus. Although the present molecular phylogenetic analyses indicated that the three Nothofagus powdery mildews are closely related, they did not group into a single clade in either the ITS or 28S trees. However none of the three statistical tests could reject the constrained trees based on the
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assumption that Nothofagus powdery mildews would form a single clade. Based on these results and the evidence that all Nothofagus powdery mildews are endemic to South America and have similar morphological characteristics except appendages, it is likely that these three species diverged from a single ancestor on Nothofagus. Genus Erysiphe consists of three sections, Erysiphe, Microsphaera and Uncinula, which were delimited by the morphology of appendages. Sections Microsphaera (appendages with dichotomously branched tips) and Uncinula (appendages with uninate to circinate tips) formed each separate group, whereas section Erysiphe (with mycelioid appendages) are polyphyletic and scattered among the groups of sections Microsphaera and Uncinula. In addition Typhulochaeta japonica and Brasiliomyces trina also were situated within the Uncinula/Erysiphe lineage. Thus the three sections of genus Erysiphe delimited by morphological characteristics do not correspond completely with the phylogenetic unit. All Nothofagus powdery mildews belong to the Uncinula/Erysiphe lineage in the phylogenetic tree. Although the spirally coiling appendage of E. nothofagi and E. patagoniaca is a unique characteristic that is never seen in species of the northern hemisphere molecular analyses indicated that the Nothofagus powdery mildews are closely related to some species of the northern hemisphere (viz. E. simulans and E. prunastri from Rosaceae, E. mori from Moraceae, E. wadae, E. gracilis, Typhulochaeta japonica and Brasiliomyces trina from Fagaceae, and E. flexuosa from Hippocastanaceae). These Erysiphe species formed a clade with the Nothofagus powdery mildews with strong supports (BS 5 74–94%, PP 5 1.0). Host families of the powdery mildews belong to the Rosales or Fagales in Eurosid I, a phylogenetic unit in the Rosid group, except for the Hippocastanaceae (APG II 2003). It is possible that these fungi achieved host expansion and genetic divergence on Rosales and Fagales of Eurosid I. Calibration of evolutionary events with molecular clocks of ITS and 28S rDNA D1/D2 domains suggests that the Nothofagus powdery mildews split from their northern hemisphere relatives 22–16 Ma in the middle Miocene. Geological and paleo-ontological data suggest that the final closure of the Isthmus of Panama took place about 3.1–2.8 Ma (Coates et al 1992, Coates and Obando 1996). Thus the molecular clock calibration suggests that the Nothofagus powdery mildews split from their northern hemisphere relatives earlier than the final closure of the Isthmus of Panama. Geological studies indicate that North America was connected to South America by an archipelago through the Central American isthmus during most of the Tertiary (Whitmore and Stewart
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FIG. 5. Phylogenetic analysis of the nucleotide sequences of the ITS region including the 5.8S rDNA for 44 sequences from genus Erysiphe including Nothofagus powdery mildews. The tree is a phylogram of the tree with the highest likelihood score among the 324 most parsimonious trees with 671 steps and was obtained by a heuristic search employing the random stepwise addition option of PAUP*. Gaps were treated as missing data. Horizontal branch lengths are proportional to the number of
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FIG. 6. Estimated dates of divergence of the Nothofagus powdery mildews based on the sequences of the divergent domains D1 and D2 sequences of the 28S rDNA and the nucleotide substitution rate of Erysiphaceae (6.5 3 10210 substitutions per site year21) reported by Takamatsu and Matsuda (2004). Ma, million years ago.
1965, Coates and Obando 1996, Coates et al 2004). The first exchange of terrestrial faunas between North and South America occurred 9.3–8.0 Ma (Coates et al 2004). Because conidia of the powdery mildews can be dispersed long distance by the wind (Yarwood 1957, Hermansen et al 1978) migration from North to South America might have been possible in the middle Miocene. Takamatsu et al (2006) reported that a South America endemic powdery mildew, Oidium reginae, split from its North American relative Golovinomyces adenophorae 10.6– 4.8 Ma. Such evidence suggests that the exchange of powdery mildew fungi was possible between North and South America in the middle Miocene and later. This study shows that the three Nothofagus powdery mildew species endemic to South America are closely related to powdery mildews of the northern hemi-
sphere based on phylogeny although they have unique morphological characteristics that are not found in the powdery mildew of the northern hemisphere. It is possible that an ancestral mildew species split from a northern hemisphere mildew in the middle Miocene, migrated from North America into South America and diverged into three species on Nothofagus. Nothofagus are distributed only in the southern hemisphere (viz. South America, Australia, New Zealand, New Guinea and New Caledonia). The occurrence of powdery mildew has been reported only in South America. One possible explanation is that the Nothofagus powdery mildews exist elsewhere but have not yet been found. However the flora of powdery mildews has been investigated in detail in Australia and New Zealand (Clare 1964, Hammett 1977, Boesewinkel 1979, Cunnington et al 2004a, b, 2005). It is unlikely that
r nucleotide substitutions that were inferred to have occurred along a particular branch of the tree. Percentage bootstrap support (1000 replications; $ 70%) and the Bayesian posterior probability value ($ 0.90) are shown on and under the branches, respectively.
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FIG. 7. Estimated dates of divergence of the Nothofagus powdery mildews based on the nucleotide sequences of the rDNA ITS region and the nucleotide substitution rate of Erysiphaceae (2.52 3 1029 substitutions per site year21) reported by Takamatsu and Matsuda (2004). Ma, million years ago.
the Nothofagus powdery mildews have not yet been found in these regions if the Nothofagus powdery mildews were present in Australia and New Zealand. Therefore an ancestral powdery mildew fungus might have acquired parasitism to Nothofagus in South America after the South American Nothofagus became isolated from those in other regions ,30 Ma (Swenson et al 2001). This assumption also is supported by the present phylogenetic analyses. Consequently our results do not support a southern hemisphere base of the Nothofagus powdery mildews.
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