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Department of Botany & Plant Pathology, Oregon State University, Corvallis, Oregon 97330
R.A. Shoemaker
Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada
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ABSTRACT |
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Phaeocryptopus gaeumannii, causal agent of the Douglas-fir foliar disease Swiss needle cast, is the only known pathogenic species of the genus. Current classifications place Phaeocryptopus in the Venturiaceae (Pleosporales), typified by the apple-scab pathogen Venturia inaequalis. All core members of this family have hyphomycetous anamorphs. We sought to confirm these relationships by means of phylogenetic analyses of the small (SSU) and large (LSU) subunits and internal transcribed spacer (ITS) region of nuclear ribosomal gene sequences (nrDNA). Analyses indicated that both the genus Phaeocryptopus and the family Venturiaceae, as currently defined, are unnatural groups. Phaeocryptopus nudus, type of the genus, is aligned in the Dothioraceae (Dothideales) and P. gaeumannii in the Mycosphaerellaceae (Capnodiales) near species of Mycosphaerella and Rasutoria. Core representatives of Venturiaceae formed an unambiguous clade but ordinal placement was unresolved. The family apparently is not included in the Pleosporales, Dothideales, Myriangiales or Capnodiales. Coelomycetous Rhizosphaera form-species are accepted generally as anamorphic states of Phaeocryptopus, however the relationship never has been established conclusively. Species of Rhizosphaera are closely related to P. nudus but not to P. gaeumannii, supporting an anamorphteleomorph connection between Rhizosphaera and Phaeocryptopus and providing further evidence that P. gaeumanii is not congeneric with P. nudus.
Key words: Capnodiales, Dothorioraceae, loculoascomycetes, Mycosphaerellaceae, nrDNA, phylogenetics, Swiss needle cast, Venturiaceae
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INTRODUCTION |
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as causing serious defoliation in Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) plantations in Europe. Although he was uncertain of the complicated taxonomy of the fungus, he stated "So far as the observations presented are concerned, our fungus...is an ascomycete belonging to the genus Adelopus, according to our knowledge, a single species is known in this genus, namely Adelopus balsamicola (Peck) Theiss". Theissen (1917) used the name Adelopus balsamicola because Saccardo (1915) considered Asterella nuda (Peck) Sacc. a synonym of Meliola balsamicola Peck (=Dimerosporium balsamicola [Peck] Ell. & Ev.) Consequently Asterina nuda Peck frequently has been confused in the literature with Meliola balsamicola Peck (now Dimerium balsamicola [Peck] Shoemaker [1965]). Hahn (1947) corrected the confusion concerning the two species, M. balsamicola and Asterina nuda, confirming that Peck had intended to erect the two distinctly different species in different genera. The combination Adelopus nudus (Peck) Theiss. resulted from a new combination attributed to Theissen by von Höhnel (1918) and perpetuated by Petrak (1925) and Rohde (1936, 1937). Peck (1885) originally described the fungus as Asterina nuda Peck on needles of Abies balsamea (L.) Mill. in New York. Because Peck’s description of this fungus did not match the description of Asterina Lév., Saccardo transferred it to Asterella nuda (Peck) Sacc. in 1891. Theissen (1914) determined that the fungus belonged neither in Asterina nor Asterella and proposed the new genus Cryptopus, typified by Cryptopus nudus (Peck) Theiss. Three years later Theissen and Sydow discovered that Cryptopus Lindl. previously had been erected as a member of the Orchidaceae and proposed Adelopus Theiss. (1917) as a replacement.
After studying new (not type) materials of Phaeocryptopus abietis Naumov (1915), Petrak (1938) determined that Adelopus nudus (Peck) Theiss. was identical to P. abietis Naumov, originally found on Abies sibirica in Perm, Ural. This resulted in the combination Phaeocryptopus nudus (Peck) Petrak. However both Hahn (1947), openly and Wilson and Waldie (1928) by implication questioned the synonymy. Petrak (1938) also considered the coelomycetous, mitosporic genus Rhizosphaera to be the anamorph of Phaeocryptopus. Although this connection was disputed by Rohde (1936), who presented experimental evidence to the contrary, Petrak dismissed Rohde’s observations, stressing ecological and morphological similarities. Rhizosphaera has been accepted generally as anamorphic Phaeocryptopus and is listed as such in the 8th and 9th editions of Ainsworth and Bisby’s Dictionary of the Fungi (Hawksworth et al 1995, Kirk et al 2001).
The causal agent of Swiss needle cast first was named Adelopus gaeumannii Rohde (Rohde 1936, 1937). Petrak (1938) proposed the new combination Phaeocryptopus gaeumannii (Rhode) Petrak. He included Adelopus balsamicola (Peck) Theiss. f. douglasii (Steiner 1937) as a synonym and did not accept Steiner’s belief that the fungus found on Pseudotsuga was merely a form of the species on Abies.
The first reference to familial placement of Phaeocryptopus was Theissen’s initial placement of ‘Cryptopus’ nudus in the Capnodiaceae (1914), but he later placed it in the Chaetothyriaceae as Adelopus nudus (1917). Petrak (1938) regarded both families as artificial and recommended their re-evaluation. Müller and von Arx (1950) erected Venturiaceae and included Phaeocryptopus within it. Barr (1968) retained Phaeocryptopus in Venturiaceae in a complete treatment of the family in North America. At that time she recognized 13 genera with 80 species, which she later modified slightly (Barr 1987b). The apple scab pathogen Venturia inaequalis was designated as type of the family. Because of the economic importance of apple scab disease, the pathology and epidemiology of V. inaequalis has been well studied. The putative familial relationship between V. inaequalis and P. gaeumannii has prompted suggestions that control measures for a new epidemic of Swiss needle cast might be adapted from apple scab management (Hansen et al 2000).
Traditional classifications have placed Venturiaceae in Pleosporales, a complex order characterized by J-, fissitunicate asci, asymmetric ascospores, asci in a basal layer, paraphysoids or pseudoparaphyses, and a pseudoparenchymatous peridium (Barr 1987b). Ascomycetous fungi with fissitunicately discharging asci that are borne in locules within ascostromatic ascomata (pseudothecia) have been variously grouped depending on which characters have been emphasized. Nannfeldt’s (1932) Ascoloculares, Luttrell’s (1951) Bitunicatae and Luttrell’s (1973) Loculoascomycetes have proposed different arrangements of 3–11 orders (Müller and von Arx 1962; Luttrell 1973; von Arx and Müller 1975; Barr 1979, 1987b). Von Arx and Müller (1975) placed all bitunicate ascomycetes in the Dothideales, in an admittedly unnatural classification.
Uncertainty regarding higher order classification of ascomycetes has led to abandonment of ranks above order (Hawksworth et al 1995) until relationships are better resolved. In the classification proposed by Eriksson (1982) class and subclass ranks were replaced by descriptive names of ascomatal types. New names based on type species more recently have been proposed for monophyletic groups recovered largely from phylogenetic analyses of DNA sequence data (Eriksson and Winka 1997, 1998; Eriksson 1999). The 11 core orders of loculoascomycetes currently are classified provisionally in Dothideomycetidae, although the limitations of molecular sequence data for this large and varied group are recognized (Kirk et al 2001). Single gene phylogentic analyses so far have failed to unequivocally establish relationships among the loculoascomycetes (Berbee 1996, Liew et al 2000, Lumbsch et al 2000, Reynolds 1998, Silva-Hanlin and Hanlin 1999, Winka et al 1998). Multigene approaches are proving more informative. Higher order relationships within the Dothideomycetes recently have been clarified by Schoch et al (2006), who recognized two subclasses and six orders, including monophyletic Pleosporales and Dothideales. Placement of Venturiaceae was unresolved because no exemplars of the family were included. Species of Venturia, Spilocea and Metacoleroa, genera traditionally classified in Venturiaceae, formed a monophyletic group in the analysis of Kruys et al (2006), based on nLSU, nSSU and mtSSU sequence data.
The purpose of this study was to use small (SSU) and large (LSU) subunits and internal transcribed spacer region (ITS) of nuclear ribosomal DNA (nrDNA) sequences to estimate phylogenies to test several hypotheses. Those explicitly tested are: (i) Phaeocryptopus nudus and P. gaeumannii are congeneric, (ii) P. gaeumannii is closely related to Venturia inaequalis, (iii) P. nudus is closely related to V. inaequalis, (iv) Rhizosphaera species are closely related to P. nudus, (v) Rhizosphaera species are closely related to P. gaeumannii. Important corollary observations not specifically tested included evaluating monophyly of the Venturiaceae and other groups of the loculoascomycetous fungi.
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MATERIALS AND METHODS |
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) were obtained either from culture collections or isolated from host tissue. Single-ascospore isolates were obtained for Apiosporina collinsii, Dibotryon morbosum, Metacoleroa dick-iei, P. gaeumannii, P. nudus, Rasutoria pseudotsugae, R. tsugae, Sthughesia juniperi and a Stomiopeltis sp. by suspending foliage bearing pseudothecia (attached to the underside of the Petri dish lids) above Petri dishes containing water agar (2%). Samples were incubated in a moist chamber at 17 C and individual ascospores removed from the agar surface with a heat-drawn Pasteur pipette. Identical methods were used to obtain single-conidium isolates of Rhizosphaera oudemansii and R. pini. Cladosporium cladosporioides, Mycosphaerella fragariae, Venturia in-aequalis and V. pyrina were isolated from host tissue surface-sterilized in 95% ethanol for 60 s and 2% NaOCl for 5 min. Representatives of Mycosphaerella pini, P. gaeumannii, P. nudus, Platychora ulmi, Plowrightia abietis, Protoventuria barriae, Rhizosphaera kalkhoffii, Venturia asperata, V. in-aequalis, V. pyrina and Xenomeris raetica were obtained from the American Type Culture Collection (ATCC) or Centraalbureau voor Schimmelcultures (CBS). Additional information regarding strain identification number and location is available on GenBank under the accession numbers provided (TABLE I). All fungal cultures were maintained on potato-dextrose agar (Difco Laboratories, Detroit, Michigan).
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PCR was performed in 50 µL reactions (1x enzyme buffer, 200 µM dNTP, 0.4 µM of each PCR primer, 2.5 U RedTaq DNA polymerase (Sigma, St Louis, Missouri), and 1 µL template DNA). Genes that code for the SSU and ITS were amplified respectively with primer sets NS1-NS4 and ITS5-ITS4 (White et al 1990) and those that code for the LSU were amplified with LROR-LR5 (Vilgalys and Sun 1994). SSU reaction conditions were 35 cycles of denaturation at 94 C for 60 s, annealing at 52 C for 60 s and extension at 72 C for 60 s. LSU and ITS reaction conditions differed only by alteration of the annealing step to 50 C and 55 C, respectively. A negative control (no DNA) was included in each set of reactions. After amplification, PCR products were prepared for direct sequencing by precipitation in one-half volume of 9 M NH4OAc and two volumes isopropyl alcohol. Cycle sequencing was performed with dye-terminator chemistry on an ABI model 377 fluorescent sequencer (PE Applied Biosystems Inc., Foster City, California) at the Central Services Laboratory of the Center for Gene Research and Biotechnology at Oregon State University. Primers NS1, NS4, NS3 (White et al 1990) and SR7 (R. Vilgalys, 5'-GTTCAACTACGAGCTTTTTAA-3') were used to sequence both strands of SSU PCR products, LROR, LR3, and LR5 the LSU (Vilgalys and Sun 1994) and ITS5 and ITS4 for the ITS. Contigs were assembled and the overlapping sequences edited with the Staden package (Staden 1996). Gapped-BLAST (Altschul et al 1997) was used to check for contaminant sequences by comparison with GenBank accessions. Sequences identified by BLAST searches with high similarity to P. nudus, P. gaeumannii and V. inaequalis were downloaded and included in analyses.
Sequence alignment and phylogenetic analyses.— –
New SSU, LSU and ITS sequences determined in this study (TABLE I
) were aligned with those obtained from nucleotide sequence libraries by means of the multiple alignment program MUSCLE v3.6 (Edgar 2004). Gaps and ambiguously aligned positions for each of the three datasets were eliminated with the software program Gblocks 0.91b with minimum block length set at 3 (Castresana 2000). The SSU and LSU datasets then were combined with missing sequence portions coded as "N".
Parsimony analysis was performed with PAUP* 4.0b10 (Swofford 2003). Maximum parsimony (MP) trees were inferred from heuristic searches with 1000 random sequence additions with the MULPARS and TBR options specified. Support for clade stability was estimated from 1000 nonparametric bootstrap replicates (Felsenstein 1985) using 5 random sequence additions with TBR branch swapping. In separate analyses we constrained parsimony searches of the combined SSU+LSU to address whether the data could support these phylogenetic hypotheses: (i) monophyletic Phaeocryptopus, (ii) Phaeocryptopus gaeumanii included in Venturiaceae (as delimited by parsimony and Bayesian analyses), (iii) Phaeocryptopus nudus included in Venturiaceae, (iv) Rhizosphaera species and P. gaeumannii monophyletic, (v) Rhizosphaera species and P. nudus monophyletic. Only the nodes of interest were resolved in the five constraint trees which were subjected to heuristic searches at the above settings in PAUP*. The most parsimonious constrained and unconstrained trees were compared by means of the Templeton (1983) and winning-sites tests in PAUP*.
Bayesian analysis was performed with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001) with the following settings. The maximum likelihood model employed 6 substitution types ("nst = 6"), with base frequencies estimated from the data ("basefreq = estimate"). Rate variation across sites was modeled using a gamma distribution, with a proportion of sites being invariant (rates = "invgamma"). The Markov chain Monte Carlo search was run with 4 chains for 3 000 000 generations and trees were sampled every 100 generations. The program Tracer v1.3 (http://evolve.zoo.ox.ac.uk/software.html?id=tracer) was used to ensure that stationarity was achieved and determine the point of burn-in. The first 2000 trees were discarded as burn-in. Phylogenetic trees were visualized with the program TreeView (Page 1996). Clade support was considered strong with Bayesian posterior probabilities (PP) 
0.95 and parsimony nonparametric bootstrap proportions (BS) 70% (Lutzoni et al 2004). Support was considered moderate with either PP 0.95 or BS 70%.
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RESULTS |
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). Culture collection sequences of P. gaeumannii, P. nudus, V. inaequalis and V. pyrina each were identical to those obtained from cultures isolated independently in this study. We therefore assumed that these taxa were identified correctly and included the consensus sequence of each species in phylogenetic analyses. The initial SSU alignment with 50 sequences obtained from GenBank (TABLE I) included 1645 nucleotide positions and 73 taxa, including the Arthoniomycetes Dendrographa leucophaea f. minor and Roccella fuciformis as the designated outgroup. Gblocks selected 971 (59%) positions as conservative enough to satisfy assumptions of positional homology. The initial LSU alignment yielded a matrix 1125 positions, 768 (68%) of which were selected by Gblocks. The combined dataset consisted of 1739 characters. Of these 1246 were constant, 122 were variable but parsimony uninformative and 371 were parsimony informative, yielding 32 equally parsimonious trees (1618 steps, CI = 0.43, RI = 0.78, RC = 0.34). Results of the parsimony analysis were similar to the Bayesian analysis (FIG. 1). The mean log-likelihood of 56 000 trees from the two runs after stationarity was achieved was –11 201.
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Phaeocryptopus nudus, type of the genus, was moderately supported (PP = –0.94, BS = 91%) as belonging to an unresolved polytomy including Rhizosphaera form-species and Plowrightia abietis (Barr) Barr in the Dothioraceae. These sequences were similar and branch lengths among them short. The Templeton and winning sites tests rejected monophyly of Rhizosphaera spp. and P. gaeumannii (P < 0.0001) but could not reject monophyly of Rhizosphaera spp. and P. nudus (Templeton, 0.0606 
P 0.1282; winning sites, 0.0636 P 0.1849).
Neither P. nudus nor P. gaeumannii grouped with core members of Venturiaceae, and both Templeton and winning sites tests rejected their placement in Venturiaceae (P < 0.0001). Whereas placement of Venturiaceae was unresolved, strong support (PP = 1.0, BS = 100%) for a monophyletic Venturiaceae included the three species of Venturia, Protoventuria barriae, Metacoleroa dickiei, Apiosporina collinsii, and Dibotryon morbosum as expected. Also included in the Venturiaceae clade was Tyrannosorus pinicola, a dothi-deomycete with uncertain affiliation. Platychora ulmi, currently assigned to Venturiaceae, grouped with strong support (PP = 1.0, BS = 100%) with Didymella cucurbitacearum (uncertain classification) within a strongly supported clade of Pleosporales (PP = 1.0, BS = 100%).
ITS rDNA.— –
Of the 612 bp multiple sequence alignment, Gblocks selected 349 positions (57%) as conservative enough to satisfy positional homology. Of these characters 199 were constant, 40 were variable but parsimony-uninformative and 110 were parsimony-informative. Two MPTs were obtained (tree length = 518, CI = 0 47, RI = 0.5996). The mean log-likelihood of the 56 000 Bayesian trees sampled after burn-in was –2973. ITS sequences of both P. gaeumannii isolates were identical, therefore a consensus sequence was used for analyses. P. gaeumannii formed a strongly supported (PP = 0.98, BS = 79%) monophyletic clade with M. crystallina, M. irregularimosa, M. heimii, M. heimioides, and M. colombiensis (FIG. 2). ITS sequences of the two Rasutoria species, R. pseudotsugae and R. tsugae, were identical. The two Rasutoria species, Sthughesia juniperi, Xenomeris raetica, and P. gaeumannii all were supported strongly within the Mycosphaerella ingroup (PP = 1.00, BS = 100%).
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DISCUSSION |
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, Stone and Carroll 1985). But there are indications that some mycologists recognized this relationship early in the taxonomic history of the fungus. When Theissen (1914) transferred Asterina nuda to the new genus Adelopus, he placed it in Capnodiaceae. However in our analyses P. nudus appears more closely related to species in Dothioraceae and Dothideaceae. It is unclear what material was examined in Theissen’s and many other taxonomic revisions based on the type material. Indeed there has been much documented confusion over which of the two loculoascomycetous fungi present in Peck’s type specimen packet that he and several following mycologists actually described (see Rohde 1936, Petrak 1938, Hahn 1947).
An additional complication arose when Petrak (1938), in an influential revision, synonymized Adelopus and Phaeocryptopus but without examining the types for either. Petrak merged the two genera in spite of the reported presence of pseudoparaphyses in Phaeocryptopus (Naumov 1915) that are absent in Adelopus (Hahn 1947). Because descriptions of the two genera differ with respect to ascospore pigmentation and hamathecial elements, Hahn (1947) remained skeptical of this treatment, although it has been generally accepted. Hahn (1947) stated that he was unable to obtain the holotype of P. abietis. The Naumov holotype collection of P. abietis since has been located and examined and compared to the type collection of Asterina nuda Peck including a redescription and illustrations (Shoemaker 1965). Comparison of the holotype collections of Phaeocryptopus abietis Naumov and P. nudus (Peck) Petrak (
Aster-Asterina nuda Peck) revealed that they are morphologically indistinguishable and the name P. abietis is a synonym of P. nudus. Comparative morphological studies of P. nudus and P. gaeumannii will be reported later. The Naumov holotype material contains several fungi, noted on the specimen label, but P. abietis is clearly distinguishable. Pseudoparaphyses or interthecial threads (shown in an illustration in Naumov (1915)) are absent, and ascospores within asci are hyaline. The structure in the original Naumov (1915) illustration might be a filiform ascospore of an unrelated fungus.
The early literature also suggests that species of Rhizosphaera are the anamorphs of Phaeocryptopus because of morphological similarities between the fruiting structures and frequent cohabitation (Petrak 1925, 1938). However developmental studies have never confirmed this view (but see Stone and Carroll 1985 associating phialide-like cells with the occurrence of P. gaeumannii). The putative connection was a matter of dispute between Rohde (1937) and Petrak (1925, 1938). Detailed comparisons of developmental and culture characteristics between P. nudus, P. gaeumannii and two Rhizosphaera species (Rohde 1937) provided empirical evidence against the connection. In particular Rohde (1937) noted that the mycelia developing from single ascospore isolates of P. gaeumannii, which never formed conidia in vitro, could not be homologous to that of a Rhizosphaera species found in frequent association with P. gaeumannii that always produced conidia in culture. Petrak (1938) however dismissed Rohde’s observations and again asserted a connection between Rhizosphaera and Phaeocryptopus based on morphological similarities. Kobayashi (1967) was unable to verify the connection between R. pini and P. nudus but reported that in culture P. nudus was similar to an undescribed Rhizosphaera species and also noted that cultures of R. kalkhoffii were different from P. gaeumannii, as reported by Rohde (1937). Despite this uncertainty, Petrak’s opinion has prevailed. Rhizosphaera was accepted as anamorphic Phaeocryptopus in the 8th edition of the Dictionary of the Fungi (Hawksworth et al 1995).
Phylogenetic analyses reported in this paper now might help to resolve this matter. The SSU+LSU analyses confirm Rohde’s (1937) finding that P. gaeumannii and Rhizosphaera are unrelated. At the same time our results support a close relationship between P. nudus and the three Rhizosphaera species examined. Explicit tests could not reject monophyly of P. nudus and the three Rhizosphaera species. Sequences of all four species were similar at both loci, displaying only two adjacent polymorphic regions in the SSU and two distant regions in the LSU. Rhizosphaera species produce Hormonema synana-morphs in culture (Funk 1985, Butin and Kehr 2000), and Hormonema is well established as anamorphic Dothioraceae (de Hoog et al 1999), in agreement with the placement of Rhizosphaera species in the SSU+LSU analyses. Of interest, sequences of Plowrightia (=Xenomeris) abietis, another Abies parasite but growing on twigs, also were similar to the P. nudus and Rhizosphaera sequences and were identical to R. oudemansii. Plowrightia also produces a Hormonema anamorph in culture (Funk 1981, Hermanides-Nijhof 1977) also in agreement with its placement in the SSU+LSU analyses as a member of Dothidiales and separate from X. raetica, currently classified in Venturiaceae, but grouping with Capnodiales in our analysis.
Regardless of the taxonomic and nomenclatural difficulties, neither of the fungi included in this study presently assigned to the genus Phaeocryptopus belong to Venturiaceae. The fungus that causes Swiss needle cast, identified here as P. gaeumannii, is placed unambiguously as a member of the Capnodiales based on SSU+LSU analysis. Based on these and two additional loci, Schoch et al (2006) provided evidence for a monophyletic Mycosphaerellaceae in Capnodiales. In our analysis P. gaeumannii groups unambiguously within this family. Morphological evidence, such as the lack of a hamathecium and the presence of superficial mycelium, further supports this placement. On the other hand P. nudus is much more closely allied with Dothideales, probably in Dothioraceae, although a more extensive sampling of other members of the family, particularly the type, would augment this argument considerably.
The ITS analysis indicates that P. gaeumannii, together with the two Rasutoria species examined here, R. pseudotsugae and R. tsugae, are incorrectly classified in separate genera and also group within Mycosphaerella. Phaeocryptopus and Rasutoria both are characterized by having separate, globose, thin-walled pseudothecia, ovoid to cylindrical, bitunicate, eight-spored asci, no pseudoparaphyses, and hyaline to pale brown, equatorially euseptate, fusoid to obovate ascospores. These characters also are shared by Mycosphaerella. Rasutoria was introduced by Barr (1987a) for Dimerosporium abietis Dearness, characterized as being hypophyllous on conifer needles and having setose ascocarps, superficial on a radiating mycelium that penetrates at the stomata. Phaeocryptopus gaeumannii ascocarps are smooth, emerging from Douglas-fir needle stomata and remaining attached to internal, intercellular mycelia by a cluster of basal hyphae in the substomatal chamber. A superficial, radiating mycelium also emerges from developing ascocarps, spreading across the needle surface and reentering the needle through unoccupied stomata (Stone and Carroll 1985, Stone et al 2007).
Barr (1987a) proposed Sthughesia typified by St. juniperi ( Xenomeris juniperi [Dearn.] M.E. Barr & E. Müll. Dimerium juniperi Dearn.) in Metacapnodiaceae of Capnodiales. Eriksson and Hawksworth (1988) considered Sthughesia to be a probable but questionable synonym of Metacapnodium Speg. In most current classifications Xenomeris is placed in Venturiaceae of Pleosporales (e.g. Eriksson 2006). With the proviso that we do not have data from the type species of Metacapnodium or Xenomeris Sydow, we found in the SSU+LSU analysis that Sthughesia juniperi occurred in a sister relationship with Trimmatostoma abietis, a dematiaceous, hyphomycetous anamorph of uncertain affiliation, and separate from Xenomeris raetica (E. Müll.) Petr. on a node with moderate support (FIG. 1). The placement of Sthughesia in the LSU/SSU analysis appears to support Barr’s (1987a) opinion that X. raetica and St. juniperi are not congeneric, as well as her placement of the genus in Capnodiales. The two species also grouped together in the ITS analysis, however with no support. Stomiopeltis sp. of Micropeltidaceae with uncertain ordinal placement (Eriksson 2006), St. juniperi and X. raetica in the SSU+LSU analyses presented here were all in a sister group of Mycosphaerellaceae within Capnodiales. In the ITS analysis, St. juniperi and X. raetica also grouped together within Mycosphaerella. Thus the combination of morphological characters represented by species found to group within Mycosphaerella suggests that the genus is much more diverse than hitherto recognized. The LSU analysis of Batzer et al (2005) also grouped Stomiopeltis spp. within a clade of Mycosphaerella spp. and Mycosphaerella anamorphs. Transfer of all these species to Mycosphaerella is anticipated but will entail some modification of the genus to incorporate these characters and so will be considered later.
In the ITS analysis P. gaeumannii occurred in a clade with several pathogenic Mycosphaerella species known to produce red crystals in culture (Crous et al 2000, Crous 2001). This well supported clade also grouped near other species which produce diffusing reddish pigments that have been connected to the polyketide phytotoxins cercosporin and dothistromin (Goodwin et al 2001, Bradshaw 2004). These include the cercosporin-producing Cercospora nicotianae and Mycosphaerella pini, another conifer foliar pathogen that produces the similar phytotoxin dothistromin (Bradshaw 2004) (FIG. 2). Isolates of P. gaeumannii also have been observed to produce diffusing red pigments in culture (Winton and Stone unpubl.). In view of the phylogenetic placement of P. gaeumannii within this group, the possibility that this pigment might be a cercosporin-like substance that might play a role in the pathology of Swiss needle cast disease must be considered.
This study has added to emerging concepts of higher level loculoascomycete systematics for several groups. Venturiaceae, as presently defined (i.e. Kirk et al 2001), is polyphyletic. Kruys et al (2006) presented evidence for a monophyletic Venturiaceae, including two species, V. inaequalis and Metacoleroa dickiei, also included in our analysis. Five additional species sequenced in this study, Apiosporina collinsii, Dibotryon morbosum, Proto-venturia barriae, V. asperata and V. pyrina, in addition to Tyrannosorus pinicola (from Schoch et al 2006) formed a strongly supported monophyletic group. However the ordinal placement of these core members of the family remains uncertain, grouping consistently with neither Pleosporales nor Dothideales. Ordinal placement of Venturiaceae also was unresolved in Kruys et al (2006). Furthermore several genera that have been classified traditionally in Venturiaceae, Phaeocryptopus, Platychora and Xenomeris clearly are not related to one another or to the core Venturiaceae clade and must be assigned elsewhere. The generic placement of Dibotryon morbosum (Schwein.) Theiss. & Syd. has been somewhat controversial. Von Arx transferred the species to Apiosporina (von Arx 1954). However Barr (1989) recommended that D. morbosum be retained in Dibotryon, based on superficial hyphae and ascoma shape. Based on the SSU+LSU analysis, separate generic placements of A. collinsii and D. morbosum appear to be justified. The sequences for the two species are more dissimilar than e.g. Venturia spp. Both species grouped in a strongly supported Venturiaceae clade.
Schoch et al (2006) provide multigene sequence evidence for recognition of Davidiellaceae and Mycosphaerellaceae as distinct families within Capnodiales, and our SSU+LSU analysis also supports this finding. There was strong support for Mycosphaerellaceae, including four Mycosphaerella species, P. gaeumannii, the two Rasutoria species, and Davidiellaceae, comprising Davidiella tassiana and Cladosporium cladosporioides. Whether a separate Euantennariaceae, as in classifications of Barr (1987b) and Luttrell (1951) in the Capnodiales should be retained is unclear and should be revisited with additional sampling of genera within the family. The results of this study also agree with those of Schoch et al (2006) that Dothioraceae and Dothideaceae form a monophyletic Dothideales which is a sister group of the Capnodiales. While this arrangement is supported by morphological characters, such as the absence of hamathecial tissue in both orders, inclusion of the species that are types for genera on which higher categories are based in future analyses would considerably bolster this placement.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Present address: USDA Agricultural Research Service, Subarctic Agricultural Research Unit, 303 O’Neil Building, University of Alaska at Fairbanks, Fairbanks, AK 99775. E-mail: lori.winton{at}uaf.edu
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LITERATURE CITED |
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Barr ME. 1968. The Venturiaceae in North America. Can J Bot 46:799–864.
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