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Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, K1A 0C6 Canada
A. Tsuneda
Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, and Northern Forestry Centre, Canadian Forest Service, Edmonton, Alberta, T6H 3S5 Canada
R. S. Currah
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
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ABSTRACT |
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Capnobotryella renispora and Scleroconidioma sphagnicola form black, irregularly shaped microsclerotia that are indistinguishable in gross morphology on leaves of Sphagnum fuscum. In culture, microsclerotia of these fungi were similar, in that mature component cells possessed thick, highly melanized cell walls, poorly defined organelles, large lipid bodies and simple septa. They were different in morphogenesis, in the way their component cells were organized and in disseminative propagules. Microsclerotia of S. sphagnicola formed phialidic conidiogenous cells on their surface, whereas in C. renispora, adjacent cells in mature microsclerotia often separated from each other by septum schizolysis and formed chlamydospores. The identification of C. renispora from Sphagnum is provisional despite a 100% ITS sequence match with data for a culture derived from the type strain. No holoblastic, reniform conidia typical of the species were formed in nature or in culture, and the SSU sequence for a separately preserved culture of the ex-type strain was markedly divergent. Parsimony analyses of nuclear ribosomal DNA sequences showed that these two fungi were related to separate orders of Dothideomycetes. Both SSU and ITS data supported a close relationship for S. sphagnicola to the Dothideales sensu stricto, while the closest ITS match was to Rhizosphaera spp. In the SSU analyses, C. renispora was nested within the Capnodiales.
Key words: black yeasts, Capnobotryella, Capnodiales, dematiaceous hyphomycetes, Dothideales, Dothideomycetes, meristematic ascomycetes, Scleroconidioma, ultrastructure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 2001b), and is a pathogen of Sp. fuscum (Tsuneda et al 2001a). The colony characteristics of the latter fungus were reminiscent of Phaeosclera dematioides Sigler, Tsuneda & Carmichael (Sigler et al 1981) and Capnobotryella renispora Sugiyama (Sugiyama and Amano 1987), dematiaceous hyphomycetes that also are cerebriform in culture. Phaeosclera dematioides is a nonsporulating taxon isolated from the pith of Pinus contorta Dougl. and is known only in vitro. Dematiaceous septate hyphae convert to thick-walled bulbil-like masses of sclerotic cells analagous to microsclerotia. Capnobotryella renispora is a sooty mold producing dark, moniliform, tapered hyphae that give rise to two-celled, reniform, blastic conidia directly from distal cells. Strains have been isolated from subicula of another sooty mold, Capnobotrys neesii Hughes, on Abies veitchii Lindl. (Sugiyama and Amano 1987) and also from roof tiles (Titze and de Hoog 1990). In the absence of more detailed morphological data, however, neither name could be applied with confidence to the cerebriform fungus.
The striking similarity between the microsclerotia of S. sphagnicola and the cerebriform fungus on Sphagnum prompted a comparative examination of the development of these structures. In addition, our inability to assign a name to the cerebriform fungus, or to suggest phylogenetic placement for both S. spagnicola and the cerebriform fungus, on the basis of morphology alone, led to a comparison of DNA sequence data derived from the nuclear ribosomal gene, specifically the small-subunit operon (SSU) and the internal transcribed spacer region (ITS). During these investigations, gene sequence comparisons indicated that the cerebriform fungus is identical to the ex-type culture of C. renispora, deposited at the Centraalbureau voor Schimmelcultures (CBS). Despite the lack of conidiation, this name is applied here to the strain of the cerebriform fungus isolated from Sphagnum. Because the development of the microsclerotia of S. sphagnicola has been reported elsewhere (Tsuneda et al 2000, 2001b), the emphasis of the developmental work reported here is on the strain of C. renispora from Sphagnum.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), while all strains of C. renispora were grown on potato-dextrose agar (PDA, Difco) or 2% malt-extract agar (MEA, Difco). Incubation was at 20 C in the dark. Methods for (cryogenic and ordinary) scanning electron and transmission electron microscopy (SEM and TEM, respectively), including methods for material preparation, were the same as those used in Tsuneda et al (2001a, b).
DNA sequencing –
DNA sequences of portions of the nuclear rRNA gene were determined for the five strains examined morphologically. Genomic DNA was extracted from mycelium grown on PDA using a FastDNA® Extraction Kit (BIO 101 Inc., Carlsbad, California) and a FastPrepTM Cell Disruptor machine (BIO 101, Carlsbad, California). DNA amplification and cycle sequencing reactions were performed on a Techne Genius thermocycler (Princeton, New Jersey). PCR reactions were performed in 25 (L volumes using Ready-To-GoTM PCR Beads (Amersham Pharmacia Biotech Inc., Piscataway, New Jersey) and 2 µL of template DNA. PCR cycling parameters included 30 cycles of denaturation at 95 C for 1.5 min, annealing at 56 C for 1 min and extension at 72 C for 2 min, with an initial denaturation of 4 min and a final extension step of 10 min. Primers NS1 and ITS4 (White et al 1990) were used to amplify more than 1700 basepairs of the small subunit (SSU) as well as the complete ITS region, including the ITS1, 5.8S and ITS2. Amplified products were purified using the WizardTM PCR Preps DNA Purification System (Promega Corp., Madison, Wisconsin), and DNA concentrations were estimated from fragments stained by ethidium bromide and separated by agarose gel electrophoresis.
Sequencing reactions were performed using the BigDyeTM Terminator Cycle Sequencing System (Applied Biosystems, Foster City, California) with the recommended cycling parameters. The reaction mixture comprised one-eighth strength BigDyeTM sequencing mix, an equal volume of the reaction booster halfBD (Bio/Can Scientific, Mississauga, Ontario, Canada), 0.5 µL sequencing primer (5(M); 20–50 ng purified DNA and sterile water to a final volume of 5 µL. Reactions were purified after bringing the volume to 20 (L, by ethanol/sodium acetate precipitation and resuspended as recommended for processing on an ABI PRISMTM 310 DNA sequencer (Applied Biosystems, Foster City, California). Sequencing primers were selected from the range of SSU and ITS primers given in White et al (1990) and Landvik et al (1997) to get sequence data for the whole region. Consensus sequences were determined from overlapping sequence data for both DNA strands using the software SequencherTM (Gene Codes Corp., Ann Arbor, Michigan).
Phylogenetic analysis –
The initial SSU data matrix included 86 small-subunit sequences retrieved from nucleotide sequence databases (GenBank; http://www.ncbi.nlm.nih.gov) and chosen to represent the phylogenetic diversity of Ascomycetes. The SSU and ITS data matrices used in the final analyses included accessions identified with Gapped-BLAST (Altschul et al 1997) as having a high sequence similarity to the two fungi under study and other taxa suspected of being closely related, based on preliminary analyses of the larger SSU dataset. Accession numbers for the GenBank sequences not listed in Table I are given on Figs. 26–28. Sequence alignments were generated with the PileUp option of GCG 10.1 (Genetics Computer Group, Wisconsin Package, Madison, Wisconsin; http:www.cbr.nrc.ca) and then optimized by eye with Se-Al (Sequence Alignment Program v1.d1; http://evolve.zoo.ox.ac.uk/software/Se-Al/main.html). Alignments were trimmed to remove the characters at each end, for which there was missing data because of minor differences in sequence length. The final ITS alignment included the complete ITS1, 5.8S and ITS2 regions. The data matrices were subjected to parsimony analysis using the heuristic search option of PAUP* version 4.0b8 (Swofford 1999) with stepwise addition of taxa, tree bisection-reconnection (TBR) branch swapping and gaps treated as missing data. Support for the branching topologies was evaluated by bootstrap analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. In CBS 214.90 and 215.90, conidium production in culture was either nil or scarce. Conidia, if present, were 4.5–6.5(–8)(3–4.5 µm in CBS 214.90 and 5–5.5 (4–5 µm in CBS 215.90, both shorter than Sugiyama's description of the species in culture, 7–10 (3–4 µm but similar to the measurements from material in vivo.
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) and were filled with large lipid bodies (Fig. 25). There was a distinct gap between the newly formed and older cell walls (Fig. 24, arrows), and cells possessing only the newly formed cell wall could be dislodged from their older cell walls upon fracturing (Figs. 23, arrows).
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The closest GenBank SSU sequence matches for S. sphagnicola were for taxa classified in the Dothideales and Capnodiales, while the closest ITS matches were to Rhizosphaera spp. with 96% similarity. The closest GenBank SSU matches for C. renispora were also to the Dothideomycetes, and our identical ITS sequences for UAMH 9870 and the ex-type strain, CBS 214.90 (IAM13014), were a 100% match for a published ITS sequence (AJ244238; de Hoog et al 1999) for CBS 214.90. Other ITS sequences retrieved in the BLAST search were for Trimmatostroma spp. and Mycosphaerella spp., but the matches were at 90% similarity or less.
Our identical SSU sequences for CBS 214.90, CBS 215.90 and UAMH 9870 unexpectedly differed from a published SSU sequence for CBS 214.90 (Y18698; Sterflinger et al 1999) at 17 nt positions (98% similarity) and were only 91% similar to a second SSU sequence (AF006723; Reynolds 1998) for this strain of C. renispora separately preserved as ATCC 64891 (ITS sequence data was not available for this culture). Because of the marked discrepancies among the sequences available for the ex-type strain of C. renispora, only the sequences determined from the CBS cultures obtained for and examined in this study were used in the analyses.
Phylogenetic analysis –
The initial SSU data matrix comprised 88 taxa and 1983 aligned characters. Of these, 182 characters corresponded to short, unalignable sequence motifs unique to individual sequences (mostly for outgroup taxa); these were treated as insertions relative to the rest of the alignment and were excluded from the analyses. Of the remaining 1801 characters, 1039 were constant, 235 were parsimony uninformative and 527 were parsimony informative. An analysis using the heuristic search algorithm, with 100 replicates of random addition sequence, resulted in 18 equally parsimonious trees (MPTs) of 2648 steps and with CI = 0.432, RI = 0.711, RC = 0.308 and HI = 0.568. A bootstrap analysis (1000 replicates) was performed using the "fast" stepwise-addition search option to determine the level of support for the topology of the inferred phylogeny. Fungi classified in seven of the 11 classes of the subphylum Pezizomycotina were included in the analysis, while representatives of the Taphrinomycotina and Saccharomycotina served as outgroup taxa. Classification follows Eriksson et al (2001).
The initial analyses confirmed the BLAST search results that both S. sphagnicola UAMH 9731 and C. renispora UAMH 9870 were related to species classified in the Dothideomycetes (Fig. 26). The monophyly of the class was supported in the strict consensus of all MPTs (data not shown). Both fungi were excluded from the other major clades in the tree, identified by class name on Fig. 26, by virtue of their high bootstrap values and/or the number of character state changes, as evidenced by branch length. While bootstrap support was less than 50% for the Dothideomycetes clade, several subclades received higher support. Group A (Fig. 26), bootstrap 96%, comprised species in five families of the Pleosporales, Mycosphaerella mycopappi (Mycosphaerellaceae, incertae sedis) and Rhytidhysteron rufulum (Patellariales) in a basal position. Within the group, several suprafamilial subgroupings also received high bootstrap support. Group B comprised representatives of the Dothideales, Capnodiales and Botryosphaeriaceae (incertae sedis). Scleroconidioma sphagnicola clustered with Dothidea insculpta U42474 (Dothideales) with moderate bootstrap support at 88%; Capnobotryella renispora grouped with Coccodinium bartschii U77668 (Capnodiales) with moderate support of 78% in a sister group to Capnodium citri AY016340.
The final SSU alignment included representatives of the Group A Dothideomycetes, Dothideales, Capnodiales and Dothioraceae (incertae sedis), as well as several dematiaceous hyphomycetes. Leotia lubrica L37536, L. viscosa AF113715, Microglossum viride U46031, Cudonia confusa Z30240 (Leotiomycetes), Peziza badia L37539, Morchella elata L37537 and Urnula hiemalis Z49754 (Pezizomycetes) served as the outgroup. The ITS alignment included the sequences available for species in the Dothideaceae and Dothioraceae and closely related anamorph genera. Based on the SSU analysis, Phaoesclera dematioides AJ244254 and Sarcinomyces crustaceous AJ244258 were chosen as outgroup taxa. Capnobotryella renispora was not included in the ITS analysis; ITS data could not be aligned with confidence to the sequence for S. sphagnicola or to those sequences retrieved from GenBank as a result of the BLAST search.
The final SSU data matrix comprised 38 taxa and 1652 aligned characters. Of these, 1285 were constant, 210 were parsimony uninformative and 157 were parsimony informative. An analysis using the heuristic search algorithm, with 100 replicates of random addition sequence, resulted in 15 MPTs of 651 steps and with CI = 0.662, RI = 0.700, RC = 0.463 and HI = 0.338. Results of a full bootstrap analysis (1000 replicates) using the heuristic search option are shown on one MPT (Fig. 27). Four sequences were short compared to the rest (Table I), but analysis of only that portion of the data matrix for which all sequences were complete did not alter the overall topology of the resulting tree (data not shown). The ITS alignment comprised 25 taxa and 569 aligned characters. Of these, 21 ambiguously aligned characters were excluded, 370 were constant, 69 were parsimony uninformative and 109 were parsimony informative. A branch and bound analysis resulted in 10 MPTs of 352 steps and with CI = 0.670, RI = 0.784, RC = 0.525 and HI = 0.330. Results of a full bootstrap analysis (1000 replicates) using the heuristic search option are shown on one MPT (Fig. 28).
Parsimony analysis of the final SSU alignment supported the placement of S. sphagnicola near the Dothideales but not within the order (Fig. 27). There was high bootstrap support (95%) for a monophyletic Dothideales sensu stricto, while S. sphagnicola clustered with Delphinella strobiligena (Dothioraceae) in an unsupported sister clade. Aureobasidium pullulans and its teleomorph Discosphaerina fagi (Dothioraceae) (Yurlova et al 1999), two identical sequences in the trimmed alignment, were in a basal position. Capnobotryella renispora UAMH 9870/CBS 214.90/215.90 grouped with an undescribed species of Capnobotryella within a large clade corresponding to the Capnodiales (Fig. 27). The clade included holomorphs in Capnodium, Scorias (Capnodiaceae) and Coccodinium (Coccodiniaceae) and the allied anamorph genera Antennariella (Antennulariaceae) and Chaetasbolisia (Metacapnodiaceae) but also the dematiaceous hyphomycete genera Hyphospora and Hortaea. Although the Capnodiales clade was retained in the strict consensus of all MPTs, in general, relationships within the order were unsupported by the bootstrap analysis, and terminal branch lengths were long, indicating a substantial genetic distance between species. The relationships of the other dematiaceous hyphomycetes included in the analysis, Phaeotheca, Phaeosclera and Sarcinomyces, were unresolved. Two species of Botryosphaeria clustered together with high bootstrap support, forming a sister clade with Sarcinomyces petricola to the well-supported clade comprising Group A Dothideomycete species.
In the ITS analysis, two major clades received high bootstrap support (Fig. 28). In the first (clade I), S. sphagnicola clustered with Rhizosphaera spp., as predicted by the BLAST search. This well-supported grouping was sister to a cluster of three groups corresponding to teleomorph genera in the Dothideaceae (Dothidea) and Dothioraceae (Dothiora and Sydowia). The coelomycetous genus Rhizosphaera has a putative teleomorph connection to Phaeocryptopus (Kirk et al 2001), yet Phaeocryptopus gaumannii (Rhode) Petrak (Venturiaceae) was excluded from the Rhizosphaera clade. Species of Hormonema clustered with Dothiora or Sydowia; both genera produce anamorphs in Hormonema (de Hoog et al 1999). Species of the anamorphic genus Kabatina were basal to the Sydowia and Dothidea clades but did not cluster together. The other major clade (II) comprised strains of the anamorphic black yeast Aureobasidium pullulans, [synonym Kabatiella lini (Lafferty) Karakulin] (Yurlova et al 1999).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Some of these fungi possess budding cells and have a close taxonomic affinity to the black yeasts of the Chaetothyriomycetes (Haase et al 1995), while others with a yeast-like phase are allied with the Dothideomycetes (Sterflinger et al 1999). Many black yeast taxa convert to meristematic growth under stressful conditions and therefore the distinction between "black yeasts" and "meristematic fungi" is not absolute (Sterflinger and Krumbein 1995, Sterflinger et al 1999, de Hoog et al 1999). As well, the term "black fungi" sometimes is used to embrace both groups of fungi (Sterflinger and Krumbein 1995). Most of these fungi occur in extreme environments, tolerating stresses caused by temperature, water availability, oxygenic action, irradiation by ultraviolet rays (UV), electrolyte content and/or scarcity of nutrients (Sterflinger et al 1999, de Hoog et al 1999). Epiphytic species occur in such genera as Aureobasidium, Coniosporium, Hormonema, Hyphospora, Phaeotheca, Sarcinomyces and Trimmatostroma (e.g., Butin et al 1995, DesRochers and Ouellette 1994, Sigler et al 1981, Sterflinger et al 1999, Yurlova et al 1999), fungi with diverse phylogenetic affinities.
Despite similarities in their morphology and habitat, molecular evidence indicates that meristematic fungi and black yeasts belong to at least three different orders, Chaetothyriales, Dothideales (sensu lato) and Pleosporales (Haase et al 1995, de Hoog et al 1999, Sterflinger et al 1999). Our parsimony analyses of small-subunit nuclear rDNA sequence data showed that S. sphagnicola and C. renispora phylogenetically were distinct from one another. Both were related to fungi classified in the Dothideomycetes but to different orders, specifically to the Dothideales and the Capnodiales, respectively, and distant from the Chaetothyriomycetes. These two dematiaceous fungi isolated from Sphagnum leaves also were distinct morphologically. Although they produced microsclerotia similar in gross morphology and simple septation (Figs. 9, 11) (Tsuneda et al 2001b), further examination showed that they differed in the mode of development, internal structure and disseminative propagules.
Morphological and ecological aspects –
In C. renispora, the dominant form of cell multiplication in microsclerotium formation is apical and polar, leading to the formation of acropetal chains of globose cells (moniliform hyphae; Fig. 4). Thus, growth is predominantly hyphal. Adjacent cells in mature microsclerotia separate by septum schizolysis. However in S. sphagnicola, growth is revealed primarily by isodiametric enlargement and subdivision of mother cells (Figs. 21–23) (Tsuneda et al 2001b), and the cells of mature microscerotia are cemented in a solid sclerotic mass. The similar, cauliflower-like macromorphology of the microsclerotia observed on the host, then, is derived from different developmental processes.
Diverse fungi, including some parasitic ones, occur on living or decomposing Sphagnum (Döbbeler 1978, 1986, Redhead 1981, Redhead and Spicer 1981, Thormann et al 2001, Tsuneda et al 2001a). Sphagnum cell walls are highly resistant to microbial degradation, and environmental conditions prevailing in Sphagnum bogs, i.e., wide fluctuations in temperature, moisture and radiation, could select for fungi producing dark pigments and having the ability to form sclerotia. It is apparent that the primary function of microsclerotia in both C. renispora and S. sphagnicola is survival on the host. Component cells of mature microsclerotia in both fungi contain a large amount of reserve material (lipid bodies) and possess thick cell walls that are densely covered and heavily impregnated with melanin granules. Further, their cell organelles are not well defined (Fig. 15) (Tsuneda et al 2001b), except in actively multiplying cells. These characteristics indicate that the microsclerotia remain in a resting state until environmental conditions become favorable for growth. Melanin appears to play an important role in stress tolerance because wall-bound melanin protects fungal cells from various stress factors, including physical (e.g., extreme temperatures and desiccation) and biological ones, such as lysis caused by cell-wall degrading enzymes released by other microorganisms (Butler et al 2001). In addition, melanin granules absorb X-, gamma and UV rays (Zhdanova et al 1973) and limit the leakage of certain metabolites (Butler et al 2001). These characteristics of melanin explain the higher incidence of melanized microbes occurring on rocks, leaf surfaces and other exposed locations (Ursi et al 1997).
Microsclerotia of S. sphagnicola function not only as survival structures but also as a reservoir of nutrients that sustains the production of conidiogenous cells and numerous conidia, when environmental conditions are favorable (Tsuneda et al 2001a, b). Dissemination of this fungus among Sphagnum stands thus is primarily by conidia. In contrast, conidia never were observed for the cerebriform fungus from Sphagnum, although the type strain of C. renispora produces holoblastic, reniform conidia, both in its natural habitat and in culture (Sugiyama and Amano 1987). Then how could C. renispora on Sphagnum disseminate itself in bogs? Figures 15–19 clearly show that many of the component cells of mature C. renispora microsclerotia separate by septum schizolysis. In this way, the microsclerotium could function as a mass of thick-walled, thallic spores (= chlamydospores). It is reasonable to assume that such chlamydospores can function to disperse the fungus when the microsclerotium is weathered and/or crushed by external force. The highly melanized outer cell walls become brittle as microsclerotia age, and thus moisture can be readily supplied to the internal cells. This characteristic enables microsclerotial cells to resume growth quickly in response to favorable environmental conditions.
A striking characteristic of both C. renispora and S. sphagnicola is that each component cell of a mature microsclerotium possesses a thin layer of newly formed cell wall (Fig. 15, arrow) (Tsuneda et al 2001b). That portion of the cell with the newly formed cell wall can be separable from the highly melanized, older cell wall (Figs. 16–18, 23, 24). In C. renispora, when re-growth takes place from these cells (= chlamydospores), the newly forming cell breaks out of the outer cell wall of the chlamydospore (Fig. 3, arrow). These features indicate that the outer cell wall is no longer an integral part of the "living" cell but serves as a physical barrier between the cell and its surroundings. The portion consisting of a protoplast and newly formed cell wall thus is analogous to an endoconidium. Endoconidiation occurs in such genera of black fungi as Aureobasidium, Coniosporium, Hyphospora, Phaeotheca, Sarcinomyces and Trimmatostroma (de Leo et al 1999, de Hoog and Hermanides-Nijhof 1977, Sigler et al 1981, Wollenzien et al 1997, Zalar et al 1999a, b). In Phaeotheca fissurella, whose microsclerotia resemble those of S. sphagnicola in ultrastructure, each cell with the newly formed cell wall subsequently is subdivided to form two to several endoconidia. These endoconidia are released by dissolution of mother-cell walls (Sigler et al 1981, Tsuneda and Murakami 1985). Microsclerotia of C. renispora and S. sphagnicola lack such an elaborate releasing mechanism.
Phylogenetic aspects –
The SSU and ITS analyses provided evidence of a close relationship between S. sphagnicola and the Dothideales sensu stricto, restricted to one family (Dothideaceae) in the Eriksson et al (2001) classification, and genera in the Dothioraceae. Based on the ITS sequences available in GenBank, its closest relatives are in Rhizosphaera, a coelomycete genus characteristically found on conifer needles functioning as a weak parasite (Butin and Kehr 2000). These genera are similar in the shape of conidiogenous cells and conidia, and in phialidic conidiogenesis. Conidiogenous cells of Rhizosphaera are produced within black pycnidia, and they form the single layer of cells of the conidiomatal wall (Sutton 1980), whereas those of Scleroconidioma form on the surface of dematiaceous microsclerotia (Fig. 23) (Tsuneda et al 2001b), giving the impression of a pycnidium of Rhizosphaera turned inside-out.
There was support from the ITS analysis for a more inclusive concept of the Dothideales, that would include the Dothioraceae (anamorphs in Hormonema), and the anamorph genus Kabatina, an acervular fungus that is Hormonema-like in pure culture (Hermanides-Nijhof 1977). Rhizosphaera and Scleroconidioma were sister taxa, while several strains of Aureobasidium, its synonym Kabatiella and by inference its teleomorph Discosphaerina (Fig. 27; Yurlova et al 1999) formed a separate well-supported monophyletic group. Strong support for such a wider concept, which also would include Aureobasidium, was provided by maximum-likelihood and neighbor-joining analyses of combined large and small-subunit data (Lumbsch and Lindemuth 2001) but not by SSU analysis alone (this study, Berbee 1996). Our analyses raised questions about phylogenetic relationships within the Venturiaceae. Although Rhizosphaera spp. are considered to be anamorphic Venturiaceae (Kirk et al 2001), Phaeocryptopus gaumannii is far removed in the ITS tree and, in the SSU analyses, Venturia liriodendri Hanlin is allied with the Pleosporales clade (data not shown, Silva-Hanlin and Hanlin 1999).
Our identical sequence data for UAMH 9870 and the ex-type strain of C. renispora (IAM 31014) preserved as CBS 214.90 strongly suggested conspecificity. Furthermore, our sequence data for a second strain used for the original description of the species (CBS 215.90, IAM 31015; Sugiyama and Amano 1987) also were identical, except for one nucleotide difference in the ITS sequence. The genus Capnobotryella (Metacapnodiaceae) clustered well within the Capnodiales in the SSU analysis, as did the two dematiaceous hyphomycetes, Hyphospora and Hortaea. In a study focused on the Capnodiales, Reynolds (1998) found that the taxa sampled formed a monophyletic group based on a neighbor-joining analysis of SSU sequences. Capnobotryella renispora and other allied anamorphic genera clustered with the teleomorph representatives but with low bootstrap support. The results of our SSU analyses, which incorporated the relevant sequences from the studies of Reynolds (1998) and Lumbsch and Lindemuth (2001), are in agreement.
Relationships among taxa within the Capnodiales clade (Fig. 27) were unresolved and difficult to interpret. Type species have not yet been sampled, two of the four families included were represented by anamorph taxa only, and there are other capnodiaceous families for which no sequence data are available. Two species of Capnodium (Capnodiaceae) clustered in different subclades as did two genera of the Metacapnodiaceae. The lack of bootstrap support for the clade and the genetic distance between taxa within the clade, as evidenced by the long terminal-branch lengths, suggest that the monophyly of the sooty mold order might not hold up with increased sampling. ITS sequences available for representatives of the Capnodiales and the other anamorph genera in the clade were highly divergent and could not be aligned with confidence to sequences of C. renispora.
The identification of the Sphagnum isolate remains provisional because it did not form the holoblastic, reniform conidia that characterize C. renispora and because three different SSU sequences have been determined for the ex-type strain (our data, Y18698 and AF006723). The morphological characters we observed for the CBS culture agreed with those in the original description of material from the host, although conidia were rare. We were confounded by the marked differences between the SSU sequences derived from the CBS and ATCC cultures. We were unable to examine the fungus preserved as ATCC 64891 for this study, but in our analyses all three SSU sequences cluster within the Capnodiales (data not shown) and therefore it is unlikely that the discrepancies are a result of culture contamination.
Two other possible explanations are that a sequencing error occurred or that two distinct sooty mold species have been preserved inadvertently as IAM 31014. Capnobotyrella renispora was isolated from the subicula of another sooty mold, Capnobotrys neesii, for which there are no cultures and no sequence data. Sooty molds are found superficially on living plants, and several taxa frequently grow together in apparent harmony in such a way that species are not readily distinguishable from each other on the host and specimens often comprise more than one species (Hughes 1976). In addition, species are highly pleomorphic, with up to three synanamorphs produced. Careful studies of more specimens are needed to clarify the extent of morphological and molecular variation exhibited by fungi in the form genus Capnobotryella. Conidiation among the strains examined ranged from prolific (CBS 572.89) to scarce or nil; the long terminal branch lengths for the three fungi included in the SSU analysis presented in Fig. 27 (C. renispora and Capnobotryella sp. nov.) suggest that genetic variation is substantial.
Other meristematic fungal genera allied with the Dothideomycetes include Aureobasidium, Botryomyces, Hormonema, Hortaea, Hyphospora, Phaeosclera, Phaeotheca and some species of Sarcinomyces (de Hoog et al 1999, Sterflinger et al 1999). Of these, only Botryomyces is related to the Pleosporales (de Hoog et al 1999) or the Group A Dothideomycetes of our analyses. The rest clustered with Group B taxa, as shown in Figs. 27 and 28. Prior molecular-based studies have examined relationships within the Dothideomycetes and have highlighted the division of the class into two groups, one consistently being the well-supported monophyletic Pleosporales (Berbee 1996, Silva-Hanlin and Hanlin 1999, Liew et al 2000, Lumbsch and Lindemuth 2001). In general, the groups correspond to the pseudoparaphysate taxa of the Pleosporales, including the Melanommatales, versus the aparaphysate taxa in the Dothideales and Capnodiales, thus Groups A and B of our analyses. Overall, the ascomycete phylogenetic hypotheses presented here concur with prior hypotheses of supra-ordinal taxonomic relationships (Tehler et al 2000, Eriksson et al 2001). They also reinforce the finding that relationships among Group B Dothideomycete taxa are not well resolved by parsimony analysis of larger datasets of small- subunit sequence data (Silva-Hanlin and Hanlin 1999).
Conclusions – Using rDNA sequence data, we have shown that two dematiaceous hyphomycetes occurring together on Sphagnum leaves are related to separate orders of ascostromatic ascomycetes, the Dothideales and Capnodiales. They are phylogenetically distinct from other taxa that exhibit meristematic growth patterns. Their classification remains inconclusive, pending the discovery of their teleomorphs and/or the availability of sequence data for a wider range of taxa from these two orders. Comparative microscopic examinations have elucidated fundamental morphological differences that corroborate the phylogenetic distinction. Scleroconidioma sphagnicola and C. renispora differ in the development and internal structure of superficially similar microsclerotia produced on the host and in culture and in disseminative propagules, which are predominantly phialoconidia and chlamydospores, respectively. Evidence is presented that the component cells of the mature microsclerotia are analogous to the endocondia produced by other genera of black fungi of diverse phylogenetic affinities. Morphological and developmental characteristics of both species are well suited to the severe fluctuating environmental conditions of Sphagnum bogs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication February 10, 2003.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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