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Mycologia, 95(5), 2003, pp. 959–975.q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897
Comparative morphology and phylogenetic placement of two microsclerotialblack fungi from Sphagnum
S. Hambleton1
Eastern Cereal and Oilseed Research Centre,Agriculture and Agri-Food Canada, Ottawa, Ontario,K1A 0C6 Canada
A. TsunedaDepartment of Biological Sciences, University ofAlberta, Edmonton, AB T6G 2E9, and NorthernForestry Centre, Canadian Forest Service, Edmonton,Alberta, T6H 3S5 Canada
R. S. CurrahDepartment of Biological Sciences, University ofAlberta, Edmonton, Alberta T6G 2E9, Canada
Abstract: Capnobotryella renispora and Scleroconidi-oma sphagnicola form black, irregularly shaped mi-crosclerotia that are indistinguishable in gross mor-phology on leaves of Sphagnum fuscum. In culture,microsclerotia of these fungi were similar, in that ma-ture component cells possessed thick, highly mela-nized cell walls, poorly defined organelles, large lipidbodies and simple septa. They were different in mor-phogenesis, in the way their component cells wereorganized and in disseminative propagules. Micros-clerotia of S. sphagnicola formed phialidic conidi-ogenous cells on their surface, whereas in C. renis-pora, adjacent cells in mature microsclerotia oftenseparated from each other by septum schizolysis andformed chlamydospores. The identification of C. ren-ispora from Sphagnum is provisional despite a 100%ITS sequence match with data for a culture derivedfrom the type strain. No holoblastic, reniform conid-ia typical of the species were formed in nature or inculture, and the SSU sequence for a separately pre-served culture of the ex-type strain was markedly di-vergent. Parsimony analyses of nuclear ribosomalDNA sequences showed that these two fungi were re-lated to separate orders of Dothideomycetes. BothSSU and ITS data supported a close relationship forS. sphagnicola to the Dothideales sensu stricto, whilethe closest ITS match was to Rhizosphaera spp. In theSSU analyses, C. renispora was nested within the Cap-nodiales.
Key words: black yeasts, Capnobotryella, Capno-diales, dematiaceous hyphomycetes, Dothideales,
Accepted for publication February 10, 2003.1 Corresponding author. E-mail: [email protected]
Dothideomycetes, meristematic ascomycetes, Sclero-conidioma, ultrastructure
INTRODUCTION
Black, irregularly shaped microsclerotia were collect-ed on the leaves of Sphagnum fuscum (Schimp.)Klinggr growing in a southern boreal bog in Alberta,Canada. While these structures appeared similar un-der a dissecting microscope, once cultured they clear-ly represented two different fungi. One fungal spe-cies produced mycelial colonies with abundant blackmicrosclerotia, many of which bore papillate conidi-ogenous cells that produced successive hyaline, ba-cilliform, conidia, while the other formed black, cere-briform colonies devoid of blastic conidia. The for-mer fungus was named Scleroconidioma sphagnicolagen. nov. & sp. nov. (Tsuneda et al 2000, 2001b), andis a pathogen of Sp. fuscum (Tsuneda et al 2001a).The colony characteristics of the latter fungus werereminiscent of Phaeosclera dematioides Sigler, Tsuneda& Carmichael (Sigler et al 1981) and Capnobotryellarenispora Sugiyama (Sugiyama and Amano 1987), de-matiaceous hyphomycetes that also are cerebriformin culture. Phaeosclera dematioides is a nonsporulatingtaxon isolated from the pith of Pinus contorta Dougl.and is known only in vitro. Dematiaceous septate hy-phae convert to thick-walled bulbil-like masses of scle-rotic cells analagous to microsclerotia. Capnobotryellarenispora is a sooty mold producing dark, moniliform,tapered hyphae that give rise to two-celled, reniform,blastic conidia directly from distal cells. Strains havebeen 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 moredetailed morphological data, however, neither namecould be applied with confidence to the cerebriformfungus.
The striking similarity between the microsclerotiaof S. sphagnicola and the cerebriform fungus onSphagnum prompted a comparative examination ofthe development of these structures. In addition, ourinability to assign a name to the cerebriform fungus,or to suggest phylogenetic placement for both S.spagnicola and the cerebriform fungus, on the basisof morphology alone, led to a comparison of DNA
960 MYCOLOGIA
sequence data derived from the nuclear ribosomalgene, specifically the small-subunit operon (SSU)and the internal transcribed spacer region (ITS).During these investigations, gene sequence compar-isons indicated that the cerebriform fungus is iden-tical to the ex-type culture of C. renispora, depositedat the Centraalbureau voor Schimmelcultures (CBS).Despite the lack of conidiation, this name is appliedhere to the strain of the cerebriform fungus isolatedfrom Sphagnum. Because the development of the mi-crosclerotia of S. sphagnicola has been reported else-where (Tsuneda et al 2000, 2001b), the emphasis ofthe developmental work reported here is on thestrain of C. renispora from Sphagnum.
MATERIALS AND METHODS
Fungal strains and microscopy. Subcultures of C. renisporaUAMH 9870 and S. sphagnicola UAMH 9731, both isolatedfrom leaves of Sp. fuscum, were used throughout this study.For comparison, three additional strains of C. renispora,CBS 214.90 (IAM13014; ex-type strain), CBS 215.90(IAM13015) and CBS 572.89, were examined by light mi-croscopy. Scleroconidioma sphagnicola was grown on corn-meal agar with dextrose (CMAD; Difco, Detroit, Michigan)(Tsuneda et al 2000), while all strains of C. renispora weregrown on potato-dextrose agar (PDA, Difco) or 2% malt-extract agar (MEA, Difco). Incubation was at 20 C in thedark. Methods for (cryogenic and ordinary) scanning elec-tron 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 nucle-ar rRNA gene were determined for the five strains exam-ined morphologically. Genomic DNA was extracted frommycelium grown on PDA using a FastDNAt Extraction Kit(BIO 101 Inc., Carlsbad, California) and a FastPrepy CellDisruptor machine (BIO 101, Carlsbad, California). DNAamplification and cycle sequencing reactions were per-formed on a Techne Genius thermocycler (Princeton, NewJersey). PCR reactions were performed in 25 (L volumesusing Ready-To-Goy PCR Beads (Amersham PharmaciaBiotech Inc., Piscataway, New Jersey) and 2 mL of templateDNA. PCR cycling parameters included 30 cycles of dena-turation at 95 C for 1.5 min, annealing at 56 C for 1 minand extension at 72 C for 2 min, with an initial denatur-ation of 4 min and a final extension step of 10 min. PrimersNS1 and ITS4 (White et al 1990) were used to amplify morethan 1700 basepairs of the small subunit (SSU) as well asthe complete ITS region, including the ITS1, 5.8S and ITS2.Amplified products were purified using the Wizardy PCRPreps DNA Purification System (Promega Corp., Madison,Wisconsin), and DNA concentrations were estimated fromfragments stained by ethidium bromide and separated byagarose gel electrophoresis.
Sequencing reactions were performed using the BigDyeyTerminator Cycle Sequencing System (Applied Biosystems,Foster City, California) with the recommended cycling pa-
rameters. The reaction mixture comprised one-eighthstrength BigDyey sequencing mix, an equal volume of thereaction booster halfBD (Bio/Can Scientific, Mississauga,Ontario, Canada), 0.5 mL sequencing primer (5(M); 20–50ng purified DNA and sterile water to a final volume of 5mL. Reactions were purified after bringing the volume to20 (L, by ethanol/sodium acetate precipitation and resus-pended as recommended for processing on an ABIPRISMy 310 DNA sequencer (Applied Biosystems, FosterCity, California). Sequencing primers were selected fromthe range of SSU and ITS primers given in White et al(1990) and Landvik et al (1997) to get sequence data forthe whole region. Consensus sequences were determinedfrom overlapping sequence data for both DNA strands us-ing the software Sequenchery (Gene Codes Corp., Ann Ar-bor, Michigan).
Phylogenetic analysis. The initial SSU data matrix included86 small-subunit sequences retrieved from nucleotide se-quence databases (GenBank; http://www.ncbi.nlm.nih.gov)and chosen to represent the phylogenetic diversity of As-comycetes. The SSU and ITS data matrices used in the finalanalyses included accessions identified with Gapped-BLAST(Altschul et al 1997) as having a high sequence similarityto the two fungi under study and other taxa suspected ofbeing closely related, based on preliminary analyses of thelarger SSU dataset. Accession numbers for the GenBank se-quences not listed in TABLE I are given on FIGS. 26–28. Se-quence alignments were generated with the PileUp optionof GCG 10.1 (Genetics Computer Group, Wisconsin Pack-age, Madison, Wisconsin; http:www.cbr.nrc.ca) and then op-timized 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 eachend, for which there was missing data because of minordifferences in sequence length. The final ITS alignment in-cluded the complete ITS1, 5.8S and ITS2 regions. The datamatrices were subjected to parsimony analysis using theheuristic search option of PAUP* version 4.0b8 (Swofford1999) with stepwise addition of taxa, tree bisection-recon-nection (TBR) branch swapping and gaps treated as missingdata. Support for the branching topologies was evaluatedby bootstrap analysis.
RESULTS
Morphological observations. FIGURES 1–9 show thetypical process of microsclerotium development inCapnobotryella renispora (UAMH 9870). Two distinctmodes of cell multiplication occur. In the first mode,cells become enlarged more or less isodiametricallywith subsequent subdivisions (FIGS. 1, 2, arrows), ac-companied by the frequent formation of bubble-likeoutgrowths or blebs (FIGS. 1, 2, arrowheads). Blebsalso were subdivided by septa. In the second mode,apical and polar cell multiplication resulted in theproduction of acropetal chains of globose cells (mo-niliform hyphae) (FIG. 4). The first formed new cellin this mode often erupted through the outer cell
961HAMBLETON ET AL: CAPNOBOTRYELLA AND SCLEROCONIDIOMA ON SPHAGNUM
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wall of its mother cell (FIG. 3, arrow), and all thesubsequently formed cells remained unicellular (FIG.4). The two modes co-occurred in the initial stagesof microsclerotium development (FIG. 3), however,the second mode became more dominant as the mor-phogenesis proceeded (FIGS. 5, 7, 8). Older cells inmicrosclerotium initials became darkly pigmented(FIG. 5, arrow) and exuded an amorphous coatingmaterial (FIG. 8, arrow) that made the black micros-clerotium shiny (FIG. 6). Well-developed microscler-otia were cerebriform (FIG. 9) and adjacent ones fre-quently fused (FIG. 6). No conidia were found in cul-ture or on microsclerotia formed in nature. Theseresults also applied to the three older strains of C.renispora (CBS 214.90, 215.90 and 572.89), exceptthat CBS 572.89 produced numerous, mostly two-celled conidia on both PDA and MEA. Morphologi-cal characters of this strain agreed well with thosedescribed by Sugiyama and Amano (1987). In CBS214.90 and 215.90, conidium production in culturewas either nil or scarce. Conidia, if present, were 4.5–6.5(–8)(3–4.5 mm in CBS 214.90 and 5–5.5 (4–5 mmin CBS 215.90, both shorter than Sugiyama’s descrip-tion of the species in culture, 7–10 (3–4 mm but sim-ilar to the measurements from material in vivo.
TEM of cerebriform colonies of C. renispora re-vealed that the apical cells of growing hyphae firstshowed apical elongation (FIG. 10) and then weresubdivided by a simple septum with Woronin bodies(FIG. 11, arrows). The actively growing cells had well-defined organelles and electron-light cell walls (FIG.10) on which melanin granules deposited rapidly(FIGS. 10, 12, arrows). In the cells delimited from thegrowing apices, small vacuoles appeared and subse-quently enlarged. Larger vacuoles usually containedelectron-dense inclusions (FIG. 13, TABLE I). Even-tually, these cells became filled with large lipid bodies(FIG. 14) and possessed poorly defined organellesand heavily melanized cell walls (FIGS. 14, 15). Lat-eral branches often occurred (FIG. 14, arrow). Ma-ture cells possessed a newly formed, electron-light,thin cell wall between the melanized, older cell walland plasma membrane. In some cases, the newlyformed and older cell walls were separated by a largenumber of melanin granules that had accumulatedbetween them (FIG. 15, arrow).
Shown in FIGS. 16–19 are cells of cryofractured,well-developed microsclerotia of C. renispora. Manyof these cells were either in the process of septumschizolysis (FIGS. 16, 17, arrows) or had separatedfrom adjacent cells (FIGS. 18, 19, arrows). Septal poresites were recognized as minute protuberances (FIG.18, arrowheads). Large lipid bodies (L) and the new-ly developed (or developing) cell wall (arrowheads)were evident in cells of near-median fracture (FIG.
963HAMBLETON ET AL: CAPNOBOTRYELLA AND SCLEROCONIDIOMA ON SPHAGNUM
FIGS. 1–9. Typical developmental process of cerebriform colonies (microsclerotia) in Capnobotryella renispora UAMH9870. 1, 2. Initial stages showing more or less isodiametric enlargement of mother cells with subdividing cells (arrows).Arrowheads indicate blebs. Bar 5 10 mm. 3, 4. Formation of acropetal chains of globose cells (moniliform hyphae) fromthick-walled darkly pigmented mother cells (chlamydospores). A new cell breaks out of the mother cell walls (arrow in 3).Bar 5 10 mm. 5. Microsclerotial initials. Older cells are darkly pigmented (arrow). Bar 5 30 mm. 6. Well-developed micros-clerotia. Adjacent ones often fuse. Bar 5 500 mm. 7–9. SEM micrographs showing later stages leading to the formation of acerebriform colony (microsclerotium). No conidia are formed. The arrow in 8 indicates amorphous coating material thatwill eventually cover the entire microsclerotium. Bar 5 20 mm in 7 and 8, 200 mm in 9.
17). In older cells, the thick, melanized outer andthe newly formed inner cell walls were readily sepa-rable (FIG. 16, arrowheads) by fracturing.
In S. sphagnicola, mode (1) of cell multiplication,mentioned above, dominated in microsclerotium de-velopment (FIGS. 20–22). Although its cells some-
times formed moniliform chains, most of them sub-sequently were subdivided by septa (FIG. 20, arrow).FIGURES 23–25 are cryofractured views of mature mi-crosclerotia showing that component cells of micros-clerotia are cemented firmly together, forming a sol-id sclerotic mass of cells. Unlike in C. renispora, sep-
964 MYCOLOGIA
FIGS. 10–15. Ultrastructure of component cells of Capnobotryella renispora microsclerotia (10, 11, 13–15, TEM; 12, SEM).10. Actively growing apex cell of a moniliform hypha. Cell organelles, including a nucleus (N), are well defined, and devel-oping cell wall is electron light and already covered with a thick layer of melanin granules (arrow). Bar 5 1 mm. 11. Simpleseptum (S) associated with Woronin bodies (arrows). M 5 mitochondrion. Bar 5 0.2 mm. 12. Chlamydospore that germinatedto form a new globose cell. Arrows indicate melanin granules. Bar 5 2 mm. 13. Maturing cells with large vacuoles containingelectron-dense inclusions (I). Note lack of large lipid bodies at this stage. Abundant melanin granules occur on the cellsurface (arrows) and between adjacent cells (arrowheads). Bar 5 2 mm 14. Mature cells of a hypha filled with large lipidbodies (L). The arrow indicates a side branch. Bar 5 4 mm. 15. Mature cell (chlamydospore) after septal schizolysis. Cellorganelles are poorly defined, and cell walls are heavily impregnated with melanin. The arrow indicates the area where newlydeveloped and older cell walls are separated by accumulated melanin granules. The newly developed wall is thin and electronlight. L 5 lipid bodies. Bar 5 2 mm.
965HAMBLETON ET AL: CAPNOBOTRYELLA AND SCLEROCONIDIOMA ON SPHAGNUM
FIGS. 16–19. Cryo-fractured views of mature microsclerotia of Capnobotryella renispora. 16. Cells in the process of septumschizolysis (arrow). Arrowheads indicate a gap between the newly formed and older cell wall layers. Bar 5 2 mm. 17. Cell ofnear-median fracture having large lipid bodies (L) and a newly developed (developing) cell wall layer (arrowheads). Thearrow points to the schizolysing septum. Bar 5 1 mm. 18, 19. Lower-magnification micrographs, showing separating andseparated cells (chlamydospores) (arrows). Arrowheads in 18 indicate septal pore areas and those in 19 point to schizolysingsepta. L 5 lipid body. Bar 5 3 mm in 18, 5 mm in 19.
tum schizolysis did not occur between adjacent cellsand microsclerotia often developed a layer of, orsmall masses of, conidiogenous cells on their surface(FIG. 23, arrowheads). As with C. renispora, however,component cells of well-developed microsclerotia
possessed an inner, newly formed cell wall (Tsunedaet al 2001b) and were filled with large lipid bodies(FIG. 25). There was a distinct gap between the newlyformed and older cell walls (FIG. 24, arrows), andcells possessing only the newly formed cell wall could
966 MYCOLOGIA
FIGS. 20–25. Typical developmental process of microsclerotia (conidiomata) in Scleroconidioma sphagnicola (UAMH 9731).20. Chain of subglobose cells that undergo subdivision (arrow). Bar 5 10 mm. 21. Microsclerotium initial consisting of activelysubdividing cells (arrow). Bar 5 10 mm. 22. SEM view of a microsclerotium initial. Bar 5 5 mm. 23–25. Cryo-fractured viewsof mature microsclerotia. 23. Small masses of conidiogenous cells (arrowheads) developed on a microsclerotium. The lowerarrowhead points to a conidium emerging from a conidiogenous cell. Septa (S) of component cells of the microsclerotiumshow no sign of schizolysis. Arrows point to cells dislodged from their older cell walls upon fracturing. Bar 5 3 mm. 24. Partof a mature microsclerotium. Distinct gap is evident between newly formed and older cell walls in many component cells(arrows). Bar 5 6 mm. 25. Mature component cells containing large lipid bodies (L). Bar 5 3 mm.
be dislodged from their older cell walls upon frac-turing (FIGS. 23, arrows).
DNA sequencing. The lengths of the SSU sequencesdetermined for S. sphagnicola UAMH 9731 and C.
renispora UAMH 9870 were 1744 basepairs (bp). TheSSU sequences were complete at the five-prime end.The length of the sequence determined for S. sphag-nicola using primers ITS1–4 was 512 bp; the ITS1 and
967HAMBLETON ET AL: CAPNOBOTRYELLA AND SCLEROCONIDIOMA ON SPHAGNUM
ITS2 alone were 176 and 163 bp, respectively. For C.renispora, the total ITS sequence was 472 bp inlength; the ITS1 and ITS2 alone were 146 and 153bp. The SSU and ITS sequences for CBS 214.90, ex-type culture of C. renispora, and CBS 215.90 wereidentical to those of UAMH 9870, except for the in-sertion of a single nucleotide (nt) in the ITS1 se-quence of CBS 215.90 (four consecutive adenine nt,rather than three, positions 143–146). The ITS se-quence for CBS 572.89 (469 bp) was only 95% similarto that of the other two strains, while the SSU se-quence differed at three nt positions. The newly de-rived SSU-ITS sequences were deposited in GenBankas AY220610 (UAMH 9731), AY220611 (UAMH9870), AY220612 (CBS 214.90), AY220613 (CBS215.90) and AY220614 (CBS 572.89).
The closest GenBank SSU sequence matches for S.sphagnicola were for taxa classified in the Dothidealesand Capnodiales, while the closest ITS matches wereto Rhizosphaera spp. with 96% similarity. The closestGenBank SSU matches for C. renispora were also tothe Dothideomycetes, and our identical ITS sequenc-es for UAMH 9870 and the ex-type strain, CBS 214.90(IAM13014), were a 100% match for a published ITSsequence (AJ244238; de Hoog et al 1999) for CBS214.90. Other ITS sequences retrieved in the BLASTsearch were for Trimmatostroma spp. and Mycospha-erella spp., but the matches were at 90% similarity orless.
Our identical SSU sequences for CBS 214.90, CBS215.90 and UAMH 9870 unexpectedly differed froma published SSU sequence for CBS 214.90 (Y18698;Sterflinger et al 1999) at 17 nt positions (98% simi-larity) and were only 91% similar to a second SSUsequence (AF006723; Reynolds 1998) for this strainof C. renispora separately preserved as ATCC 64891(ITS sequence data was not available for this culture).Because of the marked discrepancies among the se-quences available for the ex-type strain of C. renis-pora, only the sequences determined from the CBScultures obtained for and examined in this studywere used in the analyses.
Phylogenetic analysis. The initial SSU data matrixcomprised 88 taxa and 1983 aligned characters. Ofthese, 182 characters corresponded to short, unalign-able sequence motifs unique to individual sequences(mostly for outgroup taxa); these were treated as in-sertions relative to the rest of the alignment and wereexcluded from the analyses. Of the remaining 1801characters, 1039 were constant, 235 were parsimonyuninformative and 527 were parsimony informative.An analysis using the heuristic search algorithm, with100 replicates of random addition sequence, resultedin 18 equally parsimonious trees (MPTs) of 2648
steps and with CI 5 0.432, RI 5 0.711, RC 5 0.308and HI 5 0.568. A bootstrap analysis (1000 repli-cates) was performed using the ‘‘fast’’ stepwise-addi-tion search option to determine the level of supportfor the topology of the inferred phylogeny. Fungiclassified in seven of the 11 classes of the subphylumPezizomycotina were included in the analysis, whilerepresentatives of the Taphrinomycotina and Sac-charomycotina served as outgroup taxa. Classificationfollows Eriksson et al (2001).
The initial analyses confirmed the BLAST searchresults that both S. sphagnicola UAMH 9731 and C.renispora UAMH 9870 were related to species classi-fied in the Dothideomycetes (FIG. 26). The mono-phyly of the class was supported in the strict consen-sus of all MPTs (data not shown). Both fungi wereexcluded from the other major clades in the tree,identified by class name on FIG. 26, by virtue of theirhigh bootstrap values and/or the number of char-acter state changes, as evidenced by branch length.While bootstrap support was less than 50% for theDothideomycetes clade, several subclades receivedhigher support. Group A (FIG. 26), bootstrap 96%,comprised species in five families of the Pleosporales,Mycosphaerella mycopappi (Mycosphaerellaceae, incer-tae sedis) and Rhytidhysteron rufulum (Patellariales) ina basal position. Within the group, several suprafam-ilial subgroupings also received high bootstrap sup-port. Group B comprised representatives of the Doth-ideales, Capnodiales and Botryosphaeriaceae (incer-tae sedis). Scleroconidioma sphagnicola clustered withDothidea insculpta U42474 (Dothideales) with mod-erate bootstrap support at 88%; Capnobotryella renis-pora grouped with Coccodinium bartschii U77668(Capnodiales) with moderate support of 78% in asister group to Capnodium citri AY016340.
The final SSU alignment included representativesof the Group A Dothideomycetes, Dothideales, Cap-nodiales and Dothioraceae (incertae sedis), as well asseveral dematiaceous hyphomycetes. Leotia lubricaL37536, L. viscosa AF113715, Microglossum virideU46031, Cudonia confusa Z30240 (Leotiomycetes),Peziza badia L37539, Morchella elata L37537 and Ur-nula hiemalis Z49754 (Pezizomycetes) served as theoutgroup. The ITS alignment included the sequenc-es available for species in the Dothideaceae and Do-thioraceae and closely related anamorph genera.Based on the SSU analysis, Phaoesclera dematioidesAJ244254 and Sarcinomyces crustaceous AJ244258 werechosen as outgroup taxa. Capnobotryella renispora wasnot included in the ITS analysis; ITS data could notbe aligned with confidence to the sequence for S.sphagnicola or to those sequences retrieved fromGenBank as a result of the BLAST search.
The final SSU data matrix comprised 38 taxa and
968 MYCOLOGIA
969HAMBLETON ET AL: CAPNOBOTRYELLA AND SCLEROCONIDIOMA ON SPHAGNUM
←
FIG. 26. One of 18 equally parsimonious trees (2648steps, CI 5 0.432) resulting from a maximum-parsimonyanalysis of the initial SSU data matrix using the heuristicsearch algorithm of PAUP* version 4.ob8. Bootstrap valuesabove 65% are given adjacent to the corresponding node.
1652 aligned characters. Of these, 1285 were con-stant, 210 were parsimony uninformative and 157were parsimony informative. An analysis using theheuristic search algorithm, with 100 replicates of ran-dom addition sequence, resulted in 15 MPTs of 651steps and with CI 5 0.662, RI 5 0.700, RC 5 0.463and HI 5 0.338. Results of a full bootstrap analysis(1000 replicates) using the heuristic search optionare shown on one MPT (FIG. 27). Four sequenceswere short compared to the rest (TABLE I), but anal-ysis of only that portion of the data matrix for whichall sequences were complete did not alter the overalltopology of the resulting tree (data not shown). TheITS alignment comprised 25 taxa and 569 alignedcharacters. Of these, 21 ambiguously aligned char-acters were excluded, 370 were constant, 69 were par-simony uninformative and 109 were parsimony infor-mative. A branch and bound analysis resulted in 10MPTs of 352 steps and with CI 5 0.670, RI 5 0.784,RC 5 0.525 and HI 5 0.330. Results of a full boot-strap analysis (1000 replicates) using the heuristicsearch option are shown on one MPT (FIG. 28).
Parsimony analysis of the final SSU alignment sup-ported the placement of S. sphagnicola near theDothideales but not within the order (FIG. 27). Therewas high bootstrap support (95%) for a monophylet-ic Dothideales sensu stricto, while S. sphagnicola clus-tered with Delphinella strobiligena (Dothioraceae) inan unsupported sister clade. Aureobasidium pullulansand its teleomorph Discosphaerina fagi (Dothiora-ceae) (Yurlova et al 1999), two identical sequences inthe trimmed alignment, were in a basal position. Cap-nobotryella renispora UAMH 9870/CBS 214.90/215.90grouped with an undescribed species of Capnobotryel-la within a large clade corresponding to the Capno-diales (FIG. 27). The clade included holomorphs inCapnodium, Scorias (Capnodiaceae) and Coccodinium(Coccodiniaceae) and the allied anamorph generaAntennariella (Antennulariaceae) and Chaetasbolisia(Metacapnodiaceae) but also the dematiaceous hy-phomycete genera Hyphospora and Hortaea. Al-though the Capnodiales clade was retained in thestrict consensus of all MPTs, in general, relationshipswithin the order were unsupported by the bootstrapanalysis, and terminal branch lengths were long, in-dicating a substantial genetic distance between spe-cies. The relationships of the other dematiaceous hy-
phomycetes included in the analysis, Phaeotheca,Phaeosclera and Sarcinomyces, were unresolved. Twospecies of Botryosphaeria clustered together with highbootstrap support, forming a sister clade with Sarci-nomyces petricola to the well-supported clade compris-ing Group A Dothideomycete species.
In the ITS analysis, two major clades received highbootstrap support (FIG. 28). In the first (clade I), S.sphagnicola clustered with Rhizosphaera spp., as pre-dicted by the BLAST search. This well-supportedgrouping was sister to a cluster of three groups cor-responding to teleomorph genera in the Dothidea-ceae (Dothidea) and Dothioraceae (Dothiora and Sy-dowia). The coelomycetous genus Rhizosphaera has aputative teleomorph connection to Phaeocryptopus(Kirk et al 2001), yet Phaeocryptopus gaumannii(Rhode) Petrak (Venturiaceae) was excluded fromthe Rhizosphaera clade. Species of Hormonema clus-tered with Dothiora or Sydowia; both genera produceanamorphs in Hormonema (de Hoog et al 1999). Spe-cies of the anamorphic genus Kabatina were basal tothe Sydowia and Dothidea clades but did not clustertogether. The other major clade (II) comprisedstrains of the anamorphic black yeast Aureobasidiumpullulans, [synonym Kabatiella lini (Lafferty) Karak-ulin] (Yurlova et al 1999).
DISCUSSION
On the host and in colony and morphogenic char-acteristics, both S. sphagnicola and C. renispora resem-ble the so-called ‘‘meristematic fungi,’’ fungi withgrowth characterized by slowly expanding, black, cau-liflower-like colonies (5 cerebriform colonies or mi-croscerotia) and reproduction by isodiametric en-largement with subdividing cells (Sterflinger et al1999). Some of these fungi possess budding cells andhave a close taxonomic affinity to the black yeasts ofthe Chaetothyriomycetes (Haase et al 1995), whileothers with a yeast-like phase are allied with the Doth-ideomycetes (Sterflinger et al 1999). Many blackyeast taxa convert to meristematic growth understressful conditions and therefore the distinction be-tween ‘‘black yeasts’’ and ‘‘meristematic fungi’’ is notabsolute (Sterflinger and Krumbein 1995, Sterflingeret al 1999, de Hoog et al 1999). As well, the term‘‘black fungi’’ sometimes is used to embrace bothgroups of fungi (Sterflinger and Krumbein 1995).Most of these fungi occur in extreme environments,tolerating stresses caused by temperature, water avail-ability, oxygenic action, irradiation by ultraviolet rays(UV), electrolyte content and/or scarcity of nutrients(Sterflinger et al 1999, de Hoog et al 1999). Epi-phytic species occur in such genera as Aureobasidium,Coniosporium, Hormonema, Hyphospora, Phaeotheca,
970 MYCOLOGIA
FIG. 27. One of 15 equally parsimonious trees (651 steps, CI 5 0.662) resulting from a maximum-parsimony analysis ofthe final SSU data matrix using the heuristic search algorithm of PAUP* version 4.ob8. Bootstrap values above 60% are givenadjacent to the corresponding node. Branches in bold were present in the strict consensus.
971HAMBLETON ET AL: CAPNOBOTRYELLA AND SCLEROCONIDIOMA ON SPHAGNUM
FIG. 28. One of 10 equally parsimonious trees (352 steps, CI 5 0.670) resulting from a branch and bound analysis of theITS data matrix. Bootstrap values above 65% are given adjacent to the corresponding node. Branches in bold were presentin the strict consensus. Bold type indicates teleomorphic taxa.
Sarcinomyces and Trimmatostroma (e.g., Butin et al1995, DesRochers and Ouellette 1994, Sigler et al1981, Sterflinger et al 1999, Yurlova et al 1999), fungiwith diverse phylogenetic affinities.
Despite similarities in their morphology and habi-tat, molecular evidence indicates that meristematicfungi and black yeasts belong to at least three differ-ent orders, Chaetothyriales, Dothideales (sensu lato)and Pleosporales (Haase et al 1995, de Hoog et al1999, Sterflinger et al 1999). Our parsimony analysesof small-subunit nuclear rDNA sequence data showed
that S. sphagnicola and C. renispora phylogeneticallywere distinct from one another. Both were related tofungi classified in the Dothideomycetes but to differ-ent orders, specifically to the Dothideales and theCapnodiales, respectively, and distant from the Chae-tothyriomycetes. These two dematiaceous fungi iso-lated from Sphagnum leaves also were distinct mor-phologically. Although they produced microsclerotiasimilar in gross morphology and simple septation(FIGS. 9, 11) (Tsuneda et al 2001b), further exami-nation showed that they differed in the mode of de-
972 MYCOLOGIA
velopment, internal structure and disseminativepropagules.
Morphological and ecological aspects. In C. renispora,the dominant form of cell multiplication in micros-clerotium formation is apical and polar, leading tothe formation of acropetal chains of globose cells(moniliform hyphae; FIG. 4). Thus, growth is pre-dominantly hyphal. Adjacent cells in mature micros-clerotia separate by septum schizolysis. However in S.sphagnicola, growth is revealed primarily by isodia-metric enlargement and subdivision of mother cells(FIGS. 21–23) (Tsuneda et al 2001b), and the cells ofmature microscerotia are cemented in a solid scle-rotic mass. The similar, cauliflower-like macromor-phology of the microsclerotia observed on the host,then, is derived from different developmental pro-cesses.
Diverse fungi, including some parasitic ones, occuron living or decomposing Sphagnum (Dobbeler 1978,1986, Redhead 1981, Redhead and Spicer 1981,Thormann et al 2001, Tsuneda et al 2001a). Sphag-num cell walls are highly resistant to microbial deg-radation, and environmental conditions prevailing inSphagnum bogs, i.e., wide fluctuations in tempera-ture, moisture and radiation, could select for fungiproducing dark pigments and having the ability toform sclerotia. It is apparent that the primary func-tion of microsclerotia in both C. renispora and S.sphagnicola is survival on the host. Component cellsof mature microsclerotia in both fungi contain alarge amount of reserve material (lipid bodies) andpossess thick cell walls that are densely covered andheavily impregnated with melanin granules. Further,their cell organelles are not well defined (FIG. 15)(Tsuneda et al 2001b), except in actively multiplyingcells. These characteristics indicate that the micros-clerotia remain in a resting state until environmentalconditions become favorable for growth. Melanin ap-pears to play an important role in stress tolerancebecause wall-bound melanin protects fungal cellsfrom various stress factors, including physical (e.g.,extreme temperatures and desiccation) and biologi-cal ones, such as lysis caused by cell-wall degradingenzymes released by other microorganisms (Butler etal 2001). In addition, melanin granules absorb X-,gamma and UV rays (Zhdanova et al 1973) and limitthe leakage of certain metabolites (Butler et al 2001).These characteristics of melanin explain the higherincidence of melanized microbes occurring on rocks,leaf surfaces and other exposed locations (Ursi et al1997).
Microsclerotia of S. sphagnicola function not onlyas survival structures but also as a reservoir of nutri-ents that sustains the production of conidiogenous
cells and numerous conidia, when environmentalconditions are favorable (Tsuneda et al 2001a, b).Dissemination of this fungus among Sphagnumstands thus is primarily by conidia. In contrast, conid-ia never were observed for the cerebriform fungusfrom Sphagnum, although the type strain of C. ren-ispora produces holoblastic, reniform conidia, bothin its natural habitat and in culture (Sugiyama andAmano 1987). Then how could C. renispora onSphagnum disseminate itself in bogs? FIGURES 15–19clearly show that many of the component cells of ma-ture C. renispora microsclerotia separate by septumschizolysis. In this way, the microsclerotium couldfunction as a mass of thick-walled, thallic spores (5chlamydospores). It is reasonable to assume that suchchlamydospores can function to disperse the funguswhen the microsclerotium is weathered and/orcrushed by external force. The highly melanized out-er cell walls become brittle as microsclerotia age, andthus moisture can be readily supplied to the internalcells. This characteristic enables microsclerotial cellsto resume growth quickly in response to favorableenvironmental conditions.
A striking characteristic of both C. renispora and S.sphagnicola is that each component cell of a maturemicrosclerotium possesses a thin layer of newlyformed cell wall (FIG. 15, arrow) (Tsuneda et al2001b). That portion of the cell with the newlyformed cell wall can be separable from the highlymelanized, older cell wall (FIGS. 16–18, 23, 24). In C.renispora, when re-growth takes place from these cells(5 chlamydospores), the newly forming cell breaksout of the outer cell wall of the chlamydospore (FIG.3, arrow). These features indicate that the outer cellwall is no longer an integral part of the ‘‘living’’ cellbut serves as a physical barrier between the cell andits surroundings. The portion consisting of a proto-plast and newly formed cell wall thus is analogous toan endoconidium. Endoconidiation occurs in suchgenera of black fungi as Aureobasidium, Coniospor-ium, Hyphospora, Phaeotheca, Sarcinomyces and Trim-matostroma (de Leo et al 1999, de Hoog and Her-manides-Nijhof 1977, Sigler et al 1981, Wollenzien etal 1997, Zalar et al 1999a, b). In Phaeotheca fissurella,whose microsclerotia resemble those of S. sphagnicolain ultrastructure, each cell with the newly formed cellwall subsequently is subdivided to form two to severalendoconidia. These endoconidia are released by dis-solution of mother-cell walls (Sigler et al 1981, Tsu-neda and Murakami 1985). Microsclerotia of C. ren-ispora and S. sphagnicola lack such an elaborate re-leasing mechanism.
Phylogenetic aspects. The SSU and ITS analyses pro-vided evidence of a close relationship between S.
973HAMBLETON ET AL: CAPNOBOTRYELLA AND SCLEROCONIDIOMA ON SPHAGNUM
sphagnicola and the Dothideales sensu stricto, re-stricted to one family (Dothideaceae) in the Erikssonet al (2001) classification, and genera in the Dothior-aceae. Based on the ITS sequences available inGenBank, its closest relatives are in Rhizosphaera, acoelomycete genus characteristically found on coni-fer needles functioning as a weak parasite (Butin andKehr 2000). These genera are similar in the shape ofconidiogenous cells and conidia, and in phialidicconidiogenesis. Conidiogenous cells of Rhizosphaeraare produced within black pycnidia, and they formthe single layer of cells of the conidiomatal wall (Sut-ton 1980), whereas those of Scleroconidioma form onthe surface of dematiaceous microsclerotia (FIG. 23)(Tsuneda et al 2001b), giving the impression of a pyc-nidium of Rhizosphaera turned inside-out.
There was support from the ITS analysis for a moreinclusive concept of the Dothideales, that would in-clude the Dothioraceae (anamorphs in Hormonema),and the anamorph genus Kabatina, an acervular fun-gus that is Hormonema-like in pure culture (Herman-ides-Nijhof 1977). Rhizosphaera and Scleroconidiomawere sister taxa, while several strains of Aureobasi-dium, its synonym Kabatiella and by inference its te-leomorph Discosphaerina (FIG. 27; Yurlova et al 1999)formed a separate well-supported monophyleticgroup. Strong support for such a wider concept,which also would include Aureobasidium, was provid-ed by maximum-likelihood and neighbor-joininganalyses of combined large and small-subunit data(Lumbsch and Lindemuth 2001) but not by SSUanalysis alone (this study, Berbee 1996). Our analysesraised questions about phylogenetic relationshipswithin the Venturiaceae. Although Rhizosphaera spp.are considered to be anamorphic Venturiaceae (Kirket al 2001), Phaeocryptopus gaumannii is far removedin the ITS tree and, in the SSU analyses, Venturialiriodendri Hanlin is allied with the Pleosporales clade(data not shown, Silva-Hanlin and Hanlin 1999).
Our identical sequence data for UAMH 9870 andthe ex-type strain of C. renispora (IAM 31014) pre-served as CBS 214.90 strongly suggested conspecific-ity. Furthermore, our sequence data for a secondstrain used for the original description of the species(CBS 215.90, IAM 31015; Sugiyama and Amano1987) also were identical, except for one nucleotidedifference in the ITS sequence. The genus Capno-botryella (Metacapnodiaceae) clustered well withinthe Capnodiales in the SSU analysis, as did the twodematiaceous hyphomycetes, Hyphospora and Hor-taea. In a study focused on the Capnodiales, Reynolds(1998) found that the taxa sampled formed a mono-phyletic group based on a neighbor-joining analysisof SSU sequences. Capnobotryella renispora and otherallied anamorphic genera clustered with the teleo-
morph representatives but with low bootstrap sup-port. The results of our SSU analyses, which incor-porated the relevant sequences from the studies ofReynolds (1998) and Lumbsch and Lindemuth(2001), are in agreement.
Relationships among taxa within the Capnodialesclade (FIG. 27) were unresolved and difficult to in-terpret. Type species have not yet been sampled, twoof the four families included were represented by an-amorph taxa only, and there are other capnodia-ceous families for which no sequence data are avail-able. Two species of Capnodium (Capnodiaceae) clus-tered in different subclades as did two genera of theMetacapnodiaceae. The lack of bootstrap support forthe clade and the genetic distance between taxa with-in the clade, as evidenced by the long terminal-branch lengths, suggest that the monophyly of thesooty mold order might not hold up with increasedsampling. ITS sequences available for representativesof the Capnodiales and the other anamorph generain the clade were highly divergent and could not bealigned with confidence to sequences of C. renispora.
The identification of the Sphagnum isolate remainsprovisional because it did not form the holoblastic,reniform conidia that characterize C. renispora andbecause three different SSU sequences have been de-termined for the ex-type strain (our data, Y18698 andAF006723). The morphological characters we ob-served for the CBS culture agreed with those in theoriginal description of material from the host, al-though conidia were rare. We were confounded bythe marked differences between the SSU sequencesderived from the CBS and ATCC cultures. We wereunable to examine the fungus preserved as ATCC64891 for this study, but in our analyses all three SSUsequences cluster within the Capnodiales (data notshown) and therefore it is unlikely that the discrep-ancies are a result of culture contamination.
Two other possible explanations are that a se-quencing error occurred or that two distinct sootymold species have been preserved inadvertently asIAM 31014. Capnobotyrella renispora was isolated fromthe subicula of another sooty mold, Capnobotrys nee-sii, for which there are no cultures and no sequencedata. Sooty molds are found superficially on livingplants, and several taxa frequently grow together inapparent harmony in such a way that species are notreadily distinguishable from each other on the hostand specimens often comprise more than one species(Hughes 1976). In addition, species are highly pleo-morphic, with up to three synanamorphs produced.Careful studies of more specimens are needed toclarify the extent of morphological and molecularvariation exhibited by fungi in the form genus Cap-nobotryella. Conidiation among the strains examined
974 MYCOLOGIA
ranged from prolific (CBS 572.89) to scarce or nil;the long terminal branch lengths for the three fungiincluded in the SSU analysis presented in FIG. 27 (C.renispora and Capnobotryella sp. nov.) suggest that ge-netic variation is substantial.
Other meristematic fungal genera allied with theDothideomycetes include Aureobasidium, Botryomyces,Hormonema, Hortaea, Hyphospora, Phaeosclera, Phae-otheca and some species of Sarcinomyces (de Hoog etal 1999, Sterflinger et al 1999). Of these, only Botry-omyces is related to the Pleosporales (de Hoog et al1999) or the Group A Dothideomycetes of our anal-yses. The rest clustered with Group B taxa, as shownin FIGS. 27 and 28. Prior molecular-based studies haveexamined relationships within the Dothideomycetesand have highlighted the division of the class intotwo groups, one consistently being the well-support-ed monophyletic Pleosporales (Berbee 1996, Silva-Hanlin and Hanlin 1999, Liew et al 2000, Lumbschand Lindemuth 2001). In general, the groups cor-respond to the pseudoparaphysate taxa of the Pleos-porales, including the Melanommatales, versus theaparaphysate taxa in the Dothideales and Capnodi-ales, thus Groups A and B of our analyses. Overall,the ascomycete phylogenetic hypotheses presentedhere concur with prior hypotheses of supra-ordinaltaxonomic relationships (Tehler et al 2000, Erikssonet al 2001). They also reinforce the finding that re-lationships among Group B Dothideomycete taxa arenot well resolved by parsimony analysis of larger da-tasets of small- subunit sequence data (Silva-Hanlinand Hanlin 1999).
Conclusions. Using rDNA sequence data, we haveshown that two dematiaceous hyphomycetes occur-ring together on Sphagnum leaves are related to sep-arate orders of ascostromatic ascomycetes, the Doth-ideales and Capnodiales. They are phylogeneticallydistinct from other taxa that exhibit meristematicgrowth patterns. Their classification remains incon-clusive, pending the discovery of their teleomorphsand/or the availability of sequence data for a widerrange of taxa from these two orders. Comparative mi-croscopic examinations have elucidated fundamentalmorphological differences that corroborate the phy-logenetic distinction. Scleroconidioma sphagnicola andC. renispora differ in the development and internalstructure of superficially similar microsclerotia pro-duced on the host and in culture and in dissemina-tive propagules, which are predominantly phialocon-idia and chlamydospores, respectively. Evidence ispresented that the component cells of the maturemicrosclerotia are analogous to the endocondia pro-duced by other genera of black fungi of diverse phy-logenetic affinities. Morphological and developmen-
tal characteristics of both species are well suited tothe severe fluctuating environmental conditions ofSphagnum bogs.
ACKNOWLEDGMENTS
We thank M. H. Chen and G. Braybrook for their technicalassistance, L. Sigler for her useful suggestions and KeithSeifert for helpful discussions of the molecular analyses.This research was financed in part by the Natural Sciencesand Engineering Research Council of Canada through avisiting fellowship in Canadian Government Laboratories toS.H. and an operating grant to R.S.C. and also by the Tot-tori Mycological Institute through a research grant to A.T.
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