Capnodiaceous sooty mold phylogeny

Don R. Reynolds

Abstract: Sequences from the 18s rDNA gene of representatives of the capnodiaceous sooty mold familiesAntennulariellaceae, Capnodiaceae, and Metacapnodiaceae as well as 14 ascomycete taxa representing thePlectomycetes, Dothideales, Pyrenomycetes, and Pleosporales, and yeast outgroups were analyzed. Sooty moldcapnodiaceous ascomycetes comprising were found to be a monophyletic group, the Capnodiales. The convergentorigin of the bitunicate ascus associated with the periphysoid sterile element is validated. The major Capnodialescharacters are the foliicolous habit, darkly pigmented hyphae, and a distinctive periphysoid sterile element associatedwith a fissitunicate type of bitunicate ascus.

Key words: ascomycetes, Capnodiales, periphysoid sterile elements.

Résumé: Les auteurs ont analysé les séquences du gène rADN 18s de divers représentants des familles de fumaginesAntennulariellaceae, Capnodiaceae et Métacapnodiaceae, ainsi que 14 taxons d’ascomycètes représentant lesPlectomycètes, les Dothidéales, les Pyrénomycètes, les Pléosporales et des levures. Les ascomycètes capnodiacéscomportant des fumagines forment un groupe monophylétique, les Capnodiales. L’origine convergente de l’asquebituniqué associé à des éléments périphysoïdes stériles est confirmé. Les principaux caractères des Capnodiales sontl’habitat foliicole, les hyphes à pigmentation fonçée, un élément périphysoïde stérile typique associé à un asquebituniqué de type fissituniqué.

Mots clés: ascomycètes, Capnodiales, éléments stériles périphysoïdes.

[Traduit par la Rédaction] Reynolds 2130

Introduction

Sooty mold is a term given to a group of ascomycetefungi because of their appearance on living plant surface. Adark, melanoid pigmentation in the cell walls of copious my-celium and reproductive structures imparts the appearance ofa sooty coating. The sooty molds are usually observed in as-sociation with scales and aphids that parasitize vascularplants; these insects exude a plant-sap derived substance thatserves as a fungal substrate.

The prototypic sooty mold isCapnodium salicinumMontagne (Reynolds 1978b). The original taxonomic con-cept ofCapnodiumincluded species described as producing“sporidia” or ascospores, as well as those known only frommitosporic reproduction (Berkeley and Desmazières 1849;Montagne 1849; Saccardo 1882).

The first major monographic review of capnodiaceoussooty molds recognized both sexual and asexual species asthe Eucapnodieae (Fraser 1935b). A later review separatedthe species into two groups, reflecting the taxonomic prac-tice of the times with the ascomycete species treated as theCapnodiales by Batista and Ciferri (1963a) and the mito-sporic sooty molds as the Asbolisiaceae (Batista and Ciferri1963b). Subsequent overviews of the Capnodiaceae sensulato followed this artificial separation as sexual and asexual

taxa (Barr 1987a; Hawksworth et al. 1995; Luttrell 1955,1973).

CapnodiumandScoriasbecame the core ascomycete gen-era of the sooty mold Capnodiaceae (Hughes 1976; Barr1987a) with the transfers of taxa out of Capnodiaceae sensulato to other families based on morphological characters.The ascocarp structure ofLimacinula(Reynolds 1971, 1975)was regarded as similar to that ofCoccodiniumand bothtaxa were classified in the Coccodiniaceae (Hawksworth etal. 1995) and the Chaetothyriales (Barr 1987b). A distinctiveascus structure was proposed forTrichomeriumand a link tothe Pleosporales as well as the basis of a new family, theTriposporipsidaceae (Hughes 1976; Reynolds 1982).Periphysoids were found inLimacinia (=Metacapnodium)(Corlett 1970; Reynolds 1985) that substantiated a transferto the Metacapnodiaceae (Hughes 1972) and the Chaeto-thyriales (Barr 1979, 1987b). Periphysoids have been re-ported in Capnodium, in Scorias, and in Trichomerium(Reynolds 1978a, 1978b, 1979, 1989a).

The occurrence of periphysoids is a somewhat unresolvedissue for sooty mold species. Fraser (1935a) found “pseudo-periphyses” inC. salicinum, as “…stroma hyphae [that]grow up under [the apical pore] simulating periphyses;” itwas noted that these structures were eventually “resorbed.”“Capnodiaceous periphysoids” were found in the type speci-men ofC. salicinum(Reynolds 1978b), and noted to have atendency to reorient during ascus discharge from a down-ward, hanging direction to extend toward the ostiolar region.Their similarity to the periphysoids as defined by Eriksson(1981) has been confirmed using herbarium specimens col-lected in Europe and Australia and fresh material collectedin California (D.R. Reynolds, unpublished data). These same

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Received December 17, 1997.

D.R. Reynolds.Natural History Museum of Los AngelesCounty, 900 Exposition Boulevard, Los Angeles, CA 90007,U.S.A. e-mail deynold@bcf.usc.edu

structures have been interpreted as “delicate periphyses inthe ostiolar region” and the ascus as being unassociated witha definitive sterile element, thus warranting the assignmentof Capnodiumto the Dothideales (Barr 1987a).

The sooty molds were utilized in a creative approach toascomycete systematics by Hughes (1976). This innovationestablished plausible monophyletic groups of capnodiaceousfungi whereby shared life history characters grouped bothmono- and pleo-morphic “holomorphs” in a family leveltaxon (Kendrick 1979; Reynolds and Taylor 1993). In es-sence, the original, polymorphic concept ofCapnodium(Berkeley and Desmazières 1849; McAlpine 1896) was ef-fected at a phylogenetic level as the Antennarulariaceae,Euantennariaceae, Capnodiaceae, and the Metacapnodiaceae.

The results of molecular sequence studies of filamentousascomycetes suggest a polyphyletic origin for the sootymolds. A sister relationship withDothidea and Aureo-basidiumas well asBotryosphaeriain the “Dothideales s.str.” was proposed for the periphysoidal bitunicate ascusspecies Coccodinium bartschii of the Coccodiniaceae(Winka et al. 1998). These authors, as well as Silva-Hanlinand Hanlin (1999), affirmed the discovery of the convergentorigin of the bitunicate ascus ofCapronia(Berbee 1996) anda sister relationship with the “black yeast” on a Plecto-mycete clade. They also recognized convergence for theperiphysoid character association with the bitunicate ascus.These findings predict a polyphyletic origin for sooty moldsspecies with a bitunicate ascus associated with a sterile ele-ment termed the periphysoid to denote its originate from theinner wall of the ascocarp cavity near the apical ostiole.

One objective of this study is a better understanding ofhomology of capnodiaceous characters. Another goal is togain insight into the cladistic relationships of capnodiaceoussooty mold species among the ascomycetes.

Materials and methods

Five species of capnodiaceous sooty mold ascomycetes, includ-ing two sexual and three asexual taxa, were utilized in this study.Partial sequences (1095 nucleotides) were utilized from the nuclearsmall subunit rDNA gene forAntennariella californica, Capno-botryella renispora, Capnodium dematium, Chaetasbolisia falcata,andScorias spongiosa. These sooty molds were compared with 14other ascomycetes. Specimen data and GenBank accession num-bers are found in Table 1.

Sooty mold isolates (Table 1) were maintained on solid yeast –maltose agar (4 g yeast extract, 10 g malt extract, 4 g glucose, 15 gagar in 1 L of distilled water) at 20°C. Mycelium for DNA extrac-tion was first grown in Petri dish cultures at room temperature for5–10 days. Agar bits, 2–3 mm in diameter, with actively growinghyphal tips, served as the inoculum for liquid CMA cultures thatwere subsequently maintained on a shake table at room tempera-ture for 5 days. The harvested mycelium was rinsed in sterile, fil-tered, and distilled water and hand pressed between layers of filterpaper to remove excess moisture. A NUNC tube was filled withthe mycelium and placed at –60°C for a minimum of 7 days andthen transferred to –10°C for curation.

To isolate DNA, up to one-quarter volume of a 950 µL eppen-dorf tube was filled with frozen mycelium and crushed with a spe-cially fitted pestle. Five hundred microlitres of 5% Chelex (Bio-Rad, Richomond, Calif.) solution was added to the pulverized ma-terial and the solution was heated for at least 30 min at 65°C. Se-rial dilutions of 1/10 and 1/100 of the DNA chelex solution were

used as template DNA solutions for polymerase chain reactions(PCR).

Macro-hair PCR amplification was carried out using the primersNS1, NS17, NS19, Basid3, NS5, and NS7 paired in various combi-nations with primers NS2, NS4, NS6, and NS8 (Gargas and Taylor1992). The 25 µL PCR reaction consisted of 1.25 µL of 20× PCRreaction buffer, 2.5 µL of 10× dNTPs (2.0 µM each), 2.5 µL eachof two primers (1 µM each), 0.625 units of TFL enzyme (EpicentreTechnologies, Madison, Wis.), 10 µL of DNA template, and sterile,filtered, and distilled water to bring the volume to 25 µL. APerkin–Elmer 9600 Thermo-Cycler was used for 40 thermal cy-cles with the following parameters: denaturization at 95°C for30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min.A final extension was made at 72°C for 5 min after thermal cy-cling.

Amplified DNA was electrophoresed on a 0.8% NuSieve GTGagarose diagnostic gel. The PCR product was prepared for se-quencing using a QIAquick PCR purification kit (Qiagen,Chatsworth, Calif.). PCR template was sequenced with cycle se-quencing using furochrome labeled dideosynucleotides (ABI se-quencing apparatus, Molecular Genetics Instrumentation Facility,University of Georgia, Athens, Ga.). Sequences from overlappingprimer pairs, for forward (5′ to 3′) and reverse (3′ to 5′) segments,were manually assembled and compared for accuracy, with refer-ence to chromatograms. An initial alignment of the GenBank se-quences was made with ClustalV (Higgins and Sharp 1989). Sootymold sequences were added as they were available, with manualadjustments using the edit function inanalyses were conducted us-ing test version 4.0d64 of PAUP written by David L. Swofford.The data set consisted of 1147 alignable sites of which 416 siteswere variable and 184 sites were phylogenetically informative. Thealignment is available from the author.

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Species Source

Alternaria alternata GB-AAU05194Antennariella californica GB-AF006722, VS, CC-LAM300855Aureobasidium pullulans GB-M55639Capnobotryella renispora GB-AF006723, VS, CC-ATCC64891Capnodium dematium GB-AF006724, VS, CC-LAM20668Capronia pilosella GB-U42473Chaetasbolisia falcata GB-AF006725, VS, CC-LAM21988Dothidea insculpta GB-U42474Dothidea hippophäeos GB-U42475Eremascus albus BG-M83258Erotium rubrum GB-U00970Rhinocladiella

(=Fonsecaea) pedrosoiGB-L36996

Hypomyces chrysopermusGB-M83259Neurospora crassa GB-X04971Ophiostoma ulmi GB-M83261Pleospora rudis GB-U00975Schizosaccharomyces

pombeGB-X54866

Scorias spongiosa GB-AF006726, CC-UAMH4777Sporormia lignicola GB-U42478Taphrina deformans GB-U00971

Note: The sources of sequence and voucher specimens are given withthe abbreviations: CC, culture collection; ATTC, American type culturecollection; UAMN, University of Alberta Microbiology Herbarium; GB,Gene Bank; VS, voucher specimen; LAM, Natural History Museum ofLos Angeles County specimen number.

Table 1. Fungal species data.

The data were analyzed using the parsimony, neighbor joining,and maximum likelihood methods found in PAUP.Schizo-saccharomyces pombeand Taphrina deformanswere the desig-nated outgroup taxa. Branch support values were determined froma 50% majority-rule consensus tree from 2000 bootstrapped datasets with the maximum parsimony optimality criterion (Felsenstein1985). Five likelihood-based Kishino–Hasegawa tests (Kishino andHasegawa 1989) were used to evaluate various hypotheses(Tables 1 and 2). Trees with the capnodiaceous species attached tothe subclade withCapronia pilosellawere prepared with the con-straint option of PAUP. A tree withCapnobotryella renisporaat-tached to the subclade withPleospora rudiswas likewise prepared.The log likelihood of trees was determined using PAUP. The treewith the highest log likelihood value was deemed the “best tree.”

A tree wasconsidered “significantly worse” and rejected if thet value (Table 2) was greater than 2.

The data are compartmentalized rather than used in a total evi-dence (Kluge 1989). The molecular data are used for cladistic anal-ysis. Select morphological character data are projected onto theDNA tree with elaboration in discussion as is done in similar re-cent studies (Berbee 1996; Kuldau et al. 1997; Reddy et al. 1998;Wedin and Tibell 1997).

Results and discussion

The analysis with the maximum parsimony optimality cri-terion and branch-and-bound search yielded nine trees with

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TreeParsimonytree-length ln L1 Difference ln L2 SD3 t value4

Significantlyworse5

01 675 –5420.97742 0.00000 0.00000 0.3640 No02 675 –5421.63341 0.65598 3.87137 0.1694 No03 675 –5425.7904 4.10161 10.92475 0.3754 No04 675 –5420.97742 0.00000 0.00000 0.0831 No05 675 –5421.65598 3.87137 3.41380 0.8655 No06 675 –5425.07904 4.10161 10.92475 0.3754 No07 675 –5369.90825 Best08 675 –5421.65598 0.65598 3.87137 0.1694 No09 675 –5425.07904 4.10161 10.92475 0.3754 No10 677 –5428.96418 7.98676 5.28245 1.5119 No11 772 –5611.96090 190.96347 30.07678 6.3499 Yes12 723 –5614.93606 193.95864 28.48396 6.8094 Yes13 725 –5626.78720 205.80977 29.15844 7.0583 Yes14 730 –5636.11778 215.14035 27.04143 7.9560 Yes15 716 –5562.48989 141.51247 23.19711 6.1004 Yes16 682 –5446.55800 25.58058 11.60356 2.2045 Yes

Note: 1, log likelihood; 2, difference in log likelihood compared with that of best tree; 3, the standard deviation of loglikelihood; 4, thet value is determined by dividing the difference in log likelihood by the standard deviation; 5, a tree isconsidered significantly worse if the difference in log likelihood is more than twice the standard deviation.

Table 2. Kishino–Hasegawa tests.

Tree 01 = (1,(2,((((3,4),(8,9)),(((5,(6,7)),((16,(18,19)),(17,20))),((13,14),15))),((10,12),11)))Tree 02 = (1,(2,((((3,4),(8,9)),(((5,(6,7)),(((16,18),19),(17,20))),((13,14),15))),((10,12),11))))Tree 03 = (1,(2,((((3,4)(8,9)),(((5,(6,7)),(((16,18),17),(19,20))),((13,14),15))),((10,12),11))))Tree 04 = (1,(2,((((3,4),(8,9)),((10,12),11)),(((5,(6,7)),((16,(18,19)),(17,20))),((13,14),15)))))Tree 05 = (1,(2,((((3,4),(8,9)),((10,12),11)),(((5,(6,7)),(((16,18),19),(17,20))),((13,14),15)))))Tree 06 = (1,(2,((((3,4),(8,9)),((10,12),11)),(((5,(6,7)),(((16,18),17),(19,20))),((13,14),15)))))Tree 07 = (1,(2,(((3,4),(8,9)),((((5,(6,7)),((16,(18,19)),(17,20))),((13,14),15)),((10,12),11)))))Tree 08 = (1,(2,(((3,4),(8,9)),((((5,(6,7)),(((16,18),19),(17,20))),((13,14),15)),((10,12),11)))))Tree 09 = (1,(2,(((3,4),(8,9)),((((5,(6,7)),(((16,18),17),(19,20))),((13,14),15)),((10,12),11)))))Tree 10 = (1,(2,(((3,4),(8,9)),((((5,6(6,7)),((16,(18,19)),(17,20))),((10,12),11)),((13,14),15)))))Tree 11 = (1,(2,(((3,4),(20,8,9)),((((5,(6,7)),((16,(18,19)),(17))),((13,14),15)),((10,12),11)))))Tree 12 = (1,(2,(((3,4),(8,9)),(((5,(6,7)),((13,14),15)),(((16,(18,19)),(17,20)),((10,12),11))))))

Note: The topology convention follows that of the PAUP manual. Trees 01–09 are parsimony trees.Tree 10 is the nearest neighbor tree. Trees 11–12 are constraint hypothesis trees based on ParsimonyTree 07. Tree 11.Capnobotryella renisporais attached toCapronia pilosella. Tree 12. TheCapnodiales and Pleosporales clades are constrained as sister groups. The numbered taxa are:1, Schoizosaccharomyces pombe; 2, Taphrina deformans; 3, Eurotium rubrum; 4, Eremascus albus;5, Aureobasidium pullulans; 6, Dothidea hippophäeos; 7, Dothidea insculpta; 8, Capronia pilosella;9, Rhinocladiella(=Fonsecaea) pedrosoi; 10, Pleospora rudis; 11, Sporormia lignicola, 12, Alternariaalternata; 13, Hypomyces chrysopermus; 14, Neurospora crassa; 15, Ophiostoma ulmi; 16, Scoriasspongiosa; 17, Capnodium dematium; 18, Antennariella californica; 19, Chaetasbolisia falcata;20, Capnobotryella renispora.

Table 3. Tree descriptions.

a length of 675, a consistency index of 0.7141, a homoplasyindex of 0.2859, and 174 parsimony informative characters.The mean base frequencies of the data set were A =0.27,T = 27, G = 0.26, and C = 0.20.

Filamentous ascomycetes clades found by Berbee (1996)and Spatafora (1995) were represented in the trees foundfrom the data set used in this study as predicted by the taxonselection, including the Plectomycetes and Chaetothyrialesclade, the Dothideales clade, a Pyrenomycete clade, and thePleosporales clade. Five agreement subtrees included 16 of18 ingroup taxa: (1) Plectomycetes:Eurotium rubrum, Ere-mascus albus; (2) Chaetothyriales:Capronia pilosella,Rhinocladiella (=Fonsecaea) pedrosoi; (3) Capnodiales(capnodiaceous sooty molds):Antennariella californica,

Capnobotryella renispora, Capnodium dematium, Chaetas-bolisia falcata, and Scorias spongiosa; (4) Dothideales:Aureobasidium pullulans, Dothidea hippophäeos, Dothideainsculpta; (5) Pleosporales:Pleospora rudis, Alternariaalternata, Sporormia lignicola; and (6) Pyrenomycetes:Hypomyces chrysospermus, Neurospora crassa, andOphios-toma ulmi.

Tree 07 was determined as the Best Tree with maximumlikelihood analysis (Table 2; Fig. 1). Branch support on Tree07 indicated by the bootstrap value (BSV) ranged from nearthe 50% majority rule minimal value for the Capnodialesclade to 75% for the Dothideales clade and over 95% for thePleosporales, Plectomycete, Chaetothyriales, and Pyreno-mycete clades (Fig. 1).

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Fig. 1. Most parsimonious Tree 07 from nine generated from the data used in this study with PAUP. Clades: 1, Plectomycetes;2, Chaetothyriales; 3, Capnodiales; 4, Dothideales; 5, Pleosporales; 6, Pyrenomycetes (clade names 1, 2, 5, and 6 are those used byBerbee (1996));S. pombeand T. deformanscomprise the outgroup. The diagrammatic figures represent an ascocarp midsection withthe silhouette of a bitunicate ascus and the Luttrellian Systematics character combinations (Luttrell 1955; Barr 1987a). The illustrationnear Clade 3 depicts apically originating, downward directed periphysoidal sterile elements and its convergent origins. The illustrationnear Clade 4 shows no sterile elements typically used to characterize the Dothideales. The basally originating and upward directedpseudoparaphyses typical of the Pleosporales are depicted near Clade 5 (the cartoons are patterned after Fig. 2 from Eriksson (1981)).Bootstrap values greater than 50 are indicated.

The neighbor-joining tree was found with the maximumparsimony optimality criterion. The tree length was 677,consistency index 0.7120, and homoplasy index 0.2880.Five agreement subclades were similar to those in Tree 07from the parsimony analysis except for the positions of thePyrenomycete and Pleosporales clades.

The constraint trees were compared with the parsimonyand the neighbor-joining trees (Table 2). All of them weredetermined as “significantly worse.” The hypothesis of deri-vation of the capnodiaceous sooty molds from a unitunicateancestor as was found for the chaetothyriaceous species,Capronia pilosella(Berbee 1996), is rejected. The hypothe-sis of a Pleosporales kinship for the Capnodiales is rejected.

The capnodiaceous sooty molds constitute a monophyleticgroup, named the Capnodiales clade (Fig. 1 (clade 3)).Sco-rias spongiosaand Capnodium dematium(Capnodiaceae),Antennariella californica(Antennulariaceae), andChaetas-bolisia falcata and Capnobotryella renispora(Metacapno-diaceae) form the clade. The Dothideales clade is a sistergroup. The BSV for the branch supporting both these twoclades was low with a higher BSV for the immediate branchof the Dothideales clade than for the Capnodiales clade(Fig. 1). Among the nine most parsimonious trees, thebranching pattern supporting the Capnodiales clade wasmade variable with the shifting of the positions ofChaetas-bolisia falcataandCapnobotryella renisporaon the reoccur-ring subclade ofScorias spongiosa, Capnodium dematium,and Antennariella californica.

The Capnodiales clade, represented in this study by bothsexual and asexual species, has come full circle in a phylo-genetic sense with the original concept of the taxonCapno-dium (Montagne 1849) and the Eucapnodieae (Fraser1935b). The sister relationship to the Dothideales and the re-jection of kinship of capnodiaceous taxa withCaproniapilosella and its associated “black yeast” ascomycetes aswell as the unlikelihood of sister status with the Pleosporalesreflect the phylogeny implied by Luttrellian taxonomic con-cepts (Luttrell 1951; Reynolds 1981).

The major shared Capnodiales characters are the foliico-lous habit and copious, darkly pigmented hyphae. Theascocarpic elements include a characteristic capnodiaceousperiphysoid sterile element. One difference with that of thesense of the periphysoid as having a downward direction, ifany at all in reality, is a flexibility of the capnodiaceousperiphysoid so that it may become redirected toward the api-cal pore. A tendency for these structures to resorb may ob-scure their observation in older herbarium material. Thecapnodiaceous periphysoid is always associated with afissitunicate type of bitunicate ascus (Reynolds 1989b). Boththese attributes are found in a presumedly stromatic fruitbody.

Acknowledgements

Acknowledgement is made to Mary Berbee, M.E.B.Bigelow, Eric Swann, Tim Szaro, and John Taylor for ad-vice. Financial support for this project was provided fromthe National Science Foundation and the Los AngelesCounty Museum of Natural History Foundation.

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