North American Fungi

Volume 8, Number 10, Pages 1-13 Published June 19, 2013

Vialaea insculpta revisited

R.A. Shoemaker, S. Hambleton, M. Liu

Biodiversity (Mycology and Botany) / Biodiversité (Mycologie et Botanique) Agriculture and Agri-Food Canada / Agriculture et Agroalimentaire Canada 960 Carling Avenue / 960, avenue Carling, Ottawa, Ontario K1A 0C6 Canada

Shoemaker, R.A., S. Hambleton, and M. Liu. 2013. Vialaea insculpta revisited. North American Fungi 8(10): 1-13. doi: http://dx.doi: 10.2509/naf2013.008.010

Corresponding author: R.A. Shoemaker: [email protected]. Accepted for publication May 23, 2013

http://pnwfungi.org Copyright © Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada

Abstract: Vialaea insculpta, occurring on Ilex aquifolium, is illustrated and redescribed from nature and pure culture to assess morphological features used in its classification and to report new molecular studies of the Vialaeaceae and its ordinal disposition. Tests of the germination of the distinctive in water containing parts of Ilex flowers after seven days resulted in the production of appressoria without mycelium. Phylogenetic analyses based on a fragment of ribosomal RNA gene small subunit suggest that the taxon belongs in .

Key words: and Vialaeaceae, (), (Diatrypales), and Hyphonectriaceae (Xylariales), Ilex, .

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Introduction: Vialaea insculpta (Fr.) Sacc. is on oatmeal agar at 20°C exposed to daylight. a distinctive species occurring on branches of Isolation attempts from several other collections Ilex aquifolium L. Oudemans (1871, tab. v, f.9) yielded only contaminants. The extractions were illustrated the asci and ascospores as did performed using the UltraClean Microbial DNA Saccardo (1896, Tav. V. fig. 10a,b) but Saccardo isolation kit (Mo BIO Laboratories, Inc. gave a misleading illustration of perithecia Carlsbad, CA USA) according to the immersed in a surrounding stroma (fig. 10c), manufacture’s protocol. A fragment of ribosomal which may have influenced later authors in the RNA gene 18S small subunit was amplified using ordinal disposition. Cannon (1995) reviewed the a primer pair NS1 and NS6 (White et al 1990). history of the various placements of V. insculpta PCR was performed in 10 µl reactions containing as follows: Amphisphaeriaceae (Diatrypales) 0.1 mM dNTPs (Invitrogen Canada Inc. (Chadefaud 1957, 1960, 1961); ‘Vialaeacéés’ Burlington, ON), 2 pmoles of each primers, 1 µl (Diatrypales) (Schrantz 1960); 10× TitaniumTm Taq buffer, 0.1 µl 50× (Sphaeriales) (Müller & von Arx 1962); TitaniumTm Taq DNA Polymerase (BD Amphisphaeriaceae (Xylariales) (Müller & von Biosciences, Mississauga, ON). Thermal cycling Arx 1973; Barr 1978; Dennis 1978; Hawksworth conditions were set as: 95°C for 3 min followed et al. 1983; Redlin 1989; Eriksson & by 36 cycles of 95°C for 30 sec, 63°C for 20 sec, Hawksworth 1993); and 72°C for 2 min, followed by a final extension (Xylariales) (Barr 1990). Cannon (1995) of 72°C for 8 min and hold at 10°C. PCR proposed the new family Vialaeaceae, which he products were direct-sequenced using BigDyeTm tentatively placed in Diaporthales. Kirk et al. Terminator v. 3.1 Cycle Sequencing Reaction Kit (2008) placed Vialaeaceae in an uncertain (ABI Prism/ Applied Biosystems, Streetsville, position within . These ON) in 10 µl sequencing reactions using the PCR dispositions were made on the basis of primers. The reaction mix included 1.75 µl 5× morphology. The current molecular study based Buffer, 0.5 µl 2.5x BDT sequencing Mix and 1.6 on a culture that produced mature perithecia in pmol primer. Amplicons were purified by vitro, and a similar study based on fresh ethanol/sodium acetate precipitation and perithecia dissected from Ilex aquifolium stems analyzed by Applied Biosystems 3130×1 Genetic adds new information on the classification of the Analyzer (AppliedBiosystems, Streetsville, ON). organism. Phylogenetic analyses based on a Sequences were edited using SequencherTM ver fragment of ribosomal RNA gene small subunit 4.8 (Gene Codes Corporation, Ann Arbor, MI). suggest that the taxon belongs to Xylariales. Cultural studies have been impeded by the lack of germination of the ascospores and, in our Phylogenetic analyses: experience, by the frequent occurrence of Sixty-seven DNA sequences of 18S small subunit contaminants: Phoma sp., Diaporthe sp., ribosomal RNA gene were downloaded from Pullularia sp., and Diplodia ilicicola Desm. GenBank by searching nucleotide sequences in generated by AFTOL. After preliminary analyses showing that Vialaea is Materials and Methods located in Xylariales, 26 more DNA sequences DNA extraction, PCR and sequencing: DNA from Xylariales were included. Finally ninety- was extracted from whole perithecia dissected four DNA sequences (Table 1) were included in a from twigs of specimen DAOM 240257, and parsimony analysis. Sequences were aligned from the perithecia and mycelium of vegetative with MAFFT version 6 isolate, DAOM 240860, that produced (http://mafft.cbrc.jp/alignment/server/index.ht perithecia, asci and ascospores in pure culture ml). Heuristic search settings were as follow:

Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 3 starting tree obtained by random stepwise and illustrations of the microscopic features of addition, one tree held at each step during asci and ascospores were given by Oudemans stepwise addition, branch-swapping by tree- (1871, p. 30, Pl. V, Figs. 9, 10) and by Saccardo bisection-reconnection (TBR), 20 replicates, (1896, p. 67, Tab. V. fig. 10 b,c) who in fig. 10c number of re-arrangements per replicate limited illustrated several perithecia immersed in to 1,000,000. For bootstrapping analysis, 200 stroma, above the wood, with the tips of replicates were conducted with heuristic search individual beaks protruding but barely evident settings as mentioned previously. above the raised surface layer of host material. He described the structure as follows. Results “Stromatibus laxe gregariis, pustulatis, Vialaea insculpta (Fr.) Sacc., Bull. Soc. Mycol. epidermide tumidula velatis, 2 mm diam., intus France 12: 66. 1896 flavidis.” From recent examinations, the typical ≡ Sphaeria insculpta Fr.: Fr., Elench. Fung. position is intraepidermal with flush beaks 2: 95. 1828 surrounded with a well developed clypeus and ≡ Zignoëlla insculpta (Fr.: Fr.) Sacc., Syll. the naked spherical part of the perithecium Fung. 2: 225. 1883 situated below without any surrounding stroma ≡ Boydia insculpta (Fr.: Fr.) Grove, J. Bot., as shown by Redlin (1989, Fig. 2) and quite London 59: 13. 1921 unlike the stromatic Diaporthales. = Boydia insculpta forma sparsa Grove, J. Bot., London 59: 15. 1921, nom. inval. Morphology: Perithecia solitary or a few in a = Boydia remuliformis A.L. Smith, Trans. group. Clypeus surrounding the upper beak, up Brit. Mycol. Soc. 6: 151. 1921 to 400 μm diam., composed of an upwardly = Cryptospora ludwigiana Kirschst., aligned pallisade of rectangular to globoid cells Hedwigia 91: 195. 1944 beneath the cuticle and above the phellem (Fig. 1). Beak immersed in clypeus, with a wall 16-20 Symptoms: The disease of Ilex aquifolium, μm wide of 2-5 layers of 4-7 μm thin-walled English holly, is a die-back of branches with or textura globosa, 300-400 μm long, 100-130 μm without conspicuous cankers but with wide. Ostiole 60-80 μm wide, filled with numerous, small, white discs of raised outer wall numerous, hyaline, periphyses, 2-3 μm wide of the epidermis, often circumscribed by a (Fig. 11). Perithecium wall in surface view a clypeus, which appears as a brown-pigmented textura globosa of 12-20 μm thin-walled cells ring. Within the clypeus are a number of very (Fig. 2); in section, 20-50 μm thick of 3-6 layers small points, which are the flush apices of of mainly rectangular cells 15-20 x 8-12 μm; perithecium beaks. The brown circular edge of body 300-450 μm. Paraphyses not observed. the clypeus appears as though engraved under See the description and illustration of short, the upper half of the split epidermis of the bark septate, tapered paraphyses by Cannon (1995). (insculpta). The Canadian collections examined Asci free-floating at maturity, unitunicate, with usually do not include conspicuous cankers. 8 bi- to triseriate spores (Fig. 8), 70-90 x 6-8 However, Atkinson & Trelawny (1962) μm. Apical apparatus a coussinet and illustrated cankers they observed at Saanichton, manubrium 5-6 μm diam distally, slightly B. C. in a commercial planting. Dennis (1968, narrowed to 3-4 μm proximally, 6-8 μm long Fig. 23c) illustrated the usual appearance of the (Fig. 7), subtended by a flat subapical ring 3 μm fungus in branches without cankers. For some diam. IKI+ (blue) (Fig. 12). Ascospores of two years, the disease was known as Boydia canker fusoid parts joined by a narrow isthmus, curved, and attributed to Boydia remuliformis A.L. 70-90 x 6-8 μm, transversely 0-1-septate (in Smith, a later synonym. An early description isthmus), rarely with two close septa, septum

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thin, wall very thin, hyaline, cell with the peak season September to December content granular, multiguttulate, later with one and March through April, if rainfall was large guttule, surface smooth, (Fig. 3), without adequate. Adrian Leuchtmann who has studied evident appendages or sheath (in India ink). many endophytic fungi suggested that spores The clypeus that raises the outer wall of the stem may have dormancy and germinate on newly epidermis is conspicuous on diseased plants but opened buds in the spring (pers. comm. 19 Sept. absent around the long cylindrical beak of 2003). Species within the Ilex are perithecia formed in agar cultures. Redlin dioecious. The ascospores might be adapted to (1989) found that a clypeus could be induced to selectively infect opening female flowers perhaps form in culture in response to contact aided by pollinating bees as vectors. Redlin (stereotropism). (1989) reported adhesion of spores by one tip to the glass wall of liquid culture vessels. Specimens examined: on Ilex aquifolium; AUSTRIA, Steirmark, Graz, Geidorf Distr. Bot. Classification based on morphology: Garden, Karl-Franzens-Univ., C. Scheuer, 21 Chadefaud (1960, p. 605-606, Fig. 454, 1) placed Oct. 2003, DAOM 232315; CANADA, BC, Vialaea in Diatrypales allied with Cromarty, Sidney, M.E. Barr 11068, 4 May 2005, . He considered the apical DAOM 240203, plus 5 collections from the same apparatus in the ascus of Vialaea very typical of location Barr 8798, 9589, 10647, 10777 and 6 the Diatrypales. He remarked on the collections from Sidney, Ardmore, Barr 8641, unique ascospores and noted some resemblance 8763, 8879, 9095, 9134, 10075; Steveston, to the ascospores seen in Richmond, S.A. Redhead, 7 Oct. 2008, DAOM (Diaporthales). However, the illustration he 240073; South of Duncan, Vancouver Island, 10 gave for Pleuroceras cordianum (Ellis & Kelsey) Oct. 2008, S.A.R., 10 Oct. 2008, DAOM 240074; Chadefaud (1960, p. 591, Fig. 466, 1) was based Vancouver, corner Locarno Cresc. & N.W. on an unpublished study by Schrantz and is not Marine Drive, S.A.R., 5 Oct. 2008, DAOM at all like the fungus described by Ellis & Kelsey. 240075; Victoria, 506 W. Burnside Road, Müller & Arx (1962, p. 753) placed Vialaea in Brenda Callan, 27 Mar. 2009, DAOM 240257, Diaporthaceae along with Pleuroceras Reiss. source of perithecia from nature used for The type species of Pleuroceras, P. cryptoderis extraction; Victoria, 506 W. Burnside Road, (Lév.) Höhn., has many of the morphological Brenda Callan, 05 Feb. 2010, DAOM 240860, features of V. insculpta as seen in the source of pure culture also used for direct illustrations by Müller & Arx (1962, pp. 752, extraction from extirpated perithecia. 753). Müller & von Arx (1962) placed V. insculpta in Diaporthaceae despite Schrantz’s Phenology: Ellis & Ellis (1985, p. 149) reported (1960) proposal of Vialaeacéés (nom. inval.). the season in Britain as Sept.-Apr. Cannon Later, Müller & von Arx (1973) placed it in (1995) provided data on 13 collections (mainly Amphisphaeriaceae and Barr (1978, p. 211) British) of which nine were collected between concurred but later, accepted a placement in Dec. and Apr. with one in June and three in Hyponectriaceae (Xylariales) (Barr 1990). August. From DAOM, 11 collections over 11 Cannon (1995) proposed Vialaeaceae, tentatively years at Sidney BC, two identifiable (mature) placed in Diaporthales. The ascus structure samples were collected in Nov., Dec. and Jan., reported here accords with that illustrated by one in Feb., three in May, and one in July. At Chadefaud (1957, 1961) and Redlin (1989) and is Saanichton, a xeric site in interior BC, the not discordant with that of members of seasonal cycle of ascospore discharge was Amphisphaeriaceae. Like Redlin, we did not studied in detail by Atkinson & Trelawny (1962) observe the “tractus” and “corps ombilique”

Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 5 reported by Chadefaud but these structures the teleomorph in pure culture on autoclaved require special staining techniques. There are twigs of Ilex and on several culture media (CMD, several unusual features: free-floating asci that MA, OA and PDA). He found that even the may slide intact up the ostiole and the distinctive ascospores that formed in culture were non- clypeus. Cannon (1995) noted pores in the germinable. Redlin (1989) reported that in vitro perithecium wall cells and gave good the ascospores accumulate in a moist droplet at illustrations of the infrequently observed the tip of the beak but that ascospores also are paraphyses that were broad at the base, closely found on the lid of Petri dishes. This might have septate and narrowed rapidly to the apex, as well occurred by forcible discharge of either whole as to many other features of the fungus. We did asci or individual ascospores. He also noted that not observe paraphyses in our material. The in liquid malt extract, the ascospores adhere to various families and orders in which Vialaea has the bottom of the Petri plate by an attachment at been placed are: Amphisphaeriaceae and one end. The nature of the attachment was not Hyponectriaceae (Xylariales), Diatrypaceae described. (Diatrypales), Valsaceae and Vialaeaceae (Diaporthales) (Cannon, 1995). Pure Cultures: DAOM 240860, a fresh collection of Ilex aquifolium was collected by On the basis of morphology with special Brenda Callen at Victoria, B.C. on 5 Feb. 2010. emphasis on the ascus apical apparatus we place Isolations of V. insculpta were accomplished by Vialaeaceae in Xylariales with the common excising basal parts of perithecia from infected features of pulvillus, manubrium demonstrable plant material. Pure cultures were obtained on if stained in blue ink and an annulus that turns 2% malt agar plates and subcultures made on 16 blue when tested with IKI. The latter feature Feb. 2010 with one central inoculum to 2% malt precludes its placement in Diatrypales or agar plates. The single colony diameter was 3.9- Diaporthales. Hyponectriaceae (Xylariales), 4.0 cm after 7 days and the mycelium was represented by buxi, has been inconspicuous and hyaline. The colony changed reported with a pulvillus and an IKI+ve inner rapidly to dark brown and initials of immersed ring (Chadefaud 1960). However, tests of H. perithecia were visible. By 1 March (22 days) the buxi DAOM 97954 (=IMI 100623) revealed only colony diameter reached 6.5-7.0 cm and did not a pulvillus without any evidence of an inner ring, expand further in the 8.5 cm plate. On 7 March either amyloid or non-amyloid, and this (28 days) a short beak appeared from some of placement is not accepted here. The closest the immersed perithecia. These perithecia had family affinities of Vialaceae are near asci and ascospores present. Amphisphaeriaceae but distinct on gross differences in ascospore form. On 16 Feb. 2010, transfers were made also to the growth restricting medium, DG18. By 6 March Biology: Lugenbühl and Müller (1980) found 2010 they had a diameter of 5-8 mm and the the fungus to be an endophyte in stems of Ilex colony was of dense black mycelium. On 6 aquifolium in Ronco, Tessin, Switzerland and March 2010, subcultures were made to 2% malt Musieges, Haute Savoie, France. Their and oat agar for further comparison. isolations were from mycelium grown from Subcultures on oatmeal agar plates, 1 inoculum surface-disinfected plant parts. Redlin (1989), in each of 4 quadrants after 2o days at 20° C had despite many attempts, was unable to get no perceptible surface mycelium but perithecia ascospores to germinate and was forced to rely had developed deep within the agar except at the on mycelial isolates of the fungus, which edge of the quadrants where a clear zone was eventually, after one month, consistently formed evident. Subcultures on 2% malt agar, 1

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inoculum in each of 4 quadrants, after 20 days at Associated fungi: Cannon (1995) noted 20° C., had dark brown surface mycelium that Diaporthe pardalota (Mont.) Fuckel on material was irregularly branched somewhat from Isle of Man and we found D. eres on dichotomously with lines of perithecia on some material from B. C. as well as a species of parts of the colony. Growth was restricted to 4 Phoma. The Diaporthe is easily segregated by cm and a clear demarcation zone was evident presence of a distinct black stroma line on the between the colonies. Redlin drew attention to bark surface. The clypeus of V. insculpta at its the sudden change of color of the colony from most extreme appears as a vague, pale brown, hyaline to brown as being characteristic of this etched line in the bark surface. The presence of species and it is useful on some media but not on other fungi makes the use of tissue isolations oat agar. suspect and increases the need for a germination Anamorph: No anamorph was observed in method for ascospores of V. insculpta as a cultures of V. insculpta. source of pure cultures.

Ascospore germination attempts: Redlin (1989, Molecular studies: Castlebury et al. (2002) p. 303) noted: “Ascospores did not germinate” sequenced all three cultures deposited as V. and “Ascospore walls disintegrated on culture insculpta at CBS: 735.68, Netherlands, van der media several days after treatment to Aa; 345.76, Switzerland, Luginbuehl; 106.89, temperature extremes.” Ascospores from pure Oregon, U.S.A., Redlin = ATCC 66008, but none culture DAOM 240860 suspended in sterile of these proved to belong in the water were added to plant parts: Ilex verticillata Sordariomycetidae. Castlebury (pers. comm. 23 young flowers, buds, stems and overwintered Sept 2003) reported CBS 735.68 gave 1331/1379 leaves collected in May as well as to mango skin, identities to a Setosphaeria monoceras LSU and banana pulp placed on sterilized slides. No sequence [96.5%]; CBS 345.76 has 1285/1370 mycelial growth was observed from the identical positions to Cainia graminis LSU ascospores on any of the substrates tested. After sequence [93.8%]; CBS 106.89 (=ATCC 66008) 7 days, when the growth of other fungi was 1328/1331 identities to a Phoma herbarum LSU becoming evident in the watery suspension, a sequence [99.8%]]. Castlebury et al. (2002) remarkable change was observed in the stated “Representatives of Vialaeaceae could not ascospores that were near the flower parts. At or be located for this study” and did not deposit near the terminus of spores a spherical body 10- sequences of these disparate isolates in 12 μm diam., with a thin wall was observed GenBank. Schoch (pers. comm.) prepared a LSU (Figs. 4,5,9,10). This structure remained sequence from CBS 345.76 as part of AFTOL, attached to the spore and adhered to the glass but did not deposit the sequence in GenBank. At slide. It might be an attachment device, a present, there are no sequences in GenBank precursor to an appressorium, and be the basis under the name V. insculpta. for the attachment of ascospores to the wall of liquid-culture vessels reported by Redlin (1989). Current molecular work is based on perithecia dissected directly from specimen DAOM On the surface of Ilex twigs naturally infected 240257, and from the vegetative pure isolate of with V. insculpta, small, brown appressoria with DAOM 240860 that sporulated in culture. PCR a central pore indicative of an infection peg were amplification using the purified DNA resulted in detected (Fig. 13). These might be appressoria a single approximate 1 kb band and clean of V. insculpta. sequence. The alignment of 94 DNA sequences

Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 7 resulted in 3281 characters. Removing large Literature cited fragments of insertion/deletion, 1750 characters Atkinson, R.G. and J.G. Trelawny 1962. A spore were retained, among which 649 characters were trap to study sporulation in tree canker diseases. parsimony informative. A heuristic search Canadian Plant Disease Survey 42(3): 151-154 resulted in 263 most parsimonious trees: L = 2982, CI = 0.608, RI = 0.706. The position of Barr, M.E. 1978. The Diaporthales in North Vialaea insculpta was within a clade composed America. Mycologia Memoir 7. 232 pages. J. of Xylariales and supported with 84% Cramer. Lehre bootstrapping value (Fig. 14). Taxon labels include GenBank Nos. and Family level Barr. M.E. 1990. Prodromus to nonlichenized classifications abbreviated as follows – AMP: pyrenomyceteous members of Class Amphisphaeriaceae, ANA: Anamorph, API: Hymenoascomycetes. Mycotaxon 39: 43-184 , BIO: Bionectriaceae, BOL: , CAL: , CER: Cannon, P.F. 1995. Studies on fungi with Ceratocystidaceae, CHA: Chaetosphaeriaceae, isthmoid ascospores: the genus Vialaea, with the CLY: , CON: description of the new family Vialaeaceae. Coniochaetaceae, COR: Cordycipitaceae, CRY : Mycological Research 99(3): 367-373 , DIP: Diaporthaceae, DIT: http://dx.doi.org/10.1016/S0953- Diatrypaceae, GNO: , GRA: 7562(09)80915-5 Graphostromataceae, HAL: Halosphaeriaceae, Castlebury, Lisa A., Rossman, Amy, Jaklitsch, HYC: Hypocreaceae, HYN: Hyponectriaceae, W.J. and Larissa N. Vasilyeva. 2002. A LAS: , LUL: Lulworthiaceae, preliminary overview of the Diaporthales based MAG: , MEL: on large subunit nuclear ribosomal DNA , MIC: Microascaceae, OPH: sequences. Mycologia 94(6): 1017-1031 Ophiostomataceae, PAP: Papulosaceae, SOR: http://dx.doi.org/10.2307/3761867 Sordariaceae, VAL: Valsaceae, VIA: Vialaeaceae, XYL: . Chadefaud, M. 1957. Les asques des Diatrypales. Comptes rendus hebdomadaires de l’Academie Discussion: In future studies, there is need to des Sciences, Paris 244: 1813-1815 explain the factors required for germination of the ascospores, to examine a possible role of Chadefaud, M. 1960. Les Végétaux non pollinating bees as spore vectors, and to test for vasculares (Cryptogamie). 1016 pp. Masson et infection of buds or female flowers leading to Cie. Paris systemic infection of seeds and otherwise test whether V. insculpta is an endophyte. Chadefaud, M. 1961. Sur un Pyrénomycète remarquable du Jardin Alpin de « La Jaysinia », Acknowledgements: Fresh collections were a Samoëns (Haute Savoie) : Le Vialaea insculpta contributed by Dr. C. Scheuer, Austria, and by (Fr. ? Oud !) Sacc. Recueil 2: 79-86 Dr. Scott Redhead and Dr. Brenda Callan, from B.C., Canada. Dr. Scott Redlin, North Carolina, Chadefaud, M. 1973. Les Asques et la supplied helpful advice regarding isolation systématique des Ascomycètes. Bulletin procedures and possible methods to induce trimestriel de la Societé Mycologique de France ascospore germination. Drs. Amy Rossman, L. 89(2): 127-170 Castlebury and Conrad Schoch contributed information on their molecular studies on CBS Dennis, R.W.G. 1978. British Ascomycetes. 585 cultures labelled Vialaea insculpta. pages. J. Cramer: Vaduz

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Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 9 and C.L. Schoch. 2006 A five-gene phylogeny of White, T. J., T. Bruns, S. Lee, and J. W. Taylor. . Mycologia 98 (6): 1018-1028 1990. Amplification and direct sequencing of http://dx.doi.org/10.3852/mycologia.98.6.1018 fungal ribosomal RNA genes for phylogenetics. Pp. 315-322 In: PCR Protocols: A Guide to Sung, G.H. and Spatafora, J.W. 2004. Cordyceps Methods and Applications, eds. Innis, M. A., D. cardinalis sp. nov., a new species of Cordyceps H. Gelfand, J. J. Sninsky, and T. J. White. with an east Asian-eastern North American Academic Press, Inc., New York distribution. Mycologia 96 (3): 658-666 http://dx.doi.org/10.1007/BF02464316 http://dx.doi.org/10.2307/3762183 Zhang, N., Castlebury, L.A., Miller, A.N., Winka, K. and E. Eriksson. 2000. Papulosa Huhndorf, S.M., Schoch, C.L., Seifert, K.A., amerospora accommodated in a new family Rossman, A.Y., Rogers, J.D., Kohlmeyer, J., (Papulosaceae, Sordariomycetes, ) Volkmann-Kohlmeyer, B. and G.H. Sung. 2006. inferred from morphological and molecular An overview of the systematics of the data. Mycoscience 41: 97-103 Sordariomycetes based on a four-gene phylogeny. Mycologia 98 (6): 1076-1087 http://dx.doi.org/10.3852/mycologia.98.6.1076

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Table 1. GenBank accession number (GB), classification (i.s. = incertae sedis) and citation for DNA sequences selected for phylogenetic analysis

GB Order Family Taxon Citation DQ471036 Boliniaceae microspora Spatafora et al 2006 DQ470989 Boliniales Boliniaceae Camarops ustulinoides Spatafora et al 2006 DQ471011 Calosphaeriaceae minima Spatafora et al 2006 AY544689 Chaetosphaeriaceae pleurothecii AFTOL unpublished DQ471007 Coniochaetaceae Coniochaeta ostrea Spatafora et al 2006 FJ176835 Diapothales Cryphonectriaceae Amphilogia gyrosa Schoch unpublished DQ862046 Diapothales Cryphonectriaceae Chromendothia citrina Zhang et al 2006 NG_013200 Diapothales Cryphonectriaceae Chromendothia citrina Zhang et al 2006 DQ862047 Diapothales Cryphonectriaceae Chrysoporthe cubensis Zhang et al 2006 DQ862048 Diapothales Cryphonectriaceae Cryphonectria parasitica Zhang et al 2006 NG_013201 Diapothales Cryphonectriaceae Cryphonectria parasitica Zhang et al 2006 DQ471023 Diapothales Cryphonectriaceae gyrosa Spatafora et al 2006 DQ471015 Diapothales Diaporthaceae Diaporthe eres Spatafora et al 2006 DQ862051 Diapothales Diaporthaceae Mazzantia napelli Zhang et al 2006 DQ862045 Diapothales Gnomoniaceae errabunda Zhang et al 2006 NG_013199 Diapothales Gnomoniaceae Zhang et al 2006 DQ836899 Diapothales Gnomoniaceae aesculi Zhang et al 2006 DQ862049 Diapothales Gnomoniaceae hypodermia Zhang et al 2006 DQ471019 Diapothales Gnomoniaceae Gnomonia gnomon Spatafora et al 2006 DQ862055 Diapothales Gnomoniaceae euphorbiae Zhang et al 2006 DQ862052 Diapothales Melanconidaceae alni Zhang et al 2006 DQ862053 Diapothales Melanconidaceae Melanconis marginalis Zhang et al 2006 DQ862043 Diapothales Melanconidaceae Melanconis stilbostoma Zhang et al 2006 DQ862054 Diapothales Melanconidaceae Melanconis stilbostoma Zhang et al 2006 NG_013198 Diapothales Melanconidaceae Melanconis stilbostoma Zhang et al 2006 DQ862050 Diapothales Valsaceae niveum Zhang et al 2006 NG_013203 Diapothales Valsaceae Leucostoma niveum Zhang et al 2006 DQ862056 Diapothales Valsaceae ambiens Zhang et al 2006 DQ862057 Diapothales Valsaceae salicis Zhang et al 2006 AY544687 Helotiales Leotiomycetes Leotia lubrica AFTOL unpublished AY544695 Helotiales Sclerotiniaceae Botryotinia fuckeliana AFTOL unpublished DQ862044 Hypocreales Bionectriaceae Bionectria ochroleuca Zhang et al 2006 NG_013131 Hypocreales Cordycipitaceae Cordyceps cardinalis Sung & Spatafora 2004 AY544693 Hypocreales Hypocreaceae Hypocrea americana Lutzoni et al 2004 NG_016487 Hypocreales Hypocreaceae Hypocrea americana Lutzoni et al 2004 DQ471033 Leotiomycetes i.s. Leotiomycetes Microglossum rufum Spatafora et al 2006 FJ176847 Lulworthiaceae obtusa Schoch unpublished DQ470994 Lulworthiales Lulworthiaceae Lindra thalassiae Spatafora et al 2006 DQ522855 Lulworthiales Lulworthiaceae grandispora James et al unpublished FJ176848 Lulworthiales Lulworthiaceae Lulworthia lignoarenaria Schoch unpublished FJ176849 Lulworthiales Lulworthiaceae Lulworthia uniseptata Schoch unpublished FJ176801 Magnaporthaceae medullaris Schoch unpublished DQ471031 Ceratocystidaceae Ambrosiella xylebori Spatafora et al 2006 FJ176845 Microascales Halosphaeriaceae Corollospora angusta Schoch unpublished FJ176846 Microascales Halosphaeriaceae Corollospora maritima Schoch unpublished DQ471026 Microascales Microascaceae longirostris Spatafora et al 2006 DQ471006 Microascales Microascaceae Microascus trigonosporus Spatafora et al 2006 DQ471020 Microascales Microascaceae setifera Spatafora et al 2006 DQ471038 mitosporic ascomycota Graphium penicillioides Spatafora et al 2006

Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 11

FJ176832 mitosporic ascomycota Graphium sp. Schoch unpublished

DQ470996 Ophiostomalates i.s. Lanspora coronata Spatafora et al 2006 DQ471003 Ophiostomatales Ophiostomataceae Ophiostoma piliferum Spatafora et al 2006 FJ176850 Ophiostomatales Ophiostomataceae Ophiostoma stenoceras Schoch unpublished DQ836897 Ophiostomatales Ophiostomataceae Ophiostoma stenoceras Zhang et al 2006 DQ471021 Lasiosphaeriaceae Bombardia bombarda Spatafora et al 2006 NG_013187 Sordariales Lasiosphaeriaceae Bombardia bombarda Spatafora et al 2006 DQ836894 Sordariales Lasiosphaeriaceae Lasiosphaeria ovina Zhang et al 2006 DQ471032 Sordariales Sordariaceae Gelasinospora tetrasperma Spatafora et al 2006 AY083815 Sordariomyetidae i.s. Apiosporaceae Apiospora sinensis Smith et al unpublished AY083817 Sordariomyetidae i.s. Apiosporaceae Appendicospora sp. Smith et al unpublished AY083816 Sordariomyetidae i.s. Apiosporaceae Arthrinium phaeospermum Smith et al unpublished DQ470998 Sordariomyetidae i.s. Papulosaceae Papulosa amerospora Spatafora et al 2006 FJ176809 Xylariales Amphisphaeriaceae Amphisphaeria umbrina Schoch unpublished AY083811 Xylariales Amphisphaeriaceae Amphisphaeria umbrina Smith et al unpublished AY083802 Xylariales Amphisphaeriaceae bambusae Smith et al unpublished AY083801 Xylariales Amphisphaeriaceae Cainia graminis Smith et al unpublished AY083814 Xylariales Amphisphaeriaceae tosta Smith et al unpublished AY083813 Xylariales Amphisphaeriaceae cupressi Smith et al unpublished AY083818 Xylariales Amphisphaeriaceae frondicola Smith et al unpublished AF104356 Xylariales Amphisphaeriaceae sp Metz et al 2000 AY083819 Xylariales Clypeosphaeriaceae Apioclypea sp Smith et al unpublished AY083812 Xylariales Clypeosphaeriaceae uniseptata Smith et al unpublished AY083807 Xylariales Diatrypaceae Cryptosphaeria eunomia Smith et al unpublished DQ471012 Xylariales Diatrypaceae Diatrype disciformis Spatafora et al 2006 DDU32403 Xylariales Diatrypaceae Diatrype disciformis Spatafora & Blackwell 1995 DQ836896 Xylariales Diatrypaceae Eutypa lata Zhang et al 2006 AY083806 Xylariales Diatrypaceae Eutypa sp. Smith et al unpublished DQ836900 Xylariales Graphostromataceae Graphostroma platystoma Zhang et al 2006 AY083808 Xylariales Graphostromataceae Graphostroma platystoma Smith et al unpublished AF130976 Xylariales Hyponectriaceae Hyponectria buxi Winka & Eriksson 2000 AF064049 Xylariales Hyponectriaceae nivalis Winka & Eriksson 2000 JN166015 Xylariales Vialaeaceae Vialaea insculpta this study DQ836895 Xylariales Xylariaceae Anthostomella torosa Zhang et al 2006 AY083804 Xylariales Xylariaceae Astrocystis cocoes Smith et al unpublished DCU32402 Xylariales Xylariaceae Daldinia concentrica Spatafora & Blackwell 1993 AY083809 Xylariales Xylariaceae Fasciatispora petrakii Smith et al unpublished AY083803 Xylariales Xylariaceae Halorosellinia oceanica Smith et al unpublished AY083810 Xylariales Xylariaceae Hypoxylon fragiforme Smith et al unpublished AY083805 Xylariales Xylariaceae necatrix Smith et al unpublished AY544719 Xylariales Xylariaceae Xylaria acuta AFTOL unpublished XCU32417 Xylariales Xylariaceae Xylaria curta Spatafora & Blackwell 1996 NG_013136 Xylariales Xylariaceae Xylaria hypoxylon AFTOL unpublished AY544692 Xylariales Xylariaceae Xylaria hypoxylon AFTOL unpublished XHU20378 Xylariales Xylariaceae Xylaria hypoxylon Spatafora & Blackwell 1993

12 Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13

Figs. 1-13 Vialaea insculpta. Fig. 1. beaked perithecium and clypeus splitting epidermis. Fig. 2. perithecium wall surface. Fig. 3, mature ascospores . Figs. 4,5. germinated ascospores. Fig. 6, clypeus splitting epidermis. Fig. 7. ascus pulvillus. Fig. 8. ascus. Figs. 9,10. ascospores germinated. Fig. 11. periphyses cotton blue. Fig. 12. ascus annulus IKI+. Fig 13. appressoria on branch surface, two showing penetration. Figs. 1-3 of 230260. Figs. 4,5,9,10,13 of 240860, Figs. 6,8 of 240073. Fig. 7 of 233690. Scale = 10 µm except for Fig. 1 = 100 µm.

Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 13

AY083811 AMP A mphisphaeria umbrina FJ176809 AMP Amphisphaeria umbrina AY083812 CLY Clypeosphaeria uniseptata AY083814 AMP Discostroma tosta 95 AY083813 AMP AF104356 AMP Pestalosphaeria sp AY083808 GRA Graphostroma platystoma AY083817 API Appendicospora sp. JN166015 VIA Vialaea insculpta AY083818 AMP Oxydothis frondicola * AF130976 HYN Hyponectria buxi DCU32402 XYL Daldinia concentrica AY083810 XYL Hypoxylon fragiforme AF064049 HYN Monographella nivalis 90 DQ471012 DIT Diatryp e disciformis DDU32403 DIT Diatrype disciformis Xylariales DQ836896 DIT Eutypa lata AY083806 DIT Eutypa sp. AY083802 AMP Arecophila bambusae AY083801 AMP Cainia graminis AY083807 DIT Cryptosphaeria eunomia AY083804 XYL Astrocystis cocoes AY083805 XYL Rosellinia necatrix AY083803 XYL Halorosellinia oceanica AY544719 XYL Xylaria acuta XCU32417 XYL Xylaria curta XHU20378 XYL Xylaria hypoxylon AY544692 XYL Xylaria hypoxylon 96NG_013136 XYL X ylaria hypoxylon 84 AY083809 XYL Fasciatispora petrakii DQ836900 GRA Graphostroma platystoma DQ836895 XYL Anthostomella torosa AY083819 CLY Apioclypea sp AY083815 API A piospora sinensis 100 AY083816 API Arthrinium phaeospermum DQ862044 BIO Bionectria ochroleuca NG_013131 COR Cordyceps cardinalis 100 AY544693 HYC Hypocrea americana Hypocreales NG_016487 HYC Hypocrea americana AY544689 CHA Carpoligna pleurothecii 100 FJ176845 HAL Corollospora angusta Chaetosphaeriales FJ176846 HAL Corollospora maritima FJ176832 ANA Graphium sp. DQ471020 MIC Petriella setifera DQ471026 MIC Microascus longirostris Microascales 98 DQ471006 MIC Microascus trigonosporus 98 DQ471031 CER Ambrosiella xylebori DQ471038 ANA Graphium penicillioides 99 DQ471003 OPH Ophiostoma piliferum DQ836897 OPH Ophiostoma stenoceras FJ176850 OPH O phiostoma stenoceras Ophiostomatales DQ470998 PAP Papulosa amerospora DQ471011 CAL Togninia minima Calosphaeriales FJ176835 CRY A mphilogia gyrosa 88 DQ862046 CRY Chromendothia citrina NG_013200 CRY Chromendothia citrina DQ862047 CRY Chrysoporthe cubensis DQ471023 CRY Endothia gyrosa DQ471015 DIP Diaporthe eres DQ862051 DIP Mazzantia napelli 92 DQ862050 VAL Leucostoman iveum NG_013203 VAL Leucostoma niveum DQ862057 VAL Valsella salicis 99 DQ862056 VAL DQ862045 GNO Apiognomonia errabunda NG_013199 GNO Apiognomonia errabunda Diaporthales DQ836899 GNO Cryptodiaporthe aesculi DQ862055 GNO Plagiostoma euphorbiae DQ471019 GNO Gnomonia gnomon 100 DQ862049 GNO Cryptosporella hypodermia 99 DQ862052 MEL Melanconis alni DQ862053 MEL Melanconis marginalis DQ862043 MEL Melanconis stilbostoma DQ862054 MEL Melanconis stilbostoma 98 NG_013198 MEL Melanconis stilbostoma DQ862048 CRY Cryphonectria parasitica 100 95 NG_013201 CRY C ryphonectria parasitica DQ471007 CON Coniochaeta ostrea DQ470996 Lanspora coronata Coniochaetales FJ176801 MAG Gaeumannomyces medullaris 100 DQ471036 BOL Camarops microspora Magnaporthales DQ470989 BOL Camarops ustulinoides Boliniales 99 99 DQ471021 LAS Bombardia bombarda 99 NG_013187 LAS Bombardia bombarda DQ836894 LAS Lasiosphaeria ovina Sordariales 72 DQ471032 SOR Gelasinospora tetrasperma FJ176847 LUL Lindra obtusa 100 97 DQ470994 LUL Lindra thalassiae DQ522855 LUL Lulworthia grandispora Lulworthiales FJ176849 LUL Lulworthia uniseptata FJ176848 LUL Lulworthia lignoarenaria AY544695 Botryotinia fuckeliana AY544687 Leotia lubrica DQ471033 Microglossum rufum Leotiomycetes (Outgroup) 10 changes Fig. 14. One of 263 most parsimonious phylograms based on 18S small subunit ribosomal RNA gene showing the position of Vialaea insculpta within a clade composed of Xylariales. L = 2982, CI = 0.608, RI = 0.706, RC = 0.430, HI = 0.392, G-fit -462.081. Taxon labels include GenBank Accession Nos. and Family level classifications (see text for key to abbreviations).