Mycological Society of America Phylogeny of Pezicula, Dermea and Neofabraea Inferred from Partial Sequences of the Nuclear Ribosomal RNA Gene Cluster Author(s): Edwin C. A. Abeln, Marian A. de Pagter, Gerard J. M. Verkley Reviewed work(s): Source: Mycologia, Vol. 92, No. 4 (Jul. - Aug., 2000), pp. 685-693 Published by: Mycological Society of America Stable URL: http://www.jstor.org/stable/3761426 . Accessed: 06/11/2011 14:32 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. Mycological Society of America is collaborating with JSTOR to digitize, preserve and extend access to Mycologia. http://www.jstor.org Mycologia, 92(4), 2000, pp. 685-693. © 2000 by The Mycological Society of America, Lawrence, KS 66044-8897 Phylogeny of Pezicula, Dermea and Neofabraea inferred from partial sequences of the nuclear ribosomal RNA gene cluster Edwin C. A. Abeln1 resulted in a rather artificial host-based classification. Marian A. de Pagter (Wollenweber 1939). Gerard J. M. Verkley Traditionally, material on conifers is identified as Centraalbureauvoor Schimmelcultures,PO. Box 273, Pe. livida and morphologically similar material on de- 3740 AG Baarn, The Netherlands ciduous trees as Pe. cinnamomea. Kowalski and Kehr (1992) reported the presence of Pe. cinnamomea as an endophyte in both deciduous and coniferous Abstract: The phylogenetic relationship between Pe- hosts (e.g., Picea abies), suggesting a wider host range zicula, Dermea and Neofabraea species (Dermatea- for this species. This was confirmed recently after the ceae, Leotiales) was investigated using sequence anal- investigation of fresh fructifications and isolations ysis of part of the 18S ribosomal DNA, the internal from various hosts. (1999). transcribed spacer 1, the 5.8S ribosomal DNA and Several taxonomic questions within or related to the internal transcribed spacer 2. DNA was isolated Pezicula remain to be answered. Ocellariawas discrim- from 44 CBS-strains (7 Dermea, 3 Neofabraea and 34 inated from Pezicula by most authors on the basis of Pezicula confirmed the species). Parsimony analysis an immersed, sessile apothecium with a white margin that Ocellaria ocellata morphology-based hypothesis while the apothecium of Pezicula is typically more should be considered as a true Pezicula Fur- species. pronounced, lacking the white margin (Wollenweber thermore, the of two taxa (Pezicula malicor- position 1939). However, Groves (1940) found it impossible ticis and Pe. alnicola) was found to be outside the to draw a clear-cut line between Ocellariaand Pezicula Pezicula main cluster. as he considered the apothecia of Pe. aurantiaca and Key Words: anthracnose, Der- Cryptosporiopsis, Pe. corni to be intermediate forms. In his compre- mateaceae, ITS, Ocellaria, rDNA, Phlyctema, Sclerope- hensive monograph of Pezicula, Verkley (1999), also zicula, taxonomy concluded that there were no grounds for maintain- ing Ocellaria as a separate genus, stressing the simi- larity between the Cryptosporiopsisanamorphs. INTRODUCTION In his monograph of the American species of Der- mea, Groves (1946) concluded that it was also impos- Pezicula Tul. & C. Tul. nom. cons. is a genus of in- sible to draw a boundary between Dermea and Pezi- operculate discomycetes classified in the family Der- cula, regarding species such as Pe. frangulae and Pe. mateaceae (order Leotiales), with Pezicula carpinea alnicola referable to either genus with (Pers.) Tul. & Tul. as the type species (Cannon and equal justifi- cation. Although these have been classified in Hawksworth 1983). Most species occur in the north- genera different subfamilies Nannfeldt (1932a) and Korf ern temperate zone growing endophytically on by (1973), the difference between certain Dermea and shrubs and trees, whereas some are parasitic. Ana- Pezicula is indeed not obvious nor is morph connections are known with Cryptosporiopsis species always their within the Bubak & Kabat and Phlyctema Desm. (Wollenweber evolutionary relationship Dermatea- ceae. 1939,Johansen 1949, Seaver 1951, Dennis 1974). Pe- zicula alba Guthrie (Guthrie 1959) is connected to Jackson (1913) proposed the generic name Neofa- braea for the Phlyctema vagabunda Desm., an anamorph with ba- newly discovered teleomorph of the ap- nana-shaped conidia, phialidic conidiogenesis and ple anthracnose fungus, until then known as Gloeos- eustromatic conidiomata (Zazzerini and Van der Aa porium malicorticis Cordley [= Cryptosporiopsiscurv- 1979, Sutton 1980). Some taxa are known producers ispora (Peck) Gremmen]. J0rgensen (1930) pro- of secondary metabolites with antibiotic activity (No- posed the name Neofabraea corticola for another ble et al 1991, Schulz et al 1995). The morphological species causing a bark disease on apple and pear. variation exhibited by many taxa in vivo and in vitro Nannfeldt (1932b) recombined both into Pezicula, considering Neofabraea a synonym of Pezicula. Most Accepted for publication January 24, 2000. taxonomists have followed this author, but many phy- 1 Email: [email protected] topathologists continued to use the name Neofabraea. 685 686 MYCOLOGIA To clarify the generic boundaries of Pezicula, Ocel- ison, Wisconsin). Sequences from the different strains were laria and Dermea, and to get a better insight in the aligned using MegAlign from the same package. Alignments evolutionary relations of these fungi, we compared a are available at TreeBASE (S464). was with a test version part of the gene coding for the 18S small ribosomal Phylogenetic analysis performed of PAUP (Swofford 1998). The data were divided into three subunit RNA, both internal transcribed spacers, and character sets. The first character set, the SSU-set (character the 5.8S gene. 79-776) consists of the 3'-end of the small subunit, the sec- ond character set (INDEL) comprises a large insert (char- acter and the third set consists of the MATERIALAND METHODS 374-757), (ITS-region) ITS1, the 5.8S, and the ITS2 (character 777-1265). Support The strains used in this study are listed in TABLE I. Strains for the branching topologies was evaluated using bootstrap were transferred from agar cultures to 2 mL liquid medium analysis (Felsenstein 1985). Parsimony analysis was per- (2% malt extract) and incubated on a rotary shaker (300 formed with exclusion of the INDEL, using a heuristic rpm) for 2-3 wk at RT. search with the following parameters: characters were un- ordered and had equal weight, gaps were interpreted as a DNA techniques.-Liquid cultures were transferred to 2-mL fifth base. The maximum number of trees was set at 20 000. tubes, centrifuged and washed twice with sterile water. Sub- The swapping algorithm was tree bisection-reconnection sequently, 0.05 g of silica (Merck, Darmstadt, Germany) and (TBR), the steepest descent option was not in effect and to 300 xLLof CTAB extraction buffer was added [200 mm eliminate unsupported branches, branches collapsed if the tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.5, 1.5 maximum branch length was zero. Bootstrap analysis was M NaCl, 20 mm EDTA, 2% hexadecyltrimethylammonium performed using 1000 replicates and with maxtrees set at bromide ]. Tissue was with a for (CTAB) ground micropestle 100. In the analysis of the SSU and the ITS-region, Sclero- 1 min, another 200 iLLof CTAB extraction buffer was add- tinia sclerotiorum (GenBank X69850 and Z73800) was se- ed and the was incubated for 10 min at 65 C. A chlo- lysate lected as outgroup. A set of extra outgroup specimens was (24:1) extraction was roform/isoamylalcohol performed selected to get a better idea about the place of Pe. alnicola. and vol of ethanol were followed an incubation 2 added, by The ITS sequences of the following species were retrieved at -20 C for 30 min. The precipitate was centrifuged 5 min from GenBank: Calycellinapunctata (U57494), Microscypha and the was washed with 70% ethanol. The was pellet pellet ellisii (U57493), Pycnopeziza sympodialis (Z81445), Myrios- allowed to and dissolved in 100 pLLTE. 2.5 FL dry Finally, clerotinia scirpicola (Z81440), Pocolum henningsianum RNase solution was added and the solution was (10mg/mL) (Z81442), Scleromitrula calthicola (Z80886), Trichopezizella incubated for 5 min at 37 C. nidulus (U57813), Neodasyscyphacerina (U57812), Prolifer- A of the ribosomal RNA clus- Sequenceanalysis. part gene odiscus alboviridis (U57990), Solenopezia solenia (U57991), ter was amplified by PCR using primers NS7 (5'-gag gca ata and Lachnum nudipes (U59003). Gaps in this alignment aca et al and LS266 ctt ggt ctg tga tgc) (White 1990) (5'-tcc were handled as missing data. tca aca att tca cg) (Masclaux et al 1995). The amplicon contains about 200 bp of the 3' end of the gene coding for the 18S rRNA, the ITS1, the gene coding for the 5.8S rRNA, RESULTS the ITS2 and about 100 bp of the gene coding for the 28S rRNA. PCR was performed in 50 FLLreaction volumes and The partial 18S region and the ITS region of 44 each reaction contained 10-100 ng of genomic DNA, 5 FLM strains of Pezicula and its anamorphs were sequenced of each 200 pFM 0.5 unit DNA primer, dNTP, Supertaq poly- for a phylogenetic analysis. Conflicts between com- merase and 5 p1L10X PCR buffer (SphaeroQ, Leiden, the puterized basecalling occurred on average at two po- Netherlands). The amplification was performed in a ther- sitions per contig; these conflicts could always be mocycler (Biomed, type 60) with the following program: I solved on basis of visual of both electro- min 95 C, 30X [1 min 95 C, Imin 58 C, 1 min 72 C] fol- comparison The automatic resulted in a lowed by a final extension of 10 min at 72 C. PCR product phorograms. alignment was cleaned with sephadex S-300 columns (Amersham Phar- good match between all sequences, but occasionally macia Biotech, Roosendaal, The Netherlands) and analyzed manual corrections were performed.