Mycologia, 94(4), 2002, pp. 641±650. q 2002 by The Mycological Society of America, Lawrence, KS 66044-8897 A new species of Cudonia based on morphological and molecular data Zheng Wang1 Mitrula, Nothomitra and Microglossum do not ®t in Manfred Binder either of these groups (Korf 1973, Spooner 1987, David S. Hibbett Verkley 1994). Department of Biology, Clark University, 950 Main Macromorphology of the ascocarps has been em- Street, Worcester, Massachusetts 01610, USA phasized at the family or subfamily level in Helotiales (Imai 1941, Mains 1956, Korf 1973, Spooner 1987). Most mycologists have been inclined to group the Abstract: A discomycete collected in western Sich- genera with clavate or spathulate ascocarps, such as uan, China, is morphologically intermediate between Geoglossum, Microglossum, and Spathularia, together Cudonia and Spathularia. The fungus has a bright in the family Geoglossaceae, but opinions have dif- yellow capitate ascigerous head, a white, ridged stalk, fered regarding the position of the genera with pi- and a well-developed membrane covering the whole leate ascocarps, such as Cudonia, Vibrissea Fr., and ascoma. The asci, ascospores, and paraphyses are sim- Leotia Pers. Cudonia was put in the Leotiaceae, and ilar to those of Cudonia and Spathularia. Based on Spathularia and Spathulariopsis in the Geoglossaceae morphology and DNA sequence analysis, a new spe- by Korf (1973) based on macromorphology. cies, Cudonia sichuanensis, is reported. Cudonia and The well-developed gelatinized layer of textura in- Spathularia are closely related to members of Rhytis- tricata in the ectal excipulum, as in the genus Leotia, mataceae, as has been suggested previously. The sim- was emphasized at family level by Lizon et al (1998). ilarity of ascoma and ascospore development between The concept of Leotiaceae in the sense of Korf these two genera and Lophodermium (Rhytismata- (1973) was restricted to a narrow sense, and Cudonia ceae) is discussed. was excluded (Lizon et al 1998). Based on ultrastruc- Key Words: Chinese fungi, Geoglossaceae, Hel- ture of the ascus apices, Verkley (1994) suggested otiales, ITS, Leotiaceae, nuc-lsu rDNA, Spathulariop- that there are two different lineages in the family sis Geoglossaceae, and that Geoglossum, Trichoglossum and Microglossum are different from Spathularia. Except for the difference in the macromorphology INTRODUCTION of the ascomata, Cudonia and Spathularia share many The Geoglossaceae (Helotiales) is traditionally com- important characters, such as gelatinously sheathed posed of taxa such as Geoglossum Pers., Trichoglossum ascospores, curved paraphyses, club-shaped asci with Boud. and Microglossum Gillet, which are commonly non-bluing pores in Melzer's reagent, and veil struc- known as earth tongues, and other genera, including tures that are present at least at an early stage of as- Mitrula Fr., Nothomitra Maas Geest., Cudonia Fr.:Fr., coma development. The relationship between Cudon- Spathularia Pers., and Spathulariopsis Maas Geest. ia and Spathularia has been supported by some mo- These taxa often have large ascocarps, so they have lecular studies (Gargas et al 1995, Landvik 1996, Suh received a lot of attention (Durand 1908, Corner and Blackwell 1999, Bhattacharya et al 2000, Platt 1929, 1930, Imai 1941, Maas Geesteranus 1964, Mains and Spatafora 2000, Gernandt et al 2001). 1940, 1955, Korf 1973). Based on the color of the The current concept of the Geoglossaceae, in ascospores and the reaction of the ascus pore in Mel- which all stipitate, capitate, or clavulate fungi includ- zer's reagent, the family can be separated into two ing Geoglossum, Trichoglossum, Microglossum, Mitrula, groups: 1) Cudonia, Spathularia, and Spathulariopsis Spathularia, Cudonia and other taxa are included, is with hyaline ascospores and a negative reaction of the controversial (R. P. Korf pers comm). More and more ascus pore in Melzer's reagent; 2) Geoglossum and Tri- data based on morphological and molecular research choglossum with dark ascospores and a positive reac- call into question the position of Cudonia and Spa- tion (bluing) of the ascus pore in Melzer's reagent. thularia either in Leotiaceae or in Geoglossaceae (Landvik 1996, P®ster and Kimbrough 2001, Ger- Accepted for publication January 9, 2002. nandt et al 2001). 1 Corresponding author, Email: [email protected] Nannfeldt (1942) suggested that Cudonia and Spa- 641 642 MYCOLOGIA thularia share characters with the members of Phac- E600). Ascus pore iodine reactions were examined using idiaceae (including Rhytisma Fr., which now is a Melzer's reagent. Anatomical terms follow Korf (1973). member of Rhytismataceae, Rhytismatales), such as Molecular techniques. DNA was isolated from dried fruiting ®liform, branched, and circinate paraphyses, and a bodies. Approximately 20±30 mg of tissue was ground in stromatic layer that covers the hymenium in the early liquid nitrogen and extracted in 600 mL of extraction buffer stage of ascoma development. This relationship was (1% SDS, 0.15 M NaCl, 50 mM EDTA) at 75 C for 1 h, supported by parsimony analyses using nuc-ssu puri®ed with phenol-chloroform-isoamyl alcohol (25:24:1), rDNA, nuc-lsu rDNA, and RPB2 (Landvik 1996, Platt and precipitated with 95% ethanol and 3 M NaCl overnight. 1999, Gernandt et al 2001, Lutzoni et al 2001). Three Crude DNA extracts showed strong pigmentation and were diluted with distilled water up to one-thousand-fold for use groups of Helotiales were presented by P®ster and as PCR templates after an additional puri®cation step with Kimbrough (2001), and one of these three groups GeneClean (Bio 101, La Jolla, California). included Cudonia, Spathularia, and probably several Partial nuclear large subunit rDNA (nuc-lsu-rDNA) was other taxa of the Rhytismatales (P®ster and Kim- ampli®ed with primers LR0R and LR5 (Vilgalys and Hester brough 2001). 1990) in 11 isolates, representing 4 species of Cudonia and Nannfeldt's suggestion about the close relationship Spathularia (TABLE I). Internal transcribed spacers 1 and 2 between Spathularia and the Rhytismataceae was par- and the 5.8S rDNA were ampli®ed with primers ITS4 and tially based on the stromatic layer of Spathularia ve- ITS5 (White et al 1990) in 9 isolates, representing 4 species lutipes Cooke & Farlow. Maas Geesteranus (1972) of Cudonia and Spathularia (TABLE I). PCR reaction mixes compared both Spathularia velutipes and S. ¯avida (Promega Corp., Madison, Wisconsin) contained 2.5 mL 10x PCR buffer, 5 mM dNTP, 12.5 pM of each PCR primer Pers. (the type species of Spathularia), and did not and 5 mL DNA in 15 mL. The ampli®cation program in- observe a stromatic layer (he called it a veil) in his cluded 40 cycles of 94 C for 30 s, 45 C for 30 s, and 72 C material of S. ¯avida. He created the monotypic ge- for 1 min. nus Spathulariopsis for S. velutipes and pointed out PCR products were puri®ed using GeneClean (Bio 101) that the developmental type in Spathulariopsis is hem- and sequenced using the ABI Prism Bigdye-terminator cycle iangiocarpous, in contrast to the gymnocarpous type sequencing kit (Applied Biosystems, Foster City, California) he found in Spathularia ¯avida (Maas Geesteranus according to the manufacturer's protocols. Primers used for 1972). sequencing were LR0R, LR3, LR3R, LR5, ITS4, and ITS5. Eight species in Cudonia, two species in Spathular- Sequencing reactions were puri®ed using Pellet Paint (Nov- ia, and one species in Spathulariopsis are estimated agen, Madison, Wisconsin) and were run on an Applied Biosystems 377XL automated DNA sequencer. Sequences (Hawksworth et al 1995). Cudonia circinans (Pers.) were edited with Sequencher version 3.1 (GeneCodes Cor- Fr., C. confusa Bres., C. helvelloides S. Ito & S. Imai, poration, Ann Arbor, Michigan). Sequences generated in C. lutea (Peck) Sacc., Spathularia ¯avida and Spathu- this study were submitted to GenBank (accession numbers lariopsis velutipes were reported from China (Zhuang AF433136-AF433155; TABLE I). 1998). Two ®eld trips to Sichuan Province, China Phylogenetic analyses. Sequences were aligned by eye in the were made in 1997 and 1998, and about 40 speci- data editor of PAUP* 4.0b (Swofford 1999). Two datasets mens of Spathularia, Cudonia, and other members of were constructed: 1) a dataset of nuc-lsu rDNA sequences, Geoglossaceae were collected. Among them, one cap- including the 11 sequences generated for this study and 12 itate fungus is morphologically intermediate between sequences of Cudonia, Spathularia, Geoglossum, Leotia, Lo- Cudonia and Spathularia. Morphological and molec- phodermium Chevall., Sclerotinia Fuckel, and Stictis Pers., ular studies suggest that this is a new species in Cu- which were downloaded from GenBank (TABLE I); 2) a da- donia. taset of nuc-lsu rDNA and ITS sequences from 9 isolates of Cudonia, Spathularia, and Lophodermium (TABLE I). Both datasets were analyzed in PAUP* using equally weighted MATERIALS AND METHODS parsimony, with gaps treated as missing data, and all posi- tions included. Specimens and morphological studies. Specimens were col- The nuc-lsu rDNA dataset was rooted using Stictis radiata lected in Sichuan Province, China, in 1997 and 1998, and (L.) Pers. A heuristic search was performed with one thou- are deposited in the Herbarium of the Institute of Micro- sand replicate searches, each with a random taxon addition biology, the Chinese Academy of Sciences (HMAS) and the sequence, MAXTREES set to autoincrease, and TBR branch Farlow Herbarium (FH) of Harvard University. Morpholog- swapping. A bootstrap analysis was performed with one ical descriptions are based on observations of dried or of thousand replicates, each with ten random taxon addition rehydrated specimens. Microscopic studies were based on sequences, MAXTREES set to 100, and TBR branch swap- squashed tissues and sections cut with a freezing microtome ping. at 20±25 mm thickness. Measurements, illustrations, and The nuc-lsu/ITS dataset was rooted using Lophodermium photographs were usually made under cotton-blue-lactic- pinastri (Schrad.) Chevall. A branch-and-bound analysis was acid using bright ®eld and phase contrast optics (Nikon performed with MAXTREES set to autoincrease.