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A new species of Cudonia based on morphological and molecular data

     Department of Biology, Clark University, 950 Main Street, Worcester, Massachusetts 01610, USA

	ABSTRACT

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A discomycete collected in western Sichuan, China, is morphologically intermediate between Cudonia and Spathularia. The fungus has a bright yellow capitate ascigerous head, a white, ridged stalk, and a well-developed membrane covering the whole ascoma. The asci, ascospores, and paraphyses are similar to those of Cudonia and Spathularia. Based on morphology and DNA sequence analysis, a new species, Cudonia sichuanensis, is reported. Cudonia and Spathularia are closely related to members of Rhytismataceae, as has been suggested previously. The similarity of ascoma and ascospore development between these two genera and Lophodermium (Rhytismataceae) is discussed.

Key words: Chinese fungi, Geoglossaceae, Helotiales, ITS, Leotiaceae, nuc-lsu rDNA, Spathulariopsis

	INTRODUCTION

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The Geoglossaceae (Helotiales) is traditionally composed of taxa such as Geoglossum Pers., Trichoglossum Boud. and Microglossum Gillet, which are commonly known as earth tongues, and other genera, including Mitrula Fr., Nothomitra Maas Geest., Cudonia Fr.:Fr., Spathularia Pers., and Spathulariopsis Maas Geest. These taxa often have large ascocarps, so they have received a lot of attention (Durand 1908, Corner 1929, 1930, Imai 1941, Maas Geesteranus 1964, Mains 1940, 1955, Korf 1973). Based on the color of the ascospores and the reaction of the ascus pore in Melzer's reagent, the family can be separated into two groups: 1) Cudonia, Spathularia, and Spathulariopsis with hyaline ascospores and a negative reaction of the ascus pore in Melzer's reagent; 2) Geoglossum and Trichoglossum with dark ascospores and a positive reaction (bluing) of the ascus pore in Melzer's reagent. Mitrula, Nothomitra and Microglossum do not fit in either of these groups (Korf 1973, Spooner 1987, Verkley 1994).

Macromorphology of the ascocarps has been emphasized at the family or subfamily level in Helotiales (Imai 1941, Mains 1956, Korf 1973, Spooner 1987). Most mycologists have been inclined to group the genera with clavate or spathulate ascocarps, such as Geoglossum, Microglossum, and Spathularia, together in the family Geoglossaceae, but opinions have differed regarding the position of the genera with pileate ascocarps, such as Cudonia, Vibrissea Fr., and Leotia Pers. Cudonia was put in the Leotiaceae, and Spathularia and Spathulariopsis in the Geoglossaceae by Korf (1973) based on macromorphology.

The well-developed gelatinized layer of textura intricata in the ectal excipulum, as in the genus Leotia, was emphasized at family level by Lizon et al (1998). The concept of Leotiaceae in the sense of Korf (1973) was restricted to a narrow sense, and Cudonia was excluded (Lizon et al 1998). Based on ultrastructure of the ascus apices, Verkley (1994) suggested that there are two different lineages in the family Geoglossaceae, and that Geoglossum, Trichoglossum and Microglossum are different from Spathularia.

Except for the difference in the macromorphology of the ascomata, Cudonia and Spathularia share many important characters, such as gelatinously sheathed ascospores, curved paraphyses, club-shaped asci with non-bluing pores in Melzer's reagent, and veil structures that are present at least at an early stage of ascoma development. The relationship between Cudonia and Spathularia has been supported by some molecular studies (Gargas et al 1995, Landvik 1996, Suh and Blackwell 1999, Bhattacharya et al 2000, Platt and Spatafora 2000, Gernandt et al 2001).

The current concept of the Geoglossaceae, in which all stipitate, capitate, or clavulate fungi including Geoglossum, Trichoglossum, Microglossum, Mitrula, Spathularia, Cudonia and other taxa are included, is controversial (R. P. Korf pers comm). More and more data based on morphological and molecular research call into question the position of Cudonia and Spathularia either in Leotiaceae or in Geoglossaceae (Landvik 1996, Pfister and Kimbrough 2001, Gernandt et al 2001).

Nannfeldt (1942) suggested that Cudonia and Spathularia share characters with the members of Phacidiaceae (including Rhytisma Fr., which now is a member of Rhytismataceae, Rhytismatales), such as filiform, branched, and circinate paraphyses, and a stromatic layer that covers the hymenium in the early stage of ascoma development. This relationship was supported by parsimony analyses using nuc-ssu rDNA, nuc-lsu rDNA, and RPB2 (Landvik 1996, Platt 1999, Gernandt et al 2001, Lutzoni et al 2001). Three groups of Helotiales were presented by Pfister and Kimbrough (2001), and one of these three groups included Cudonia, Spathularia, and probably several other taxa of the Rhytismatales (Pfister and Kimbrough 2001).

Nannfeldt's suggestion about the close relationship between Spathularia and the Rhytismataceae was partially based on the stromatic layer of Spathularia velutipes Cooke & Farlow. Maas Geesteranus (1972) compared both Spathularia velutipes and S. flavida Pers. (the type species of Spathularia), and did not observe a stromatic layer (he called it a veil) in his material of S. flavida. He created the monotypic genus Spathulariopsis for S. velutipes and pointed out that the developmental type in Spathulariopsis is hemiangiocarpous, in contrast to the gymnocarpous type he found in Spathularia flavida (Maas Geesteranus 1972).

Eight species in Cudonia, two species in Spathularia, and one species in Spathulariopsis are estimated (Hawksworth et al 1995). Cudonia circinans (Pers.) Fr., C. confusa Bres., C. helvelloides S. Ito & S. Imai, C. lutea (Peck) Sacc., Spathularia flavida and Spathulariopsis velutipes were reported from China (Zhuang 1998). Two field trips to Sichuan Province, China were made in 1997 and 1998, and about 40 specimens of Spathularia, Cudonia, and other members of Geoglossaceae were collected. Among them, one capitate fungus is morphologically intermediate between Cudonia and Spathularia. Morphological and molecular studies suggest that this is a new species in Cudonia.

	MATERIALS AND METHODS

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Specimens and morphological studies – Specimens were collected in Sichuan Province, China, in 1997 and 1998, and are deposited in the Herbarium of the Institute of Microbiology, the Chinese Academy of Sciences (HMAS) and the Farlow Herbarium (FH) of Harvard University. Morphological descriptions are based on observations of dried or of rehydrated specimens. Microscopic studies were based on squashed tissues and sections cut with a freezing microtome at 20–25 µm thickness. Measurements, illustrations, and photographs were usually made under cotton-blue-lactic-acid using bright field and phase contrast optics (Nikon E600). Ascus pore iodine reactions were examined using Melzer's reagent. Anatomical terms follow Korf (1973).

Molecular techniques – DNA was isolated from dried fruiting bodies. Approximately 20–30 mg of tissue was ground in liquid nitrogen and extracted in 600 µL of extraction buffer (1% SDS, 0.15 M NaCl, 50 mM EDTA) at 75 C for 1 h, purified with phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated with 95% ethanol and 3 M NaCl overnight. Crude DNA extracts showed strong pigmentation and were diluted with distilled water up to one-thousand-fold for use as PCR templates after an additional purification step with GeneClean (Bio 101, La Jolla, California).

Partial nuclear large subunit rDNA (nuc-lsu-rDNA) was amplified with primers LR0R and LR5 (Vilgalys and Hester 1990) in 11 isolates, representing 4 species of Cudonia and Spathularia (Table I). Internal transcribed spacers 1 and 2 and the 5.8S rDNA were amplified with primers ITS4 and ITS5 (White et al 1990) in 9 isolates, representing 4 species of Cudonia and Spathularia (Table I). PCR reaction mixes (Promega Corp., Madison, Wisconsin) contained 2.5 µL 10x PCR buffer, 5 µM dNTP, 12.5 pM of each PCR primer and 5 µL DNA in 15 µL. The amplification program included 40 cycles of 94 C for 30 s, 45 C for 30 s, and 72 C for 1 min.

Phylogenetic analyses – Sequences were aligned by eye in the data editor of PAUP* 4.0b (Swofford 1999). Two datasets were constructed: 1) a dataset of nuc-lsu rDNA sequences, including the 11 sequences generated for this study and 12 sequences of Cudonia, Spathularia, Geoglossum, Leotia, Lophodermium Chevall., Sclerotinia Fuckel, and Stictis Pers., which were downloaded from GenBank (Table I); 2) a dataset 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 parsimony, with gaps treated as missing data, and all positions included.

The nuc-lsu rDNA dataset was rooted using Stictis radiata (L.) Pers. A heuristic search was performed with one thousand replicate searches, each with a random taxon addition sequence, MAXTREES set to autoincrease, and TBR branch swapping. A bootstrap analysis was performed with one thousand replicates, each with ten random taxon addition sequences, MAXTREES set to 100, and TBR branch swapping.

The nuc-lsu/ITS dataset was rooted using Lophodermium pinastri (Schrad.) Chevall. A branch-and-bound analysis was performed with MAXTREES set to autoincrease. A bootstrap analysis was performed with one thousand branch-and-bound replicates with MAXTREES set to autoincrease. Alignments are available at TreeBASE (study accession number S702).

	RESULTS

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Morphology – Twenty-two specimens were examined, including four collections of the new species, Cudonia sichuanensis (Figs. 1–10). The ascomata of C. sichuanensis are distinctly capitate, and the broken membrane forms a collar-like structure connected with the stalk along the margin of the ascogenous head (Figs. 4, 5, 8). The membrane is about 50 µm thick and is composed of several layers of angular cells. The outermost layer is composed of smaller cells (3–5 µm in diam) and is covered with duff or soil debris (Figs. 9–10). Such a well-developed membrane is not found in the other species of Cudonia and Spathularia from the Chinese collections. However, sometimes a kind of cortex remains on the surface of the stalk of Chinese Cudonia and Spathularia collections, which is composed of hyaline to brownish and angular to globose cells, coated with some plant debris and sand. Remnants of veils are observed at the juncture between the stipe and the hymenium from the North American species of Cudonia and Spathularia (D. Pfister pers comm). A gelatinous sheath and cap-like structure were observed on the ascospores of all the collections in rehydrated specimens (Figs. 6–7). Similar structures were observed by Johnson (1994) in Lophodermium pinastri. The size of the ascospores in C. sichuanensis ranges from 46–66 x 2.0–2.2 µm. Globose ascoconidia are produced by the ascospores in asci from all the collections of Cudonia and Spathularia, and the paraphyses show no morphological difference among those collections.

View larger version (31K): [in this window] [in a new window]    FIGS. 1–4. Cudonia sichuanensis HMAS 75140. 1. Ascospores. 2. Ascus and paraphyses. 3. Ascoconidia. 4. Ascocarps. Scale bars: 1–2 = 10 µm, 3 = 2 µm, 4 = 2 mm

View larger version (44K): [in this window] [in a new window]    FIG. 5. Longitudinal section through the fruitbody of HMAS 75140 shows ascigerous portion (AP), stalk portion (SP), subhymenium (SH), hymenium (H), stromatic membrane (SM), and Medullar layer in both stalk and ascigerous potion. Scale bar 5 = 50 µm

View larger version (65K): [in this window] [in a new window]    FIG. 8. Dry apothecia of Cudonia sichuanensis, HMAS 75140, shows ascigerous portion (AP), stalk portion (SP), and the broken stromatic layer over the hymenium and along the margin of the hymenium. A and B indicate the parts which are shown in Figs. 8. Scale bar = 20 mm.  FIGS. 9–10. Stromatic layer at different parts of the ascomata of Cudonia sichuanensis HMAS 75140. 9. The collar like structure along the margin of the hymenium (arrow). 10. Part of the stromatic layer of textura angularis along the stalk (arrows). Scale bars: 9–10 = 50 µm

View larger version (20K): [in this window] [in a new window]    FIGS. 6–7. Ascospores of Cudonia sichuanensis HMAS 75140. 6. Spores in black ink show the gelatinous sheaths and caps. 7. The gelatinous sheaths surrounded by the ink debris dissolved quickly in water. Scale bars: 6–7 = 50 µm

The nuc-lsu rDNA dataset included 962 aligned positions, with 189 variable positions and 95 parsimony-informative positions. Parsimony analysis of the nuc-lsu rDNA dataset resulted in twelve equally parsimonious trees of 297 steps (CI = 0.771, RI = 0.724; Fig. 11). Cudonia plus Spathularia form a strongly supported monophyletic group (bootstrap = 91%; Fig. 11). Lophodermium pinastri is strongly supported as the sister group of the Spathularia-Cudonia clade (bootstrap = 100%; Fig. 11). Within the Spathularia-Cudonia clade there is little resolution or support, except for three groups that include putatively conspecific isolates of C. circinans, C. lutea, and S. flavida. Neither Spathularia nor Cudonia is resolved as monophyletic (Fig. 11). The two isolates of C. sichuanensis were weakly supported as conspecific (bootstrap < 50%; Fig. 11).

View larger version (28K): [in this window] [in a new window]    FIGS. 11–12. Phylogenetic relationships of Cudonia and Spathularia inferred from molecular sequences. 11. Relationships inferred from nuc-lsu-rDNA. One of twelve equally parsimonious trees (297 steps, CI = 0.771, RI = 0.724). Nodes that collapse in the strict consensus tree are marked with an asterisk above the branch. Bootstrap values greater than 50% are indicated along nodes. Isolates used in ITS-nuc-lsu-rDNA analyses (Fig. 12) are underlined. 12. Relationships inferred from combined nuc-lsu-rDNA and ITS sequences. Single most parsimonious tree (203 steps, CI = 0.823, RI = 0.748). Bootstrap values greater than 50% are indicated along nodes. Habit illustrations of Cudonia sichuanensis, C. lutea, and Spathularia flavida are shown at the corresponding clades

	TAXONOMY

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A morphological comparison between Cudonia, Spathularia, Geoglossum, Cryptohymenium, and Lophodermium is shown in Table II. (Data were based Mains 1940, Imai 1941, Samuels and Kohn 1986, Spooner 1987, Johnston 1989, and pers obs).

Cudonia sichuanensis

Zheng Wang, sp. nov. (Figs. 1–10)

Ascomata solitaria vel gregaria, stipitata, capitata 17–35 mm longitudine; caput ascomatis globosum, luteum vividum sed siccatum fulvum, 1.5–3 mm diametro; semper adsunt fragmenta structurae membranaceae in parte superiore stipitis et secundum marginem conjunctam stipiti. In stipite corticis textura angularis, 30–50 µm crassitudine; asci clavati, ad basem certe angustiores; porus iodo non caerulescens, 136–152 x 12–14 µm; octosporae in asco; ascosporae clavato-filiformes vel aciculares, hyalinae, non-septatae, vaginatae in gelatin, 46–66 x 2–2.2 µm; paraphyses filiformes, apice curvat vel circinat. Holotypus: HMAS 75140.

Ascomata scattered to gregarious, capitate, stipitate, 17–35 mm in height. Ascigerous portion capitate, with remains of a membrane-like structure on the upper part and along the margin connecting to the stalk (Figs. 4, 5, 8), bright yellow when fresh, brownish when dry, 1.5–3 mm in diam, slightly swollen compared with the stalk. Stalk white, slender, smooth or slightly tomentose, striate to ridged, 15–25 mm in length. The interior of the stalk and ascigerous portion of textura intricata, loosely interwoven hyphae becoming more compact and parallel toward the outside (Figs. 5, 9–10). The stromatic membrane of regular textura angularis, well-developed, sometimes broken into pieces as the hymenium expands, about 40–50 µm thick over the stalk and 30–40 µm thick over the hymenium, outermost layer composed of smaller cells encrusted with soil and debris, brownish. Cells of the stromatic layer array at a right angle to the axis of the stalk, angular to globose from inner side to outer surface, 3 x 5–15 x 25 µm. Hymenium about 120–150 µm thick. Asci clavate, narrower towards the base, 8-spored, apical pore J-, 136–152 x 12–14 µm. Ascospores hyaline, acicular, rounded above, acuminate below, multiguttulate, nonseptate, 46–66 x 2.0–2.2 µm, the wall with a gelatinous layer swelling in water to 1.5–2 µm thick, prominent gelatinous cap present at the wider end of the ascospore, and sometimes at both spore poles. Ascoconidia subspherical, irregular ellipsoid or obovoid, 1–2 x 1–2 µm, 1-celled, hyaline, sometimes replacing the ascospores and filling the asci. Paraphyses filiform, simple or branched below, not or irregularly branched above, strongly curved to circinate or straight above, hyaline, about 2 µm in diam.

Specimens examined. CHINA. SICHUAN PROVINCE: Xiangchenxian, Daxuesan Mountains, on duff of Abies sp., 24 Jul 1998, Zheng Wang WZ0178 (HOLOTYPE HMAS75140, ISOTYPE FH-WZ0178); between Xiangchenxian and Dongwangxian, on duff, 26 Jul 1998, Zheng Wang WZ202 HMAS 75139; between Xiangchenxian and Dongwangxian, on duff, 26 Jul 1998, Zheng Wang WZ187 HMAS 75141; Xiangchenxian, Wumingsan, on duff, 12 Jul 1998, Zheng Wang WZ96 HMAS 75143.

	DISCUSSION

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Cudonia sichuanensis is distinct from other species in the genus in its capitate ascomata (Figs. 8, 12) and well-developed membrane covering the whole fruitbody. Subglobose or even globose pilei have been reported in Cudonia, but generally the ascigerous portions expand widely and the margins of the hymenia are free from the stalk in these species (Fig. 12). The form of the ascigerous areas was a character used to separate Cudonia from Spathularia. Several ascomata of C. sichuanensis also show expansion along the margin of ascogenous head, and the membrane can be seen stretched between the hymenium and the stalk.

The color of the hymenium and the size of the ascospores are key characters in the delimitation of species in Cudonia. The bright yellow hymenium and ascospores of 46–66 x 2.0–2.2 µm of the new species are similar to those of Cudonia helvelloides, which has large (25 to 70 mm) pileate ascomata (Mains 1940, Imai 1941). Cudonia convoluta Lloyd is the only species in the genus so far reported to have capitate ascomata, and was considered as an intermediate between Cudonia and Mitrula by Lloyd (Lloyd 1916). However, Mains (1940) examined the type specimen of C. convoluta and found the species was distinctly pileate, and could be treated as a synonym of C. orientalis Yasuda.

Stunted and degenerate growth forms are occasionally seen in Spathularia flavida. Spathularia pilatii Velen., reported as having a globose ascogenous head, turned out to be a misshapen form of S. flavida (Mass Geesteranus 1972). Nothomitra species have capitate ascocarps and are placed in the family Geoglossaceae. Nothomitra, unlike Cudonia sichuanensis, has ascospores lacking gelatinous sheaths and amyloid ascus pores (Mass Geesteranus 1964).

Phylogenetic analysis suggests that Cudonia sichuanensis is a unique species (Figs. 11, 12). However, sequences of several other species of Cudonia were not available in this study, including C. confusa and C. helvelloides, which have both been found in China (Zhuang 1998).

Some recent molecular studies using 18S nuc-ssu rDNA, 25S nuc-lsu rDNA or RPB2 supported the close relationship among Cudonia, Spathularia, and the Rhytismataceae (Landvik 1996, Platt 1999, Gernandt 2001, Lutzoni et al 2001). In the present study, data from nuc-lsu rDNA also strongly support the clade including Cudonia, Spathularia, and Lophodermium (Fig. 11). In addition, the intron of about 300 bp in nuc-lsu rDNA of Chinese collections of S. flavida shows nearly 70% similarity to that of Lophodermium pinastri.

Cudonia sichuanensis represents an intermediate between the pileate species of Cudonia and the spathulate species of Spathularia. In young ascomata of Spathularia the hymenium is covered by an ephemeral stromatic membrane that appears before the stalk develops, and is destroyed as the stalk expands (Z. Wang unpubl). In S. flavida the membrane disappears early in the fruitbody development, but in C. sichuanensis the membrane is persistent and forms a distinctive cortex structure (Figs. 5, 9–10). Thus, the ascomata of Cudonia and Spathularia are hemiangiocarpous, as suggested by Nannfeldt (1932). Johnston (1988) described a mode of ascocarp development in the non-coniferous species of Lophodermium, in which a layer of vertically oriented cells between the ascocarp wall and the developing hymenium present at all stages of a Lophodermium ascocarp maturity. The layer described by Johnston is similar to the membrane structure in Cudonia sichuanensis. We suggest that the term "stromatic layer" introduced by Nannfeldt (1942) be used to refer to the membrane covering the whole ascomata of Cudonia and Spathularia. The stromatic layer is a developmental character that unites Cudonia, Spathularia, and members of Rhytismataceae, as suggested by Nannfeldt (1942). Maas Geesteranus described in detail an outermost tissue on the stalk of Spathularia velutipes as "The outer cells of the textura globulosa 8–15 x 6–12 µm, angular, globose, obovoid, with thick brown cell-walls, becoming largely detached one from another and forming short chains at right angels to the axes of the stipe"(Maas Geesteranus 1972). He thought that the outermost tissue is continuous with a veil, which encloses the whole fruitbody in the very beginning of the ascomata, and this presents a type of development called hemiangiocarpous by Nannfeldt (1942). Maas Geesteranus did not see any similar structure on the stalk of other Spathularia species, the development type of which he thought to be gymnocarpous, and he also thought there is difference between Spathularia velutipes and Spathularia flavida on the hyphal construction of the medulla layer in the stalk (Maas Geesteranus 1972). These are the two main reasons Maas Geesteranus erected a new genus Spathulariopsis based on Spathularia velutipes.

Our observation of several specimens of Spathularia flavida and S. velutipes (type materials deposited in FH) did not reveal any striking difference in the hyphal construction of the medulla layer in the stalks between these two species. We interpret the membrane found in S.velutipes as homologous with that of Cudonia sichuanensis. We accept Spathularia velutipes rather than Spathulariopsis velutipes because the lack of a distinct cortex in the mature ascomata of Spathularia cannot be regarded as a different development type to that of Spathulariopsis. In fact, membrane-like structures were found frequently in the ascomata in both Spathularia and Cudonia (Sever 1951, Mains 1955, 1956), and usually on the surface of the hymenia and at the juncture between the stipe and the hymenium.

Samuels and Kohn (1986) described an unusual, capitate discomycete, Cryptohymenium pycnidiophorum from New Zealand, in which the hymenial portion of the ascoma is produced below a multiloculate pycnidium. The hymenium of C. pycnidiophorum is covered by a cellular epithecium, and in this regard it resembles C. sichuanensis and other Rhytismatales (R. P. Korf, pers comm). However, it differs from Cudonia, Spathularia, and Rhytisma in having fusoid to subfusoid ascospores and a positive reaction of the ascus pore in Melzer's reagent, as well as the presence of pycnidia above the hymenium and other characters (Samuels and Kohn 1986; Table II).

	ACKNOWLEDGMENTS

 
We are indebted to R. Korf, D. Pfister, K. O'Donnell and J. Platt for their valuable comments and suggestions, P. R. Johnston for providing some important references, and I. Sun in the Department of Foreign Languages, Clark University, for providing the Latin diagnosis. Thanks are also due to the members of the 1997–1998 field expeditions, including D. Boufford, M. Donoghue, Y. Jia, R. Ree, and Z.-L. Yang, for their encouragement and support, which made the field and herbarium work possible. This research was supported by National Science Foundation grants DEB-9903835 (to D. Hibbett) and DEB-9705795 (to M. Donoghue), and a grant from the Chinese Academy of Sciences to Z. Wang.

	FOOTNOTES

 
¹ Corresponding author, Email: zwang{at}clarku.edu

Accepted for publication January 9, 2002.

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