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Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Ladkrabang, Bangkok 10520, Thailand
Rajesh Jeewon
Centre for Research in Fungal Diversity, Department of Ecology & Biodiversity, The University of Hong Kong, SAR, PR China
Dennis E. Desjardin 2
Department of Biology, San Francisco State University, San Francisco, California
Kasem Soytong
Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Ladkrabang, Bangkok 10520, Thailand
Kevin D. Hyde
Centre for Research in Fungal Diversity, Department of Ecology & Biodiversity, The University of Hong Kong, SAR, PR China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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Phylogenetic relationships within the genus Cyathus (bird’s nest fungi) were investigated with neighbor joining, maximum likelihood, weighted maximum parsimony and MrBayes analyses of ITS and LSU ribosomal DNA sequences datasets. Twenty-two taxa of Cyathus were used in the analyses based primarily on type and authentic specimens. The current infrageneric classification system of Brodie recognizes seven infrageneric groups based on morphological characters, including peridium plications and variations in peridium hair anatomy, peridiole structure and fruit-body color. These groups are not supported by molecular data. Instead the ITS and LSU datasets support recognition of three infrageneric groups herein named the ollum, pallidum and striatum groups. Morphological characters useful in distinguishing these groups include basidiospore size, fruit-body coloration and peridium anatomy. Cyathus africanus var. latisporus is considered a synonym of Cyathus jiayuguanensis, and a new combination Cyathus lanatus (Brodie) R.L. Zhao is proposed based on morphological and molecular data.
Key words: bird’s nest fungi, gasteromycetes, Nidulariaceae, phylogenetics, taxonomy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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]). Lloyd (1906) monographed Cyathus and accepted five infrageneric groups (viz. two sections in the eucyathus group and three sections in the olla group). No formal names were provided for Lloyd’s sections. Later H.J. Brodie published more than 50 papers and two monographs on Cyathus, describing 62 species and subdividing Cyathus into seven groups (for references refer to Brodie 1975, 1984, and for a key to species accepted by Brodie refer to Brodie 1977).
In Brodie’s taxonomic system distinct plications on the peridium of the fruiting body were considered to be a major diagnostic character for separating groups and species (Brodie 1975), following Tulasne and Lloyd before him. Species with plications were placed into the "striatus" and "poeppigii" groups, while species lacking plications were placed in the "olla", "pallidus", "triplex", "gracilis" and "stercoreus" groups (Brodie 1975, 1984). There are, however, several discrepancies with this system. One example occurs in the "olla" group, typified by C. olla (Batch) Pers., wherein species reputedly lack plications. Cyathus olla f. anglicus (Lloyd) H.J. Brodie possesses a distinctly sulcate mouth (Lloyd 1906, Brodie 1952). In addition C. olla f. brodiensis Shinners & J.P. Tewari, described based on Random Amplified Polymorphic DNA and morphological data, has a remarkably striate inner peridium (Shinners and Tewari 1998). Finally in Brodie’s (1975) notes on an isotype specimen of C. hookeri Berk., another member of the olla group, fruiting bodies were faintly plicate internally. In another example C. griseocarpus H.J. Brodie & B.M. Sharma was placed by Brodie (1984) in group "striatus" because of distinct striations reported on the inner peridium. Examination of the holotype specimen of C. griseocarpus however revealed that some fruiting bodies are smooth and lack plications. The importance of plications as a taxonomic character in the systematics of Cyathus therefore is questionable and this issue is addressed herein.
The two major groups based on plications were separated further into groups based on the type of peridial hairs, the structure of peridioles and the color of fruiting bodies (Brodie 1975, 1977, 1984). In general the hairs on the outside of the peridium of Cyathus fruiting bodies can be of three types: (i) a tomentum characterized by fine, short and soft hairs; (ii) hairs that are long and aggregated into small tufts or mounds; and (iii) hairs that are long and aggregated resulting in a shaggy or woolly appearance. Characteristics of the peridial hairs are affected by the environment and by the age of fruiting bodies, and its taxonomic significance therefore is questionable.
Brodie’s taxonomic organization has been followed by most mycologists. Cyathus comprises 44 species (Kirk et al 2001) and is the most speciose genus in the family Nidulariaceae (Nidulariales). Cyathus is distinguished from the other three genera in the Nidulariaceae (Crucibulum Tul., Nidula V.S. White and Nidularia Fr.) based on gray to black peridioles with funicular cords and peridia composed of three layers of tissues.
The application of DNA sequence data in phylogenetic analyses of basidiomycetous fungi have let mycologists test taxonomic constructs based on morphology (Binder and Hibbett 2002, Hibbett and Binder 2002, Moncalvo et al 2002, Zhang et al 2004). Previous phylogenetic studies based on LSU datasets and including gasteromycetous fungi have included only a single species of Cyathus (e.g. C. striatus in Hibbett et al 1997, Hibbett and Thorn 2001, C. stercoreus in Moncalvo et al 2002). In those studies Cyathus nested within the euagarics clade with numerous lamellate genera and with members of the Lycoperdales. The most recent treatment of the agaricoid clade of basidiomycetes (Matheny et al 2006) included only Crucibulum laeve and C. striatus as representatives of the Nidulariaceae. In this research the Nidulariaceae was sister of the Cystodermataceae (represented by Cystoderma amianthinum) and together these two families formed a well resolved clade with the Agaricaceae sensu stricto. In a previous study (Zhao et al 2004) RAPD analyses performed on 43 Cyathus isolates representing 18 Cyathus taxa resulted in the establishment of two new species (Zhao et al 2004). However this study failed to provide a clear picture of species relationships within Cyathus. In addition all isolates used in this study originated from China, thus the results lack worldwide applicability. To date no molecular phylogenetic studies investigating evolutionary relationships among Cyathus species have been published. Consequently the current morphological classification proposed by previous researchers has never been tested.
The objectives of this study are to reexamine the genus Cyathus with morphology and sequences analyses to determine the phylogenetic importance of various characters used in Cyathus taxonomy. We also test whether the classification scheme proposed by Brodie based on plications and hairs on fruiting bodies is phylogenetically supported. Our sampling strategy was to include as many type specimens and authentic material studied by Brodie as was available.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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). Each specimen was examined morphologically following Brodie’s protocols (Brodie 1975). Twenty-two taxa (23 strains) of Cyathus and two other bird’s nest fungi, Crucibulum laeve (Huds.) Kambly and Nidula niveotomentosa (Henn.) Lloyd, were sequenced (TABLE I). Sequences of Cystoderma amianthinum (Scop.) Fayod were retrieved from GenBank and used as outgroup for rooting propose (TABLE I) (cf. Matheny et al 2006). Cyathus samples included 13 holotypes or paratypes, two samples identified by J.H. Brodie, five samples identified by R.L. Zhao and three cultures borrowed from the Mycological Herbarium of Southwest Forestry College, China (SWFC).
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, 2004) and Cai et al (2005). The second extraction method involved the use of a commercial DNA extraction kit (E.Z.N.A. Forensic Kit, D3591-01, Omega Bio-Tek) for dried fungal specimens. PCR reactions were performed in a 50 µL volume (0.3 mM primers [LROR: 5'-ACCCGCTGAACTTAAGC-3' and LR5: 5'-TCCTGAGGGAAACTTCG-3';or ITS4: 5'-TCCTCCGCTTATTGATATGC-3' and ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3'], 10–20 ng DNA template, 1x buffer, 0.2 mM dNTPs, 1.5 units Taq and sterile water). The thermal cycles consisted of 94 C for 3 min, 30–35 cycles of 94 C for 1 min, 52 C for 50 sec and 72 C for 1 min, with a final extension step of 72 C for 10 min.
PCR products were checked by electrophoresis gels (1% agarose) stained with ethidium bromide in 1x Tris-boric acid EDTA buffer. They were purified with minicolumns according to the manufacturer’s protocol (GFX PCR DNA and Gel Band Purification Kit, 27-9602-01, Amersham Biosciences). Primers ITS4, ITS5, LROR and LR5 were used to sequence both strands of the DNA molecule in an automated sequencer at the Genome Research Centre, University of Hong Kong.
Sequence alignment and phylogenetic analysis.— –
All sequences initially were aligned with Clustal x with default settings (Thomson et al 1997). Then they were manually adjusted in BioEdit and gaps were introduced to improve alignments. The alignments were submitted to TreeBase (Submission ID: SN2794).
Phylogenetic analyses were performed with PAUP* 4.0b10 (Swofford 2004). Heuristic searches of the ITS, LSU and combined (ITS + LSU) datasets were performed under four optimality criteria: weighed parsimony (WP), maximum likelihood (ML), neighbor joining (NJ) and MrBayes (Huelsenbeck and Ronquist 2001, Huelsenbeck et al 2001). Unordered characters, random taxon addition sequences, gaps treated as missing data and the tree bisection-reconnection (TBR) branch swapping were used in the analyses. For weighted maximum parsimony MAXTREES was limited to 5000 trees with 1000 replications. The weighted parameters were produced with Stmatrix (François Lutzoni and Stefan Zoller, Duke University) as described in Miadlikowska et al (2002). The best nucleotide substitution models for maximum likelihood were chosen with MrModeltest2.2 (Nylander 2004). All characters were weighted equally for neighbor joining. Bootstrap values (BS) were obtained from 1000 replicates. Unconstrained trees (WP, ML and NJ trees) were compared in PAUP* with Kishino-Hasegawa and Shimodaira-Hasegawa tests (Kishino and Hasegawa 1989). Bayesian posterior probability (PP) was calculated with MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001). One million generations were run for four Markov chains and sampled every 100th generation, resulting in 10 000 trees. The first 2000 trees were discarded as part of the burn-in phase, and the remaining 8000 trees were used to calculate posterior probabilities in a 50% majority rule consensus tree. Trees were viewed in TreeView and exported to graphics programs (Page 1996).
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RESULTS |
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Stmatrix was used to assign appropriate parameters and gaps were treated as missing data for weighted parsimony analysis. This yielded one equally parsimonious tree with a length of 468 steps (CI = 0.776, HI = 0.224, RI = 0.746). For maximum likelihood analysis the model selected by MrModeltest 2.2 was GTR + G, and the resulting tree has a likelihood score of 2969.71694. Results of the Kishino-Hasegawa and Shimodaira-Hasegawa tests among topologies obtained from ML, NJ and WP re provided (TABLE II). The ML tree (FIG. 1) was the best tree and therefore chosen to represent ITS phylogenies.
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), although there were slight differences in the topological arrangement within the clades. Clade A includes C. africanus (type specimen and a second specimen), C. africanus var. latisporus, C. colensoi, C. hookeri, C. jiayuguanensis, C. olla and C. olla f. brodiensis with 100% PP and 98% BS supports. Clade B is composed of C. annulatus, C. crassimurus, C. renweii, C. setosus, C. stercoreus and C. triplex but had PP and BS supports of less than 50%. Clade C comprises C. berkeleyanus, C. gansuensis and C. pallidus with 100% PP and 83% BS supports.
In the ML ITS tree (FIG. 1) C. hookeri nested with C. olla and C. olla f. brodienensis with 95% PP and 67% BS supports, and of 776 aligned nucleotide sites C. hookeri, C. olla and C. olla f. brodienensis differed by only five base pairs.
Phylogeny based on LSU sequence data.— – In the LSU dataset 19 sequences were included, consisting of 16 Cyathus taxa, Crucibulum laeve, Nidula niveotomentosa, and the trees were rooted with Cystoderma amianthinum. This dataset consisted of 797 characters of which 10 characters were excluded and 69 were parsimony informative.
Weighted parsimony analysis treated gaps as missing data and yielded a single tree with length of 193 steps (CI = 0.736, HI = 0.264, RI = 0.669). Maximum likelihood analysis with likelihood settings with the best-fit model (GTR + G) resulted in a tree with a score of 2020.58832. Results of the Kishino-Hasegawa and Shimodaira-Hasegawa tests (TABLE II) indicated that the ML analysis yielded the best tree (FIG. 2), although not topologically different from the WP results.
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Phylogeny of the combined datasets.— – The Cyathus taxa that successfully provided both ITS and LSU sequences were used to construct the combined dataset. In this data matrix there were 10 Cyathus taxa, Crucibulum laeve, Nidula niveotomentosa and out-group Cystoderma amianthinum with a total of 1567 characters out of which 216 were parsimony informative and 138 characters were excluded.
WP analysis resulted in a single tree with a length of 594 steps (CI = 0.798, HI = 0.202, RI = 0.709). Maximum likelihood settings were from the best-fit model (GTR + I + G) and ML analysis yielded a tree with the score of 4781.34621. The Kishino-Hasegawa and Shimodaira-Hasegawa tests indicate that WP, ML and NJ trees were not significantly different and ML tree (FIG. 3) is the best (TABLE II). The MrBayes tree also resulted in three major clades with strong PP support and had the same internal topologies as the WP, NJ and ML trees, although the relationships among the clades were not strongly supported. Species in Clade A grouped with 100% PP and 100% BS support, species in Clade B grouped with 100% PP and 69% BS support, while those in Clade C grouped with 100% PP and 99% BS support. Cyathus setosus was outside all three major clades. In all three analyses (ITS, LSU, combined ITS-LSU) species of Cyathus formed a monophyletic clade with 100% PP and 100% BS support, although the relationship with other bird’s nest fungi was unresolved.
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–3). However the position of C. olla f. lanatus in the LSU tree (FIG. 2) was in Clade C with 93% PP and 73 BS supports.
Phylogeny of Cyathus jiayuguanensis, C. africanus and C. africanus var. latisporus. The results of ITS, LSU and the combined dataset (totaling 1567 bases) analyses using sequences from holotype specimens showed that C. jiayuguanensis and C. africanus var. latisporus have identical sequences. Cyathus africanus (SWFC 20782) nested with C. jiayuguanensis and C. africanus var. latisporus in the ITS tree (FIG. 1) but was distant from the holotype of C. africanus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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, Lloyd 1906, Brodie 1975, 1984). These systems relied heavily on the importance of a plicate peridium and at which rank this character was used to separate taxa. To date Brodie’s system is the most widely accepted one, although its phylogenetic significance has been untested. In this study we make an important step toward forming a more natural classification of Cyathus by combining morphological data with phylogenies generated from rDNA sequence datasets. Taxa incorporated in this study included representatives of all seven groups in Brodie’s classification system and most data were obtained from either type specimens or authentic Brodie-determined materials (TABLE I). Phylogenetic trees generated from the ITS, LSU and combined datasets are essentially similar. Our data indicate that the seven-group morphologically based system as proposed by Brodie (1975, 1984) is not concordant with the molecular phylogenies proposed here (FIGS. 1–3).
The presence or absence of plications was the primary character for partitioning groups in traditional taxonomic constructs. The presence or absence of a tunica on the peridioles, types of hairs and fruiting body shape were of secondary importance (Brodie 1975, 1984). However results here indicate that plications on the peridium do not appear to be a phylogenetically informative character. Species possessing this character are distributed in all three major clades in our phylogenies (FIGS. 1–3).
In contrast the size of basidiospores is a significant morphological character for distinguishing the major clades. In Clade B, there are eight Cyathus species (C. annulatus, C. crassimurus, C. helenae, C. poeppigii, C. renweii, C. setosus, C. stercoceus and C. triplex). These species are distributed among five groups in Brodie’s system: "triplex", "gracilis", "stercoreus", "poeppigii" and "striatus". All members of Clade B have basidiospores with a length greater than 15 µm. In comparison members of Clade A (C. africanus, C. africanus f. latisporus, C. conlensoi, C. griseocarpus, C. guandishanensis, C. hookeri, C. jiayuguanensis, C. olla, C. olla f. anglicus and C. olla f. brodiensis), and Clade C (C. berkeleyanus, C. gansuensis, C. olla f. lanatus and C. pallidus) form basidiospores shorter than 15 µm. In previous morphological studies (Tulasne 1844, Lloyd 1906, Brodie 1975, 1984) spore size has been used only in delimitations at the species rank, and no publications reported segregating Cyathus species into two major groups based on spores longer or shorter than 15 µm. It should be noted that clades A and C cannot be distinguished from each other solely on spore measurements.
Fruiting body color has been considered useful in the partitioning of species groups (Brodie 1975, 1984). Our morphological studies indicate that species in Clade B have peridia that are brown, reddish-brown or dark brown on the outside while species in clades A and C have peridia that are much lighter, typically yellow, gold or gray. As with basidiospore size, distinguishing among members in clades A and C based solely on peridium coloration is problematical.
Our morphological studies revealed that the best characters for distinguishing members of Clade A from Clade C are the thickness of the tomentum covering the peridium and the outline of fruiting bodies. Species in Clade C have a thick, felt-like tomentum, usually aggregating into shaggy or woolly hairs covering the peridium. Their fruiting bodies are crucible-shaped without a distinct stipe. In comparison species in Clade A have a thin tomentum of fine hairs covering the peridium and the fruiting bodies are funnel-shaped with a constricted base or a distinct stipe.
The problematical taxon Cyathus setosus has spores that are 17–24 µm long, has a dark-colored peridium and nests within Clade B in the ITS analysis (FIG. 1). In the analyses of LSU and combined datasets, however, this species was isolated and sister of clades A, B and C. The most conspicuous diagnostic feature for C. setosus is the long setae at the mouth of the fruiting body. Whether C. setosus represents a distinct group within Cyathus will require broader sampling and further testing.
In our analyses Cyathus resulted as a monophyletic lineage with 100% PP and 100% BS support in ITS, LSU and combined ITS-LSU trees. In addition, in all three analyses, Nidula niveotomentosa was sister of Cyathus but with low statistical support. To resolve the relationships among the genera within the Nidulariaceae additional members of the other genera will need to be included; however that was not the focus of this research.
Phylogeny of Cyathus olla and its forms.— –
Cyathus olla is the type species of the "olla" group in Brodie’s system. In addition to the diagnostic characters of Clade A mentioned above C. olla fruiting bodies also have flared mouths, large peridioles, peridia lacking plications and broadly ellipsoid basidiospores. A number of infraspecific taxa have been accepted by various authors, including C. olla f. olla, C. olla f. lanatus, C. olla f. brodiensis and C. olla f. anglicus (Brodie 1952, 1975, 1978, Shinners and Tewari 1998). In Lloyd’s (1906) monograph of the Nidulariaceae, C. anglicus was described as distinct because of its large fruiting body and markedly sulcate mouth. Later Brodie (1952) accepted it as a form of C. olla because single-spore mycelia of typical C. olla were found to be sexually compatible with those of C. anglicus. Cyathus olla f. brodiensis, published by Shinners and Tewari (1998), differs from other forms in having distinct plications on the inside of the peridium and a unique RAPD fingerprint. Cyathus olla f. lanatus (Brodie 1978) is characterized by having a thick tomentum that aggregates into shaggy or woolly hairs on the outside of the fruiting body. Results from our LSU analyses suggest that C. olla f. lanatus should be recognized as an independent species. Cyathus olla f. lanatus is phylogenetically closer to C. pallidus and C. gansuensis (Clade C) than to C. olla and related species (Clade A). This form therefore is redescribed and a new combination is proposed below.
Phylogeny of Cyathus hookeri and C. olla.— –
The specimens SWFC 20799 of C. hookeri and SWFC 21137 of C. olla f. brodiensis were identified based on the descriptions of Brodie (1975) and Shinners and Tewari (1998), while the specimen BPI 727227 of C. olla was identified by Brodie. The ITS sequence analyses reveal a close relationship among C. hookeri, C. olla and C. olla f. brodiensis. These taxa form a subclade within Clade A with 95% PP and 67% BS supports. All three taxa are strikingly similar morphologically and are characterized by light-colored peridia, faint plications, and they have the same basidiospore shape and size. These data suggest that C. hookeri might represent a synonym of C. olla. Before any taxonomic amendment is proposed, further morphological and molecular studies incorporating topotypical isolates of these taxa should be conducted.
Phylogeny of C. jiayuguanensis, C. africanus and C. africanus var. latisporus.— – A similar result was obtained for the species C. jiayuguanensis (type), C. africanus var. latisporus (type) and C. africanus (SWFC 20782). The ITS sequences of all three specimens are identical, and in addition they share similar morphologies. The data suggest that they should be treated as one species. However the correct epithet is not C. africanus. The phylogenetic placement of the holotype specimen of C. africanus (DAOM 200370) is distant from specimen SWFC 20782 that was identified as C. africanus. We consider the latter specimen as misidentified. We accept C. jiayuguanensis as the correct name for this species and accept C. africanus var. latisporus as a synonym below.
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TAXONOMY |
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the new groups are named herein the "ollum" group (Clade A), "striatum" group (Clade B) and "pallidum" group (Clade C). We tentatively place Cyathus setosus in the striatum group based on the ITS analysis, basidiospores >15 µm long, and the dark-colored peridium.
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Synonymy and new combination.. Cyathus jiayuguanensis J. Yu, T.X. Zhou et L.Z. Zhao, Mycosystema 21:313. 2002.
= Cyathus africanus var. latisporus Y.H. Chen et J. Yu, Mycosystema 22:345. 2003.
Fruiting bodies 6–8 mm tall x (4–) 5–6 mm wide at the mouth, cup-like, obconical, with slender stipe; mouth smooth. Peridia thin, exterior light brown to grayish-orange and covered with a fine tomentum aggregated into tufts or nodules; inside of peridium brownish-gray or gray, smooth. Peridioles 1.5–2 x 1.5–1.8 mm, subcircular, circular or broadly ellipsoid, grayish-brown or lighter; cortex a single layer 20–25 µm thick; tunica 10–20 µm thick. Basidiospores 8.5–12.5 x 6.5–8.2 µm, ovoid.
Specimens examined: PR CHINA, GANSU PROVINCE: Jiayuguan, on soil, 12 Oct 1999, Zhou, T.X. & Zhao, L.Z. (SWFC 20802; holotype of C. jiayuguanensis); PR CHINA, NEIMENGGU PROVINCE: Helingeleqi, on soil, 13 Sep 2000, Zhou, T.X. (SWFC 21187; holotype of C. africanus var. latisporus).
Notes: Because the fruiting bodies of C. jiayuguanensis lack plications and are covered with shaggy hairs Yang et al (2002) treated it as the member of the "pallidus" group. The fruiting bodies of C. africanus var. latisporus lack plications and are covered with tufts of fine hairs that are shorter than in C. jiayuguanensis, which resulted in its being treated as a member of the "olla" group (Chen et al 2003). Morphological examinations of the holotype specimens of C. jiayuguanensis and C. africanus var. latisporus showed that they share many morphological characters, such as similar color and size of fruiting bodies, the lack of plications, thin peridial walls, a single cortex with thin tunica and importantly similar basidiospore shape and size. The only distinction between the species is that C. jiayuguanensis has longer hairs on the outside of the peridium than those of C. africanus var. latisporus. This feature is one of main differences between the "olla" and "pallidus" groups of Brodie (1975). Based on overall morphological similarity and identical ITS sequences we accept C. africanus var. latisporus as a synonym of C. jiayuguanensis.
Cyathus lanatus (H.J. Brodie) R.L. Zhao, comb. et stat. nov.
Cyathus olla f. lanatus H.J. Brodie, Bot. Notiser 131:31–34, 1978.
Fruiting bodies 5–7 mm high x 5–6 mm wide at mouth, crucible-shaped, without a distinct stipe, broadly attached to a firm thickened base, some flared at the mouth, lip distinctly fimbriate. Peridia light gray, light buff or grayish-yellow, externally covered with upward-pointing tufts or radiating tufts formed from thick and fine hairs; interior smooth, lacking plications, gray and shiny. Peridioles 1.2–3.5 mm diam, lenticular or irregular in outline, some plump, gray to light buff, shiny. Funiculus stout and short. Tunica thick, cortex a single layer, together 80–100 µm thick. Basidiospores 9–13 x 7–8 µm, mostly ovoid, occasionally provided with an apiculus, thick-walled.
Specimens examined: USA, IDAHO: Owyhee County, Reynold’s Creek, on dead twigs of Artemisia sp., 4 Nov 1976, T. Trueblood (DAOM 200703, HOLOTYPE); USA. IDAHO: Owyhee County, West Rabbit Creek, on dead twigs of Sarcobatus vermiculatus, 2 Nov 1969, T. Trueblood (DAOM 200704, PARATYPE).
Notes: Examination of the holotype specimen revealed some minor differences from those published in the protologue (ibid.). In Brodie’s description the tunica was described as up to 60 µm thick with a cortex of 60–80 µm thick (in total 120–140 µm), whereas we found the tunica plus cortex to be 80–100 µm thick. The spores were smaller in our analysis, 9–13 x 7–8 µm vs. 12–15 µm x 7.5–9 µm in the protologue.
Brodie (1978) was reluctant to include this new taxon as a form of C. olla because the fruiting bodies were "very small", "often short, thick-walled and bleached." He indicated that at first glance they might be mistaken easily for Crucibulum laeve. Brodie included the taxon as a form of C. olla based mainly on fruiting-body shape (some fruit bodies were flared at the mouth), on basidiospore size, the lack of plications on the peridium and because C. olla "exists in many variations." He stated further that "no test of possible fertility between C. olla f. lanatus and C. olla has been carried out because all attempts to germinate spores of C. olla f. lanatus have been unsuccessful."
In our analysis based on the LSU dataset C. lanatus nested with C. pallidus and C. gansuensis, species with thick and fine peridial hairs and members of the pallidum group (Clade C). Cyathus olla and its forms nest with other species and comprise the group ollum (Clade A). The differences between C. lanatus and other species of the pallidum group are mainly plication characters, thickness of fine hairs and basidiospore shape.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Additional addresses: Mushroom Research Foundation, 128 Moo3, Bahn Pha Deng, T. Pa Pae, A. Mae Taeng, Chiang Mai 50150, Thailand; and Faculty of Conservation Biology, Southwest Forestry College, Kunming, Yunnan Province 650224, PR China. 
2 Corresponding authors. E-mail: ded{at}sfsu.edu and zhao_ruilin{at}yahoo.com.cn
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LITERATURE CITED |
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Brodie HJ. 1952. Infertility between two distinct forms of Cyathus olla. Mycologia 44:413–423.
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———. 1978. A hitherto un-named form of Cyathus olla (Nidulariaceae). Bot Notiser 131:31–34.
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Chen YH, Yu J, Zhou TX. 2003. A new species, a new variety and a new Chinese record of Cyathus. Mycosystema 22: 345–348.
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