A new species of bird’s nest fungi: characterisation of Cyathus subglobisporus sp. nov. based on morphological and molecular data

Recent collections of bird’s nest fungi (i.e. Crucibulum, Cyathus, Mycocalia, Nidula, and Nidularia species) in northern Thailand resulted in the discovery of a new species of Cyathus, herein described as C. subglobisporus. This species is distinct by a combination of ivory-coloured fruiting bodies covered with shaggy hairs, plications on the inner surface of the peridium and subglobose basidiospores. Phylogenetic analyses based on ITS and LSU ribosomal DNA sequences using neighbour-joining, maximum likelihood and weighted maximum parsimony support Cyathus subglobisporus as a distinct species and sister to a clade containing C. annulatus, C. renweii and C. stercoreus in the Striatum group.


InTRoduCTIon
The genus Cyathus along with the genera Crucibulum, Mycocalia, Nidula, and Nidularia are known as the bird's nest fungi because of their small vase-shaped or nest-like fruiting bodies containing lentil-shaped or egg-like peridioles. Cyathus is the most speciose genus in the family Nidulariaceae (Agaricales). Cyathus is distinguished from the other three genera in the Nidulariaceae based on grey to black peridioles with funicular cords and peridia composed of three layers of tissues (Brodie 1975). Historically, Cyathus was monographed by Lloyd (1906) and Brodie (1975Brodie ( , 1984, and their species concepts, especially those of Brodie (1975), are followed by most mycologists (Liu & Li 1989, Ren & Zhou 1992, Yang et al. 2002, Chen et al. 2003, Zhou et al. 2004). Recognition of Cyathus species is based on morphological characters such as fruiting body shape, coverings and plications of peridia, anatomy of peridioles, and the size and shape of basidiospores (Brodie 1975).
Molecular phylogenetic studies based on rDNA sequence data including several Cyathus species and other gasteromycetous fungi (e.g., C. striatus in Hibbett et al. 1997, Hibbett & Thorn 2001and C. stercoreus in Moncalvo et al. 2002 showed that Cyathus nested within the euagarics clade. The most recent treatment of the Agaricales by Matheny et al. (2006) based on sequence analyses of six loci included Crucibulum laeve and Cyathus striatus as representatives of the Nidulariaceae. Their phylogenetic reconstruction indicated that the Nidulariaceae was sister to the Cystodermateae (represented by Cystoderma amianthinum). Together these two clades appear sister to the Agaricaceae s.l. but without bootstrap support. A phylogenetic study of the genus Cyathus by Zhao et al. (2007) using ITS and LSU ribosomal DNA sequence datasets, and based primarily on type and authentic specimens of 22 taxa of Cyathus, indicated that the genus was monophyletic and included three infrageneric groups recognisable by morphological characters.
There has been a recent interest in studies of basidiomycetes in Thailand, particularly around the area of the Mushroom Research Centre (Le et al. 2007a-c, Wannathes et al. 2007. Some Cyathus species have been reported previously from Thailand (Brodie 1975, Ellingsen 1982, Soytong 1994, Desjardin et al. 2004. However, no new species have been described from Thailand prior to this paper. In this study Cyathus subglobisporus sp. nov. is described and its phylogenetic position is investigated based on ITS and LSU rDNA sequence data.

Morphological studies
Macromorphological characters of freshly collected material were documented as follows. Colour terms follow Kornerup & Wanscher (1978). Peridioles were sectioned by hand, mounted in distilled water, and examined using a light microscope. Spore statistics include average dimensions ± SD; Q, the quotient of spore length and spore width in any one spore; and Q m , the mean of Q-values ± SD. Duplicates are deposited in the BIO-TEC Bangkok Herbarium (BBH), Bangkok, Thailand, the H.D. Thiers Herbarium (SFSU) at San Francisco State University, San Francisco, California, USA, and the herbarium of the Mushroom Research Centre (MRC), Chiang Mai, Thailand.

Molecular phylogenetic studies
DNA extractions were made from gleba using a commercial DNA extraction kit (E.Z.N.A. Forensic Kit, D3591-01, Omega Bio-Tek). PCR reactions performed with primer pairs LROR and LR5 and ITS4 and ITS5. Sequencing protocols follow those of Zhao et al. (2007).
Newly generated sequences (ITS and LSU sequences from the new species), and those retrieved from GenBank (20 ITS and 19 LSU sequences; Table 1) were initially aligned using Clustal X with default settings (Thomson et al. 1997 1 Refers to the type specimen. 2 Indicates that H.J. Brodie determined the specimen. adjustments were made in BioEdit v. 7.0.4 and gaps were introduced to improve alignments. The ITS and LSU alignments were separately submitted to TreeBASE (accession number: SN3455). All sequences used in this study were derived from type or paratype specimens or from authentic material determined by us or by H.J. Brodie (cf. Zhao et al. 2007).
Phylogenetic analyses were performed using PAUP v. 4.0b10 (Swofford 2003). Heuristic searches of the ITS, LSU, and ITS+LSU datasets were performed separately under three optimality criteria: weighed parsimony (WP), maximum likelihood (ML), and neighbour-joining (NJ). Unordered characters, random taxon addition sequences, gaps treated as missing data, and tree bisection-reconnection (TBR) branch swapping were used in all analyses. For weighted maximum parsimony, maxtrees was limited to 5 000 trees with 1 000 replications. The weighted parameters were produced using Stmatrix (François Lutzoni & Stefan Zoller, Duke University) as described in Miadlikowska et al. (2002). The best nucleotide substitution models for maximum likelihood were chosen by using MrModeltest v. 2.2 (Nylander 2004). Bootstrap values (BS) were obtained from 1 000 replicates. Unconstrained trees (WP, ML, and NJ trees) were compared in PAUP using Kishino-Hasegawa and Shimodaira-Hasegawa tests (Kishino & Hasegawa 1989). Trees were viewed in TreeView v. 1.6.6 (Page 1996) and exported to graphics programmes.
The informal infrageneric group names Ollum, Pallidum, and Striatum in Cyathus follow the phylogenetic nomenclature established by Zhao et al. (2007). Although these names constitute improper Latin and do not match the specific epithets olla, pallidus, and striatus, they were established by Zhao et al. (2007) to distinguish the clades from similarly named infrageneric groups used by Brodie (1975), viz., Striatus, Pallidus, and Olla, that contain different subsets of species.

DNA alignment and phylogeny
The ITS dataset consisted of 776 characters of which 371 characters were constant, 109 variable characters were parsimonyuninformative, and 171 characters were parsimony-informative. One hundred and twenty four characters were ambiguous and were excluded. The sequences represent 16 Cyathus species (18 strains), Crucibulum laeve, and Nidula niveotomentosa, while Cystoderma amianthinum was used as the outgroup for rooting purposes based on the previous result that C. amianthinum (Cystodermateae) is sister to Nidulariaceae (Matheny et al. 2006). In all phylogenies under different optimality criteria (NJ, ML, and WP), the genus Cyathus is monophyletic with 100 % bootstrap and all trees have similar topologies. ITS results indicate that Cyathus species were partitioned into three main clades as shown by Zhao et al. (2007). Our new taxon, Cyathus subglobisporus, belongs to the Striatum group, albeit with weak statistical support (Fig. 1).
Fruiting bodies clavate when young, then opening, extending and becoming obconic to infundibuliform, with relatively straight sides in side view; 7-10 mm high, 5 -8 mm wide at the top, with the quotient of height by width 1-1.4; external peridium covered by hairs aggregated into shaggy or hirsute clusters, ivory, pale yellow or buff when young, sometimes with a hint of pale orange, then darkening to pale brown with age and the hairs remaining pallid. Inner peridium surface grey to brownish grey, darkening with age, distinctly plicate when young, becoming striate to smooth with age. Epiphragm membranous, white, covered by buff to pale orange-white hairs similar to those on the external surface of the peridium, cracking irregularly during maturation and disappearing or leaving a minutely fimbriate lip along the top edge of the peridium. Base of the fruiting bodies narrower than the rest of the fruiting body but lacking a distinct stipe. Peridioles 1.5 -2 mm diam, lenticular, greyish brown to pale brown; peridiole covering composed of two layers: a black inner cortex layer, 15 -25 μm thick, and a yellowish brown or dark brown outer tunica layer, 25 -50 μm thick. Basidiospores 13-18 × 12 -16 μm (av. = 15.8 ± 2.8 × 14.1 ± 2.1, Q = 1-1.31, Q m = 1.12 ± 0.19, n = 50), subglobose or rarely broadly ellipsoid, hyaline, smooth, thick-walled (1.5 -2(-3) μm). Basidia not observed. Clamp connections present.
Habitat -On rotten bamboo stems in moist forest.
The spores of Cyathus olivaceobrunneus (named after its olivebrown peridium) are similar to those of C. subglobisporus in shape and size, and the former species has been suspected to be a synonym of C. poeppigii (Brodie 1975). Examination of the type specimen of C. olivaceobrunneus showed the colour of its fruiting bodies to be much darker and to have longer spores (av. = 17.88 ± 8.75 × 13.2 ± 2, Q m = 1.36 ± 0.34, from holotype) than those of C. subglobisporus. We were unable to generate quality sequence data from available material of C. olivaceobrunneus for comparison with C. subglobisporus.
The genus Cyathus was originally subdivided into seven groups based on morphological characters (Brodie 1975(Brodie , 1984). Brodie's classification system is not supported by phylogenetic analysis of molecular data, and the recognition of only three infrageneric groups (Ollum, Pallidum, and Striatum) was established based on morphological and molecular data (Zhao et al. 2007). In this study, the combined ITS and LSU phylogenies indicate that C. subglobisporus belongs to the Striatum group.