Morphological and DNA-based Classification of Cyathus sp. Isolates from Thailand (Basidiomycota, Nidulariaceae)

Cyathus sp. isolates from three areas in Thailand (Khon Kaen University, Nam Nao National Park and Phu Ruea, Loei Province) were morphologically characterized by their peridiocarp, peridioles and basidiospores. This allowed to assign most isolates to five Cyathus species: C. berkeleyanus, C. earlei, C. pallidus, C. stercoreus and C. striatus. Phylogenetic analysis of ribosomal ITS sequence data yielded three groups of Cyathus isolates and unidentified species group. The Pallidum group including KKUNN1, is closely related to C. berkeleyanus (DQ463355.1), KKUITN2 (KU202745) and KKUITN3 (KU202751) are closely related to C. pallidus (DQ463356.1). The Ollum group includes C. africanus and C. hookeri. the Striatum group, such as KKUITP2 (KU202744) and KKUITP3 (KU202743) are closely related to C. stercoreus (DQ463356.1). The LSU sequence data suggest that KKULN2 and KKULN3 are closely related to C. pallidus (DQ463336.1), whereas KKULP2 and KKULP3 are closely related to C. stercoreus.


INTRoDUCTIoN
T h e b i rd 's n e s t f u n g i ( p hy l u m Basidiomycota, agaricoid clade, family Nidulariaceae) comprize five genera, viz. Crucibulum, Mycocalia, Nidularia, Nidula and Cyathus [1][2][3][4] . Typically, these genera produce small fruiting bodies on soil, animal droppings or organic matter 5 . The presence of gray to black peridioles with funicular cords distinguishes Cyathus from the other nidulariacean genera 6 . Cyathus species are inedible mushrooms that absorb nutrients into their cells by releasing enzymes to decompose organic matter. The basidiocarp of Cyathus sp. is funnel shaped with peridioles and a funicular cord. The basidiocarp are off-white color covered with fluffy hair and the reason that makes it different from other species because of the peridium surface is folded and contains subspherical basidiospores 7,8 . There are 45 species in this genus 3 , which have spread around the world, especially in warm and hot areas 5 . There are doubts about the propriety of the old classification system based on the traditional taxonomy. Basidiocarps examination presented a large amount variation between the various collections, and morphological identification. Observations show that all culture collections were classified using morphology of basidiocarp. So, Martin et al. 9 collected Cyathus from Cape Verde and identified by using morphology and molecular data. They obtained a new species, Cyathus lignilantanae sp. nov. Whereas, Amazonian Cyathus species have a new species such as Cyathus albinus with light color hirsute exoperidium contrasting with a dark brown emplacement and basidiospores ovoid to elliptical and the other three such as C. amazonicus, C. earlei and C. triplex, are recorded for the first time from their localities 10 . The extinct category of the Red List of Threatened Fungi of Japan found that Cyathus badius and C. boninensis are included. The last and the only collections of both species were made in 1936, and no additional samples have been collected since then. Nowadays the samples were collected and morphological comparisons with the holotype and phylogenetic analyses based on ribosomal ITS, LSU and concatenated dataset placed C. badius in a highly contributed clade with C. parvocinereus 11 . In northern Thailand, 16 species of the five nidulariacean genera have been collected 6 , including the new species Cyathus subglobisporus which on the basis of ITS and LSU nucleotide sequence data was assigned to the Striatum species group 6 . Yet, despite these few previous studies, the species diversity of the family Nidulariaceae, and of the genus Cyathus in particular, in Thailand remains poorly documented. Therefore, we report here on the identification of 12 specimens of Cyathus from NE Thailand, based on morphological data combined with ITS and LSU nucleotide sequences.  14,15 .

DNA sequence alignment and phylogenetic analysis
ClustalX2 was used to align nucleotide sequences according to its default settings 20,21 . Cyathus sp. comprise of 10 species for ITS, 12 species for LSU and four other bird's nest fungi obtained from GenBank (Table 1) with. PAUP v.4.0b101 were used for phylogenetic analysis following Swofford 22 , Felsenstein 23 , Huelsenbeck and Ronquist 24 and Page 25 methods.

Morphological characterization
The morphological identification of Cyathus sp. isolates was based on Fungi Keys, Mushroom Expert 2,12,13 . The isolates were classified as follows:
Our phylogenetic analyses showed that KKULN2 and KKULN3 are closely related to C. pallidus    (Fig. 7).

DISCUSSIoN
Bird's nest fungi identification is a matter of careful, sometimes microscopic, inspection of the morphological features. In this study we identified Cyathus sp. using morphological characters, including peridiocarp, peridiole and basidiospore, under stereo-and compoundmicroscopes. The results showed that KKUNN1 was C. berkeleyanus; KKUPR1 was C. earlei; KKUITN2 and KKUITN3 were C. pallidus; KKUITP2 and KKUITP3 were C. stercoreus and KKU1, KKU2, KKU3, KKU4, KKU5 and KKU6 were C. striatus. Similarly, Cyathus morelensis with taxonomic opinion, basidiocarp images and peridiole, basidiospores and peridiocarp illustrations. Additionally, the four species that registered from Brazil including C. intermedius, C. montagnei, C. setosus and C. triplex was first reported from northeastern Brazil 2 . Inspection of holotype of C. setosus, found that spore dimensions different from explained in the original publication.
The DNA sequence data of ITS and LSU, were consistent with the morphological identifications of KKUITN2 (C. pallidus), KKUITN3 (C. pallidus), KKUITP2 (C. stercoreus) and KKUITP3 (C. stercoreus). DNA identification may remain undecided if there are insufficient well-identified reference sequences in GenBank and BOLD. For example, BLAST searches in GenBank shown 72-82% identity with C. striatus. In addition, we amplified the SSU sequence of all isolates when compared to the GenBank database it showed that all of them were closely related with C. striatus with 99% identity.
Hence, the DNA data were not informative in this study. Alternately, three infrageneric groups consist of Ollum, Pallidum and Striatum was classified by using datasets of ITS and LSU. Still these three species groups can be separated on morphological basis. In northern Thailand, bird's nest fungi have been collected before and this leaded even to the description of a new