Three New Anamorph of Ceramothyrium from Fallen Leaves in Vietnam

Three new anamorph of Ceramothyrium aquaticum sp. nov., Ceramothyrium exiguum sp. nov., and Ceramothyrium phuquocense sp. nov. are described and illustrated. These fungi were isolated from submerged decaying leaves collected from Phu Quoc National Park, Phu Quoc province, Viet Nam. The phylogeny based on ITS region and D1/D2 of the 28S rDNA gene showed that these fungi nested in the Ceramothyrium. Morphologically, C. aquaticum, C. phuquocense sp. nov. and C. exiguum sp. nov. are characterized; they were different from known anamorph species of Ceramothyrium by having one main axis and two lateral arms with 70 90, 33.5 72.5 and 70 130 μm long main axis, respectively. The table to compare Ceramothyrium anamorph is also given.


Introduction
The genus Ceramothyrium was erected by Bat.& Maia based on type species Ceramothyrium paiveae; it was characterized by the lack of setae and by the hyaline, transversely pluriseptate ascospores [1].No anamorph stage of this fungus was discovered until the study of [2].In this study, they reported Stanhughesia as anamorph of the Ceramothyrium.There were 38 species of Ceramothyrium were reported; among them, three species C. carniolicum, C. linnaeae and C. lycopodii had Stanhughesia asexual stage [2].More two anamorph of Ceramothyrium were reported under the older telemorph name: C. melastoma and C. podocarpi.While C. melastoma has Trisulcosporium morph, C. podocarpi has its own anamorph stage [3].

Morphological Study
The isolates were cultured at 25˚C on a potato carrot agar medium (PCA, extract from 20 g/L potato, extract from 20 g/L carrot, 15 g/L agar), LCA and potato dextrose agar (PDA, Nissui, Japan) for morphological observations.Observations were made under a differential interference contrast microscope (DIC: Axioplan 2, Zeiss, Jena, Germany) and a scanning electron microscope (JSM-6060: JEOL, Tokyo, Japan).

DNA Isolation and PCR Amplification
A Small pieces of a colony (3 × 3 mm) grown on malt extract agar (MEA) medium at 25˚C for 10 d were put into 2 mL Cryo tubes.DNA was extracted using the PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA).PCR was performed by using KOD-Plus Kit (Toyobo, Osaka, Japan), following the manufacturer's protocol.The nrDNA large subunit region (LSU D1/D2) was amplified with primers NL1 and NL4 [5].To amply the ITS region, the combination ITS1 and ITS4 [6] were used.Amplification of the DNA fragments was performed using the GeneAmp PCR System 9700 (Applied Biosystems) under the following thermal cycling programme: an initial denaturation at 94˚C for 2 min, 35 cycles of denaturation at 94˚C for 15 s, annealing at 56˚C for 30 s, extension at 68˚C for 1 min 30 s, a final extension at 68˚C for 10 min, and a 16˚C soak.PCR products were checked by agarose gel electrophoresis, and were purified by using AMPureKit (Agencourt Biosciences, Beverly, MA, USA).Sequencing reactions were performed by using the Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems) and the primers of the PCR.The newly generated sequence data were deposited in GenBank.

Phylogenetic Analysis
Sequences were assembled and edited manually using BioEdit ver.7.09 (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA).Sequences were aligned with Gen-Bank sequence data obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/) by using Clustal X [7].A phylogenetic tree was inferred with neighbor-joining (NJ) method [8] and the Knuc value [9] by using Clustal X.The topology of the tree was evaluated by the bootstrap resembling method [10] with 1000 replicates.The NJplot programme [11] was used for plotting the phylogenetic tree.

Morphology
All of three fungal isolates were slow growth on LCA and PDA media, spores are easily produced when submerging in water 3 -4 days.Conidiopphores absent.
Conidiogenous cells intercalary in hyphae.Conidia in cultures are holoblastic, tri-radiate.The spore arises from a cell of the mycelium as a lateral bud.This bud is initially unicellular and constricted where it joints the parent hypha, it grows into the main axis of the spore.When the main axis reaches to three-or four-celled, one or two lateral arms are budding out from opposite side of basal cell of the main axis.At first arms are unicellular, and then it extend away from the main axis at near 120˚ until to become three-to six-celled as the main axis itself extends farther to become eight-to ten-celled.Eventually the main axis becomes constricted off from the parent cell and the spore become detached.
The aligned ITS region sequences of approximately 600 bases were obtained from isolates were aligned with the ITS region of the sequences obtained from Gen-Bank.The manually adjusted ITS alignment contained 21 sequences (including the one out group sequence).In NJ analysis, the phylogenetic hypothesis showed that VTCCF-1206 (LC360297); VTCCF-1209 (LC360298) and VTCCF-1210 (LC360299) cluster within the Ceramothyrium carniolicum clad, with bootstrap support of 98% (Figure 2).The genus Ceramothyrium has Stanhughesia asexual morphs [2] [3] and Trisulcosporium [3] represents a genus of epiphyllous ascomycetes in the Chaetothyriales for which DNA data has been lacking until the recent study of Chomnunti   [3], [14], [15] and [16].Phylogeny based on ITS region and D1/D2 of the 28S rDNA gene analysis showed that these new Ceramothyrium anamorph were nested in Ceramothyrium clad with 98% and 75% bootstrap value, respectively.In this study, only the asexual morph of Ceramothyrium aquaticum, C. minima and C. phuquocense were observed, we choose to name it in the older sexual genus, Ceramothyrium which consisted of 41 taxa, accepting Stanhughesia carniolica, S. linnaeae, S. lycopodii, S. nipponica, C. melastoma, C. podocarpi [2], [3], [17] and our three new anamorph having existing names in Ceramothyrium as synonym.

Figure 2 .
Figure 2. Phylogeny of Stanhughesia spp.and their relative species base on ITS rDNA sequence.MP bootstrap value ≥ 75%.
had a truncate basal cell, 3-5 µm wide, subsequent ones gradually tapered to a 2 -3 µm wide, apical cell ending in a c. 1 -1.5 µm wide beak, thinand smooth-walled, markedly constricted at the main septa, but slightly or not constricted at secondary, very thin septa which sometimes divide the cells.While our Ceramothyrium anamorph, septa is constricted at all cells, makes the main axis and arms look like chains of sausages.Furthermore, size of main axes of our new Ceramothyrium differs from each other and all of other known Ceramothyrium anamorph.Main axes of Ceramothyrium aquaticum, C. minima and C. phuquocense are 70 -90, 33.5 -72.5 and 70 -130 µm, respectively; while Stanhughesia carniolica, S. linnaeae and C. melastoma's main axes were 70 -120, 25 -40 and 15 -30 µm, respectively (Table1