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Additions to Chaetothyriaceae (Chaetothyriales): Longihyalospora gen. nov. and Ceramothyrium longivolcaniforme, a new host record from decaying leaves of Ficus ampelas

Abstract A novel ascomycete genus, Longihyalospora, occurring on leaf litter of Ficus ampelas in Dahu Forest Area in Chiayi, Taiwan is described and illustrated. Longihyalospora is characterized by dark mycelium covering the upper leaf surface, elongate mycelial pellicle with ring of setae, pale brown to brown peridium, broadly obovoid, short pedicellate asci and hyaline, fusiform, elongated (tapering ends) and multi-septate ascospores with a thin mucilaginous sheath. Phylogenetic analyses of combined ITS, LSU and SSU sequence data revealed Longihyalospora as a distinct genus within the Chaetothyriaceae with high bootstrap support. Moreover, based on morphological similarities, Chaetothyrium vermisporum transferred to the new genus. In addition, Ceramothyrium longivolcaniforme is reported for the first time on Ficus ampelas. Newly added species are compared with other similar species and comprehensive descriptions and micrographs are provided.


Introduction
The family Chaetothyriaceae was established by Hansford (1946) with the generic type Chaetothyrium Speg., and the members of this family are characterized by a loose network of dark mycelium over the substrate, ascomata produced beneath a mycelial pellicle, and forming beneath an external hyphal mat with or without setae (Batista and Ciferri 1962;von Arx and Müller 1975;Hughes 1976;Pereira et al. 2009;Chomnunti et al. 2012;Tian et al. 2014;Zeng et al. 2016). Due to some morphological similarities (i.e. bitunicate asci), Eriksson (1982) referred this family to the order Dothideales in Dothideomycetes, but subsequently, taxonomic studies have established its placement in Eurotiomycetes with support of molecular data (Chomnunti et al. 2012Tian et al. 2014;Crous et al. 2015;Maharachchikumbura et al. 2018;Yang et al. 2018 (Wijayawardene et al. 2018).
During our survey of the taxonomy and diversity of leaf litter microfungi, two interesting fungal species were collected from Dahu forest, Chiayi in Taiwan. Morphological and multi-gene phylogenetic analyses were performed to establish their taxonomic placement.

Sample collection, morphological studies and isolation
Decaying leaf litter samples of Ficus ampelas Burm.f. were collected from Dahu forest area in Chiayi, Taiwan and brought to the laboratory in plastic bags. The samples were incubated in plastic boxes at 25-30 °C for 3 days and examined following the methods described by Tian et al. (2014). Morphological observations were made using an Axioskop 2 Plus compound microscope and images were taken with an Axioskop 2 Plus compound microscope equipped with a Canon Axiocam 506 Color digital camera. Permanent slides were prepared by mounting fungal material in lactoglycerol and sealed by applying nail-polish around the margins of cover slips. All measurements were made with ZEN2 (blue edition) and images used for figures were processed with Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, USA).
Isolates (for Ceramothyrium longivolcaniforme Zeng, T.C. Wen & K.D. Hyde) were obtained from single ascospores following the methods described in Chomnunti et al. (2014). Germinated ascospores were transferred to potato dextrose agar (PDA) and incubated at 25 °C in normal light. Subsequent sub culturing was done carefully to ensure no contaminants are used to generate DNA sequence data. Culture characteristics were observed after two weeks. Type specimens were deposited in the Mae Fah Luang University Herbarium (MFLU) and living cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC). Faces of Fungi and Index Fungorum numbers were provided as in Jayasiri et al. (2015) and Index Fungorum (2019).

DNA extraction and PCR amplification
Fresh mycelia were scraped (for Ceramothyrium longivolcaniforme) using a sterile scalpel from pure cultures growing on PDA medium at 25 °C and kept in a 1.5 ml microcentrifuge tube and used as starting material for DNA extraction. When fungi failed to germinate in a culture medium, DNA was extracted directly from ascomycete fruiting bodies (for Longihyalospora ampeli) by following a modified protocol of Zeng et al. (2018) protocol: 15-20 fruiting bodies (> 500 µm diam., 10 fruiting bodies) were removed from the host substrate using a sterilized needle and transferred to a drop of sterile water, placed in a sterile Eppendorf tube (1.5 mL) under aseptic conditions.
The genomic DNA was extracted using a DNA extraction kit (E.Z.N.A Fungal DNA Mini Kit, D3390-02, Omega Bio-Tek) following the manufacturer's protocol. The DNA product was kept at 4 °C for DNA amplification and maintained at -20 °C for long-term storage. DNA was amplified by Polymerase Chain Reaction (PCR) for three genes, the large subunit (28S, LSU), small subunit (18S, SSU) and internal transcribed spacers (ITS1-5.8S-ITS2). The LSU gene was amplified by using the primers LR0R and LR5 (Vilgalys and Hester 1990;Rehner and Samuels 1994); SSU gene was amplified using the primers NS1 and NS4 (White et al. 1990); nuclear ITS was amplified by using the primers ITS5 and ITS4 (White et al. 1990). The amplification reactions were performed in 25µl of total reaction that contained 9.5 µl of sterilized water, 12.5 µl of 2×Power Taq PCR MasterMix (Tri-I Biotech, Taipei, Taiwan), 1 µl of each forward and reverse primers and 1 µl of DNA template. PCR thermal cycle program for ITS, LSU and SSU were as detailed by Tian et al. (2016). The PCR products were analyzed by 1.5% agarose gels containing the Safeview DNA stain (GeneMark, Taipei, Taiwan) to confirm the expected molecular weight of a single amplification product. PCR products were purified and sequenced with primers mentioned above by Tri-I Biotech, Taipei, Taiwan. Nucleotide sequences were deposited in GenBank (Table 1).

Phylogenetic analysis
Phylogenetic analyses were performed based on a combined ITS, LSU and SSU DNA sequence data. Newly generated sequences were subjected to a standard BLAST search of GenBank to aid in phylogenetic taxon sampling. Other sequences used in the analyses (Table 1) were obtained from GenBank based on recently published data Maharachchikumbura et al. 2018;Yang et al. 2018). The multiple alignments were made with MAFFT v. 7 at the web server (http://mafft.cbrc.jp/alignment/server), using default settings (Katoh and Standley 2013). The alignment was refined manually with BioEdit v. 7.0.5.2 (Hall 1999) where necessary. The tree topologies obtained from a single gene sequence data were compared prior to the combined gene analysis for checking the incongruence in overall topology of the phylogenetic tree. Maximum likelihood trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008;Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using GTRGAMMA model with 1,000 bootstrap replicates. Maximum parsimony analysis (MP) was performed in PAUP v. 4.0b10 (Swofford 2002), with the heuristic search option and 1,000 random replicates. Maxtrees was set to 1,000 and branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics for parsimony ( (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo sampling (BMCMC). Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 100 th generation, thus 10,000 trees were obtained. The suitable burn-in phases were determined by inspecting likelihoods and parameters in Tracer version 1.6 (Rambaut et al. 2014). Based on the tracer analysis, the first 1,000 trees representing 10% were discarded as the burn-in phase in the analysis. The remaining trees were used to calculate posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic set to 0.01). Phylograms were visualized with FigTree v1.4.0 (Rambaut 2012) and annotated in Microsoft Power Point (2010). The final alignment and trees were deposited in TreeBASE, submission ID: 24826.

Description.
Epiphytic on the upper surface decaying leaves, appearing as small black dots. Covering the upper leaf surface with dark mycelium without penetrating host tissues. Mycelial pellicle elongate, subiculum-like, comprising hyphae that are mostly narrow, dense, dark brown. Mycelial setae broad, dark brown, scattered, discrete, arranged as a ring around the pellicle, unbranched, formed on dense, dark hyphae. Sexual morph: Ascomata superficial, solitary, dark brown to black, globose to subglobose, coriaceous, uni-locular, somewhat flattened when dry, covered by a mycelial pellicle. Peridium pale brown to brown, with compressed, hyaline, inner cells of textura angularis and dark brown outer cells of textura angularis, fusing and indistinguishable from the host tissues. Asci 8-spored, bitunicate, broadly obovoid, slightly stalked, apically rounded, with a well-developed ocular chamber. Ascospores overlapping, irregularly triseriate, hyaline, fusiform, elongated, multi-septate, slightly constricted at the septa, tapering to the ends, smooth-walled, surrounded by a thin mucilaginous sheath. Asexual morph: Not observed.
Type species. Longihyalospora ampeli Tennakoon, C.H Kuo & K. D Hyde.  Etymology. Species name based on the host Ficus ampelas, from which it was collected.

Discussion
Sooty molds are an interesting group of fungi in tropical and temperate regions in worldwide Hongsanan et al. 2015;Farr and Rossman 2019;Kwon et al. 2019). Their morphology has been well-studied but their phylogenetic relationships are poorly understood due to the difficulty of obtaining good-quality DNA samples (Chomnunti et al. 2011Zeng et al. 2016;Zeng et al. 2019 (Reynolds 1998;Winka et al. 1998;Hughes and Seifert 2012;Hyde et al. 2013;Chomnunti et al. 2014;Hongsanan et al. 2016).
Recent studies have revealed that Ceramothyrium is a species rich genus. For instance, in the last few years, numerous Ceramothyrium species have been described. Ceramothyrium longivolcaniforme, C. menglunense were introduced by Zeng et al. (2016) and Hyde et al. (2016) respectively. Yen et al. (2018) introduced three Ceramothyrium species, viz. C. aquaticum, C. phuquocense and C. exiguum. Currently, there are 41 Ceramothyrium epithets in Index Fungorum (2019).
Most previous Chaetothyriaceae studies have been based on brief descriptions with line drawings and without DNA sequence data (i.e. Actinocymbe, Beelia, Ceratocarpia, Chaetothyriomyces, Euceramia, Microcallis, Stanhughesia, Treubiomyces and Yatesula). Therefore, it is essential to focus on DNA sequence data to clarify the phylogenetic affinity of above genera in Chaetothyriaceae in future studies. Thus, it is necessary to collect more fungi similar to Chaetothyriaceae in different geographic regions and hosts, isolate them into cultures, describe their morphology, analyze their DNA sequences and investigate their phylogenetic relationships for a better identification and classification.