Mendogiadiffusa sp. nov. and an updated key to the species of Mendogia (Myriangiaceae, Dothideomycetes)

Abstract Background Mendogia belongs to Dothideomycetes and its members are epiphytic on living bamboo culms or palms and distributed in tropical regions. Currently, the genus comprises seven species. Another collection resembling Mendogia was collected from the leaves of Fagales sp. in Thailand. Morphological characteristics and multilocus phylogenetic analyses, using ITS, LSU and SSU sequences, showed that the fungus is new to science, described herein as Mendogiadiffusa. Mendogiadiffusa is characterised by apothecial ascostromata, a carbonised epithecium, dark brown setae on the ascostromatal surface, hyaline paraphysoids, ovoid to clavate asci and oblong to elliptical, muriform ascospores. The fungus has a dark pigmented surface and is occasionally facultatively associated with patches of green algae, but not actually lichenised. Instead, the fungus penetrates the upper leaf surface, forming dark pigmented isodiametric cells below the epidermis. New information Re-examination of specimens of M.chiangraiensis, M.macrostroma and M.yunnanensis revealed the absence of algal associations. The status of Mendogiaphilippinensis (= M.calami) and M.bambusina (= Uleopeltisbambusina) was established, based on morphological comparisons and previous studies. Comprehensive morphological descriptions with phylogenetic analyses support M.diffusa as a novel species in Myriangiaceae. An updated key to the known species of the genus is also provided.

This study introduces a new species of Mendogia that appeared unusual due to its growth on leaves and its occasional, facultative association with patches of green algae. We conducted a detailed investigation to resolve the identity of our newly-collected material, including morphological and chemical assessments. The phylogenetic position of the taxon was investigated, based on Maximum Likelihood and Bayesian analyses of combined ITS, LSU and SSU sequences. We further re-examined herbarium collections of Mendogia chiangraiensis, M. macrostroma and M. yunnanensis to test potential associations with algae. Additionally, morphological comparisons between closely-related taxa have led to reclassify several species in Mendogia (M. philippinensis (= M. calami) and M. bambusina (= Uleopeltis bambusina)). We, therefore, provided an updated key to the genus.

Morphological analysis
The fungal material was collected in Phayao, Thailand. Herbarium specimens of Mendogia chiangraiensis, M. macrostroma and M. yunnanensis were loaned from Mae Fah Luang University Herbarium (MFLU), Chiang Rai, Thailand. Fungal structures on the substrate were observed with a stereomicroscope and micro-morphological features were examined and photographed using a Nikon Eclipse E600 fluorescence microscope with a Canon 750D digital camera. Hand sections of the ascomata were mounted in water, 5% potassium hydroxide (KOH), 5% Lugol's solution and Trypan blue. All microscopic measurements were measured in water and images were made with Tarosoft Image Frame Work (0.9.0.7) and processed with Adobe Photoshop CS6 Extended 10.0 software (Adobe Systems, San Jose, CA, USA). The newly-proposed synonymies were established, based on revision of available data from previous studies. The holotype specimen of M. diffusa was deposited in the Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand.

DNA extraction, PCR amplification and sequencing
The E.Z.N.A. Forensic DAT (D3591 -01, Omega Bio-Tek, Guangzhou, China) kit was used to extract DNA, following the manufacturer's instructions. DNA samples that were intended for use as a template for PCR were stored at 4°C for use in regular work; longterm storage was at -20°C. The small and large subunits (SSU, LSU) of the nuclear ribosomal RNA gene, as well as the internal transcribed spacer (ITS) region were amplified with primer pairs NS1/NS4 (White et al. 1990), LR0R/LR5 (Vilgalys andHester 1990, Hopple 1994) and ITS5/ITS4 (White et al. 1990), respectively. PCR amplification was performed using a final volume of 25 µl, comprised of 2.0 µl of DNA template, 1 µl of each forward and reverse primer, 12.5 µl of Taq PCR Super Mix and 8.5 µl of sterilised water. Cycling conditions were as follows: initial denaturation at 94°C for 3 min; followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 50 s, elongation at 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were examined on 1% agarose electrophoresis gels and stained with ethidium bromide. Purification and DNA sequencing were performed at Shanghai Sangon Biological Engineering Technology and Services Co. (Shanghai, P.R. China). Forward and reverse sequence reads were assembled and manually edited in Bioedit. Generated sequences were submitted to NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Alignments and phylogenetic trees were submitted to TreeBASE with Submission ID: 28050.

Phylogenetic analyses and species recognition
The newly-generated sequences were BLAST-searched against the NCBI GenBank standard nr/nt database (https://blast.ncbi.nlm.nih.gov/BLAST.cgi). Sequences of closelyrelated taxa for Myriangiales were downloaded from GenBank. We failed to generate sequences for the translation elongation factor 1-alpha (TEF1) using the primer pair EF1-983F/EF1-2218R with the PCR conditions recommended in . As a result, our phylogenetic analyses were carried out using ITS, LSU and SU sequences (Table 1) for 5,000,000 generations and the trees were sampled every 100 generation. The first 10% of trees that represented the burn-in phase were discarded and only the remaining 90% of trees were used for calculating posterior probabilities (PP) for the majority rule consensus tree.

Etymology
Referring to the morphology of the fungus with ascostromata that are diffuse and spread extensively on the leaves.

Habitats and Distribution:
On dead leaves of Fagales sp. Thus far, only known from Thailand, Phayao Province, Phu Sang District.

Notes
Mendogia diffusa is the first reported species in the genus from dead dicotyledonous leaves. Other species were mostly reported from bamboo culms, with the exception of M. manaosensis that is reported from palm leaves (Vitória 2012) and  . In those species, ascostromata do not penetrate the leaf surface and they also differ from M. diffusa in the sharply delimited ascostromata; and M. philippinensis further differs in the smaller ascospores. The new taxon shares morphological characteristics with Mendogia bambusina: carbonaceous peridium, paraphysoid-like filaments, similar asci and ascopores. However, M. diffusa differs in the absence of ascostromata, presence of setae , the type of habitat (Fagales leaves vs. bamboo or palms culms) and its distribution (Thailand vs. Indonesia) (Hyde et al. 2013 ( Sydow and Sydow 1917). This synonymy needs further testing with molecular data as previous studies on palms have shown that the taxa on different palm species differ (Konta et al. 2016, Konta et al. 2017) as they may have derived from endophytes.

Notes
Uleopeltis was introduced to accommodate U. manaosensis and later the second species U. bambusina added to this genus (Hennings 1904. Uleopeltis manaosensis was synonymised under Mendogia, while U. bambusina remained in Uleopeltis which was collected from bamboo culms in he Philippines (Hennings 1904, von Arx and Müller 1975. The species lacks molecular data and shares similar morphological characteristics with the type species of Mendogia (von Arx and Müller 1975).  gave spores of the type material of Mendogia bambusina as 13.5-25 × 5-8 μm, but mature ascospores in the photographs are 15-21 × 7-9 μm. Raciborski (1900) gave the ascospores as 17-19 × 8 μm for M. bambusina. This supports the assessment of von Arx and Müller (1975) that M. bambusina and Uleopeltis bambusina are conspecific. The synonymisation is formalised here. The report of M. bambusina from Brazil on palm leaves (Vitória 2012) has been documented with morphological and anatomical photographs and agrees well with the material from the Paleotropics. The African Pleiostomella halleriae ( Doidge 1921) will also key out close to M. bambusina and may represent another synonym. It is the only other species described in Pleiostomella, a synonym of Mendogia, but has apparently never been dispositioned. Unfortunately, no type was indicated and a total of six collections on two host species (leaves of Halleria elliptica and H. lucida) were listed. The ascus and ascospore dimensions (50-70 × 20-33 μm; 22-24 × 9-10 μm) partly fit M. bambusina, but Doidge described two types of asci, one ovate and ca. 50 × 30 μm and the other clavate and ca. 65-70 × 20-25 μm. The latter fits M. bambusina, whereas the former does not conform to any of the species recognised here. Revision of all paratypes is necessary to assess the taxonomic status of this material (Sydow and Sydow 1917).

Identification keys
Key to the species of Mendogia

Analysis Phylogenetic analyses
The genera of Myriangiaceae were well recovered, as studied in . The final alignment comprised 50 strains including the new strain and 2469 nucleotide positions. The topologies of the single gene markers tree and the tree topology obtained from the combined five-locus (SSU, LSU, ITS1, 5.8S, ITS2) dataset were congruent. Our phylogenetic analyses supported the placement of Mendogia diffusa within Mendogia. The average standard deviation of split frequencies at the end of total MCMC generations was calculated as 0.0024 in the Bayesian analysis.

Discussion
Mendogia has previously been recorded from monocotyledons, but, in the present case, was collected on a dicotyledon, indicating many more species are likely to be discovered. Other species currently recognised in Mendogia (see key above) differ from the new species in the sharply delimited ascostroma , which renders the diffusely delimited ascomata (Fig. 1) as the most diagnostic feature of M. diffusa. In terms of ascospore size, M. bambusina, M. macrostroma and M. yunnanensis are closely related to M. diffusa. Apart from the sharply delimited ascostromata and the usually bambusicolous habit of all three species, M. bambusina has narrower asci and M. macrostroma differs in the much larger ascostromata (Raciborski 1900. The internal anatomy of the ascomata of M. diffusa is also distinctive, with easily discernible paraphysoids (Fig. 2). Mendogia manaosensis and M. philippinensis (= M. calami) also form paraphysoid-like interascal hyphae, whereas in M. bambusina, these are less distinctive and, in M. chiangraiensis, M. macrostroma and M. yunnanensis, the interascal hyphae form a textura angularis (Raciborski 1900, Hennings 1904, Sydow and Sydow 1917, von Arx and Müller 1975). This variation in morphology and internal anatomy of such closely-related species is remarkable, especially given that, in our phylogenetic analysis, M. diffusa and M. chiangraiensis formed a sister clade to M. macrostroma and M. yunnanensis (Fig. 3), although without support. The new taxon shows more than 2% nucleotide differences in the ITS region compared to other Mendogia species. This, along with the discussed morphological differences, supports recognition as a new species ( . The latter also forms ascomata scattered in dense groups instead of sharply delimited ascostromata, but differs in the broad, globose asci and the much thicker covering layer of the ascomata. Mendogia diffusa was found on dead leaves and the fungal structures penetrate the upper epidermis of the leaf surface, turning the epidermal cells into a dark pigmented layer (Fig. 2). Such dark pigmented cells are absent where the ascomata are not observed.
Some ascostromata observed were found to loosely associate with algal colonies (Fig. 1). The algae are probably trentepohlioid, 3-5 µm thick, rounded to slightly elongate and greenish. However, since these are absent from most of the ascostromata and no closer anatomical associations or penetration structures were detected, we assume that this association is opportunistic, the algae is taking advantage of the microrelief formed by the ascostromata to colonise the otherwise smooth leaf surface. While the ascostromata were detected on dead leaves, it is unclear whether the fungus is also present on living leaves and how common is the observed opportunistic association with algae. It is possible that M. diffusa indirectly benefits from the presence of the algae as an additional carbon source, through leaching or by decomposing dead algal cells. Similar cases of loose associations have been reported from saxicolous biocoenoses where rock-inhabiting fungi are often growing together with algae or cyanobacteria . Muggia et al. (2016) found alpine rock lichens to be associated with members of Myriangiales. Phylogeny of Myrangiales reconstructed from a multilocus dataset with SSU, LSU, ITS1, 5.8S and ITS2. The topology is the result of ML inference performed with IQ-TREE. ML bootstrap support values ≥ 65% and Bayesian posterior probabilities ≥ 0.95 are presented above each branch. Ex-type strains are shown in black bold; the new species is highlighted in blue bold font.