Two novel species of Neoaquastroma (Parabambusicolaceae, Pleosporales) with their phoma-like asexual morphs

Abstract The monotypic genus Neoaquastroma (Parabambusicolaceae, Pleosporales) was introduced for a microfungus isolated from a collection of dried stems of a dicotyledonous plant in Thailand. In this paper, we introduce two novel species, N. bauhiniae and N. krabiense, in this genus. Their asexual morphs comprise conidiomata with aseptate and hyaline conidia. Neoaquastroma bauhiniae has ascomata, asci and ascospores that are smaller than those of N. krabiense. Descriptions and illustrations of N. bauhiniae and N. krabiense are provided and the two species compared with the type species of the genus, N. guttulatum. Evidence for the introduction of the new taxa is also provided from phylogenetic analysis of a combined dataset of partial LSU, SSU, ITS and tef1 sequence data. The phylogenetic analysis revealed a distinct lineage for N. bauhiniae and N. krabiense within the family Parabambusicolaceae.


Introduction
Thailand is a highly biodiverse country in the tropics with hot and humid climate (MacKinnon et al. 1986, Marod andKutintara 2012). Although the fungal diversity in Thailand has been relatively well-studied (Rostrup 1902, Schumacher 1982, Hyde 1989, Jones 2000, Jones et al. 2006, Suetrong et al. 2009), the number of species being discovered is steadily growing due to increasing activities in studying microfungi in a large variety of terrestrial and aquatic ecosystems (Mapook et al. 2016, Dai et al. 2017, Doilom et al. 2017, Phukhamsakda et al. 2017. The family Parabambusicolaceae was introduced for a distinct phylogenetic lineage in the suborder Massarineae (Pleosporales) (Tanaka et al. 2015). Species of Parabambusicolaceae are characterised by pseudothecioid ascomata with or without stromatic tissues, papillate to apapillate ostioles, clavate to fusiform asci and hyaline or brown phragmospores (Liu et al. 2015, Tanaka et al. 2015. The asexual morphs are sporodochial or Monodictys-like (Tanaka et al. 2015. Currently, there are seven known genera in this family; Aquastroma, Monodictys-like spp., Multilocularia, Multiseptospora, Neoaquastroma, Parabambusicola (with P. bambusina as generic type) and Pseudomonodictys , 2018. The genus Neoaquastroma Wanas., E.B.G. Jones & K.D. Hyde, has been introduced from a dead twig of a herbaceous plant collected in Northern Thailand and been typifed with N. guttulatum Wanas., E.B.G. Jones & K.D. Hyde ). The genus is characterised by immersed, glabrous pseudothecia, short, papillate, fissitunicate, clavate asci and ellipsoidal to subfusiform, multi-septate hyaline phragmospores, surrounded by a mucilaginous sheath . Molecular phylogenetic analysis using ribosomal DNA (LSU, SSU and ITS) and translation elongation factor 1-alpha (tef1) sequence data support it as a distinct genus in Parabambusicolaceae.
The purpose of this study is to describe two new species of Neoaquastroma from collections of dicotyledonous plants in Thailand. Phylogenetic analysis of combined of LSU, SSU, ITS and tef1 sequence data are provided.

Sample collection, morphological study and isolation
Fresh specimens were collected from northern and southern part of Thailand during 2015-2017. The specimens were packed into brown paper bags for transport to the laboratory. Pure cultures were obtained from single ascospores on malt extract agar (MEA; 62 g/l) in distilled water following the method of Chomnunti et al. (2014). Cultures were incubated at 25 °C for up to 8 weeks. Induction of asexual reproduction has been adapted from  by placing agar squares with mycelia on water agar placed with sterile rice straw pieces. The plates were incubated at room tempera-ture (25 °C) with the standard light cycles, 12 hrs in the light followed by 12 hrs in the dark for about eight weeks until the fructifications were produced. Type specimens are deposited in Mae Fah Luang University (MFLU) herbarium and isotypes are deposited at the Kunming Institute of Botany, Academia Sinica Herbarium (HKAS), China. Ex-type living cultures are deposited at the Mae Fah Luang Culture Collection (MFLUCC) and duplicates at the International Collection of Microorganisms and Plants (ICMP), New Zealand. Faces of fungi numbers  and MycoBank number (http://www.MycoBank.org) are provided. Samples were examined under a Nikon ECLIPSE 80i compound microscope and photographed with a Canon 600D digital camera fitted to the microscope. Measurements were made using Tarosoft (R) Image Frame Work programme and photo-plates were made by using Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, United States).

DNA extraction, amplification and sequencing
DNA was extracted from mycelium by using Biospin Fungus Genomic DNA Extraction Kit (BioFlux) (Hangzhou, P. R. China) and gene extraction kit (Bio Basic Inc., Canada). PCR amplification was carried out using primers LROR/LR5 for the nuclear ribosomal large subunit 28S rDNA gene (LSU), NS1/NS4 for the nuclear ribosomal small subunit 18S rDNA gene (SSU) and ITS5/ITS4 for internal transcribed spacer rDNA region (ITS1, 5.8S rDNA and ITS2); partial fragments of the translation elongation factor 1-alpha (tef1) gene region was amplified using primers EF1-983F and EF1-2218R (Vilgalys and Hester 1990, White et al. 1990, Carbone and Kohn 1999. Primer sequences are available at the WASABI database at the AFTOL website (aftol. org). Amplification reactions for LSU, SSU and ITS followed . The PCR thermal cycle programme for EF1-983F and EF1-2218R (Carbone and Kohn 1999) for translation elongation factor 1-alpha (tef1) was set for denaturation at 96 °C for 2 min, followed by 40 cycles of denaturation at 96 °C for 45 sec, annealing at 54 °C for 30 sec and extension at 72 °C for 1.30 min, with a final extension step at 72 °C for 5 min. Genomic DNA and PCR amplification products were checked on 1% agarose gel. PCR products were purified as described in Wendt et al. (2017), sequences were generated by Shanghai Sangon Biological Engineering Technology & Services Co. (Shanghai, P.R. China) and sequencing services at Helmholtz Centre For Infection Research (HZI, Braunschweig, Germany).

Sequence alignment and phylogenetic analysis
SeqMan v. 7.0.0 (DNASTAR, Madison, WI) was used to assemble consensus sequences. Sequences of closely related strains were retrieved using BLAST searches against GenBank (http://www.ncbi.nlm.nih.gov). We also included the strains from Wanasinghe et al. (2017) and these are listed in Table 1. Sequences were aligned with MAFTT version 7.220 (Katoh et al. 2013) online sequence alignment tools (mafft.cbrc.jp/ alignment/server), with minimal adjustment of the ambiguous nucleotides by visual examination and manually corrected in AliView programme (Larsson 2014). Leading or trailing gaps exceeding from primer binding site were trimmed from the alignments prior to tree building and alignment gaps were treated as missing data. The concatenation of the multigene alignment was created in MEGA 6 (Tamura et al. 2013). Maximum likelihood analyses (ML), including 1,000 bootstrap replicates, was performed using RAxML (Stamatakis 2014) as implemented in raxmlGUI version v.1.3.1 (Silvestro and Michalak 2011). The search strategy was set to rapid bootstrapping. The analysis was carried out with the general time reversible (GTR) model for nucleotide substitution and a discrete gamma-distributed with four rate categories (O'Meara et al. 2006, Stamatakis et al. 2008. The bootstrap replicates were summarised on to the best scoring tree. The best fitting substitution model for each single gene partition and the concatenated data set was determined in MrModeltest 2.3 (Nylander 2004) for Bayesian inference posterior probabilities (PP). In our analysis, GTR+I+G model was used for each partition. The Bayesian inference posterior probabilities (PP) distribution (Zhaxybayeva and Gogarten 2002) was estimated by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.2 (Huelsenbeck and Ronquist 2001). 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 traces inspected in Tracer version 1.6 (Rambaut et al. 2014). Based on the tracer analysis, the first 1,000 trees representing 10% of burn-in phase of the analyses were discarded. While the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic set to 0.01).
Phylogenetic trees and data files were visualised in FigTree v. 1.4 (Rambaut and Drummond 2008). The phylogram with bootstrap values and/or posterior probabilities on the branches are presented in Fig. 1 by using graphical options available in Adobe Illustrator CS v. 6. All sequences generated in this study were submitted to GenBank. The finalised alignment and tree were deposited in TreeBASE, submission ID: 22419 (http://www.treebase.org/). Maximum likelihood bootstrap values equal to or greater than 70% with Bayesian Posterior Probabilities (PP) equal or greater than 0.90 are presented below or above each node (Fig. 1).
Culture characteristics. Colonies on MEA, reaching 50 mm diam. after 4 weeks at 25 °C. Culture dark olive-green with black centre, with dense mycelia, circular, flat, umbonate, rough surface, dull, fimbriate, radially furrowed, covered with white aerial Figure 2. Neoaquastroma bauhiniae (MFLU 17-0002, holotype) a Appearance of ascomata on host surface b Close up of ascoma c Section of ascoma d Ostiolar canal e Section of partial peridium layer f Pseudoparaphyses g-j Development state of asci j Asci produced in culture k-p Development state of ascospores; (n, o Senescent spores m, p ascospores in 5% of KOH reagent); q Ascospores stained with India ink, sheath surrounding the entire ascospore r Germinated ascospore s, t Culture character on MEA u Conidiomata forming on agar on rice straw media after 8 weeks v Immature conidiomata w Conidiomatal wall x, y Conidiogenous cells and developing conidia z Conidia j, m Asci and ascospore in culture (on rice straw). Scale bars: 500 μm (b); 100 μm (c, v); 50 μm (d-j); 20 μm (k-r, w); 5 μm (x-z). mycelium; mycelium strongly radiating into agar, yellow pigment diffusing in the agar; reverse black with radiating brown mycelium. Sexual and asexual morphs formed in culture. Morphology of sexual phase similar to those on substrate.
Culture characteristics. Colonies on MEA, reaching 50 mm diam. after four weeks at 25 °C. Culture grey, becoming dark-olive brown after four weeks, of dense mycelia, colonies circular, flat, umbonate, raised from the agar in the centre, surface rough, dull, covered with aerial mycelium, white mycelium radiating into the agar, pale orange pigment diffusing in the agar; reverse black, dense, circular, with irregular, fimbriate margin. Sexual and asexual morphs formed in culture. Morphology of sexual phase similar to those on the substrates.
Distribution. Krabi Province, Thailand Notes. Neoaquastroma krabiense was collected in the southern part of Thailand on dead twigs of Barringtonia acutangula. It is placed in Neoaquastroma based on its morphology of both sexual and asexual morph and close phylogenetic affinity to other species of Neoaquastroma. Neoaquastroma krabiense is distinct in that it has a flattened ascomata base and larger and more slender asci and ascospores than N. guttulatum and N. bauhiniae. The species formed an asexual morph in culture (Fig. 3, m) as pycnidial conidiomata with hyaline conidia (Fig. 3, x-ae).

Discussion
In the present study, we introduce two new species of Neoaquastroma, as N. bauhiniae and N. krabiense. The descriptions were made from fungi isolated from dicotyledonous plants in Thailand. The new species are introduced based on multi-locus phylogeny coupled with morphology that support their placement within Parabambusicolaceae.
Parabambusicolaceae is typified with Parabambusicola Tanaka & K. Hiray. The type of the genus was described originally as Massarina bambusina Teng (Teng, 1936) from bamboo. The family is characterised by ascomata surrounded by stromatic tissues and multiseptate, clavate to fusiform and hyaline ascospores Harada 2003, Tanaka et al. 2015). The asexual morph in Parabambusicolaceae can be coelomycetous or hyphomycetous. Sporodochia or pycnidia with hyaline conidia are formed in Parabambusicola and Neoaquastroma (Tanaka et al. 2015, this study), while hyphomyceteous structures are known from Pseudomonodictys and Monodictys spp. , Tanaka et al. 2015.
Neoaquastroma was introduced as a distinct genus in Parabambusicolaceae, with N. guttulatum as the type species . The genus resembles Parabambusicola and Multiseptospora, but form distinct lineages in phylogenetic studies (Liu et al. 2015, Tanaka et al. 2015. Parabambusicola and Neoaquastroma are similar in their morphology. The differentiation between Multiseptospora, Neoaquastroma and Parabambusicola is predominantly based on the morphology of ascospores, particularly with the size and number of septa. In the phylogenetic analyses of Wanasinghe et al. (2017), Parabambusicolaceae clustered into three clades, where Neoaquastroma guttulatum (MFLUCC 14-0917) clustered with Aquastroma magniostiolata (KT 2485), Multilocularia bambusae (MFLUCC 11-0180), Monodictys sp. (JO 10, KH 331) and Pseudomonodictys tectonae (MFLUCC 12-0552) with high statistical support. In this study, Neoaquastroma forms a separate clade, sister to Multiseptospora and Parabambusicola. This is probably due to limited taxon sampling.