Fissuroma (Aigialaceae: Pleosporales) appears to be hyperdiverse on Arecaceae: evidence from two new species from southern Thailand

Thailand and other tropical regions have high fungal diversity. Our investigation and examination of microfungi on palms (Arecaceae) revealed two new ascomycetous species of Fissuroma . Fissuroma arengae and F. wallichiae spp. nov. are introduced using morphological and phylogenetic evidence. The novel species have coriaceous ascomata, cylindrical-clavate asci and ascospores with a distinct and thin mucilaginous sheath. Fissuroma arengae is similar to F. wallichiae but can be distinguished by minor morphology, host substrate and gene base-pair differences. Phylogenetic analyses of combined LSU, ITS, SSU, tef1- α and rpb2 sequence data showed that these strains grouped within Fissuroma , further confirming this genus as monophyletic. The two new species are described and illustrated to support their taxonomic placement. Fissuroma appears to be a highly diverse genus often occurring on palms. It is likely that more research will result in numerous new taxa being discovered.


Introduction
To help understand the diversity of microfungi, we have been investigating the fungi on palms, Pandanaceae and grasses (Taylor & Hyde 2003;Hyde et al. 2007;Whitton et al. 2012;Thambugala et al. 2017;Goonasekara et al. 2018;Tibpromma et al. 2018). Our studies have constantly revealed new taxa, and would suggest that the estimated 2.2-3.8 million fungal species (Hawksworth & Lücking 2017) is certainly not an excessive number. In Thailand, the diversity has been shown to be extremely high with up to 96 % of species collected being new . The high diversity of novel fungi on palms have been revealed in several studies (Hyde 1997;Fröhlich & Hyde 1999;Yanna et al. 2001;Pinnoi et al. 2006;Pinruan et al. 2007;Konta et al. 2017;Zhang et al. 2019). In one study, taxa on two adjacent palms of different genera had less than 6 % of overlapping species indicating the remarkable diversity likely to be harbored by different genera (Jones et al. 2014).
In this study, Fissuroma arengae and F. wallichiae are introduced as new species, from dead petioles of Arenga and Wallichia (Arecaceae), respectively. Their phylogenetic position was determined based on maximum likelihood, maximum parsimony and Bayesian inference of a combined LSU, ITS, SSU, tef1-α and rpf2 sequence dataset. Descriptions, illustrations and molecular data are provided to compare the new species with related taxa.

Collection and isolation
Decayed rachides/petioles were collected from Thailand (Krabi and Phang-Nga Provinces) in 2014 on Arenga pinnata and Wallichia sp. Information on the environment, geographic location and host was recorded. The fungal taxa were identified based on morphological characteristics and phylogenetic analyses. Isolations and specimen examinations were conducted following the methods provided by Konta et al. (2016). Samples were taken to the laboratory in ziplock bags and morphological characteristics were observed using a Motic SMZ 168 series stereo-microscope. Free-hand sections were made using a razor blade and placed on a droplet of water on a glass slide. Morphological characters were observed and photo-micrographed using a Nikon ECLIPSE80i compound microscope with a fitted Canon 600D digital camera. Measurements were determined using an Image Framework program. Photo-plates were made by Adobe Photoshop CS6. Pure cultures were obtained using single ascospores isolation method (Chomnunti et al. 2014). Ascospore mass was transferred to a drop of sterile water on a flame-sterilized slide. The ascospore suspension was spread on a petri-dish containing malt extract agar (MEA) and incubated at 25-28 °C overnight. Germinating ascospores were transferred to fresh MEA dishes. Two palm samples were collected and three isolates were obtained from each sample. Holotype specimens and ex-type cultures were deposited in the herbarium of Mae Fah Luang University (MFLU) and Mae Fah Luang Culture Collection (MFLUCC) at Mae Fah Luang University, Chiang Rai, Thailand. Facesoffungi and Index Fungorum numbers were registered as outlined in Jayasiri et al. (2015) and Index Fungorum (2020).
The total volume of PCR mixtures for amplification were 25 μl containing 8.5 μl ddH 2 O, 12.5 μl 2× Easy Taq PCR SuperMix (mixture of Easy Taq TM DNA Polymerase, dNTPs and optimized buffer (Beijing Trans Gen Biotech Co., Beijing, P.R. China), 2 μl of DNA template, 1 μl of each forward and reverse primers (10 pM). The quality of PCR products was checked on 1 % agarose gel electrophoresis stained with 4S green nucleic acid (Life Science Products & Services, Shanghai, P.R. China). Purification and sequencing of PCR products were carried out by Sangon Biotech Co., Shanghai, P.R. China. The resulting fragments were sequenced in both forward and reverse directions. The DNA sequences generated were analysed. Consensus sequences were computed using SeqMan software. The new sequences generated in this study were deposited in GenBank (Tab. 2).

Phylogenetic analyses
The sequences generated in this study were subjected to BLAST search in GenBank to identify closely related sequences. Sequence data retrieved from GenBank and recent publications were used as references (Tennakoon et al. 2018;Wanasinghe et al. 2018;Zhang et al. 2020). Sequences of the LSU, ITS, SSU, tef1-α and rpb2 were analysed individually and in combination. A total of 44 taxa were used for the phylogenetic analyses. Astrosphaeriella fusispora

Sirinapa Konta, Kevin David Hyde, Prapassorn Damrongkool Eungwanichayapant, Mingkwan Doilom, Danushka Sandaruwan Tennakoon, Chanokned Senwanna and Saranyaphat Boonmee
(MFLUCC 10-0555) and A. neofusispora  were selected as the outgroup taxa. Absent sequence data (i.e. ITS, tef1-α, rpb2 sequence data) in the alignments were treated as missing data. Sequence alignments were carried out with MAFFT v.6.864b (Katoh & Standley 2013) and were manually improved where necessary. The single gene datasets were combined using Mega7 (Kumar et al. 2016). Data were converted from fasta to nexus and PHYLIP format with Alignment Transformation Environment online, https://sing.ei.uvigo.es/ALTER/ (Glez-Peña et al. 2010). The tree topologies obtained from single gene sequence data were compared prior to the combined gene analysis for checking the incongruence in the overall topology of the phylogenetic tree. Maximum likelihood (ML) analysis was performed using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008;Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) with GTRGAMMA model Table 2. GenBank accession numbers of sequences used in phylogenetic analysis.

Species
Strains GenBank accession numbers Notes: Newly generated sequences are in bold; T denotes the type species of the genus; E denotes the ex-type culture.
Fissuroma (Aigialaceae: Pleosporales) appears to be hyperdiverse on Arecaceae: evidence from two new species from southern Thailand and set as 1,000 bootstrap replicates. Bayesian analysis was performed at CIPRES using Bayesian analysis on XSEDE (v.3.2.6) as part of the "MrBayes on XSEDE" tool (Huelsenbeck & Ronquist 2001;Miller et al. 2010). GTR+I+G model was selected by using MrModelTest 2.2 (Nylander 2004) under the Akaike information criterion (AIC) as the best-fit models of the combined dataset for maximum likelihood and Bayesian analysis (Nylander 2004). Bayesian posterior probabilities (BYPP) were determined by Markov chain Monte Carlo sampling (MCMC) in MrBayes on XSEDE v.3.2.6. Six simultaneous Markov chains were run for 815,000 generations and trees were sampled every 1,000 th generation. MCMC heated chain was set with a "temperature" value of 0.20. All sampled topologies beneath the asymptote (25 %) were discarded as part of a burn-in procedure; the remaining trees (816) were used for calculating posterior probabilities in the majority rule consensus tree. Maximum parsimony (MP) analysis was carried out with PAUP v 4.0b10 (Swofford 2002). Statistical supports for branches of the most parsimonious tree were estimated using maximum parsimony bootstrap analysis with 1,000 bootstrap replicates (Felsenstein 1985). All characters were unordered and of equal weight, and gaps were treated as missing data. Descriptive tree statistics

Fissuroma arengae
Culture characteristics: Colonies on MEA reaching 20 mm diam. after two weeks at 25-30 °C, colonies medium dense, circular, convex, surface slightly rough with edge entire, effuse, velvety to hairy, margin well-defined, colony from above, white to cream at the margin, brown at the center, not producing pigments in culture.