﻿Taxonomy and phylogeny of the novel rhytidhysteron-like collections in the Greater Mekong Subregion

﻿Abstract During our survey into the diversity of woody litter fungi across the Greater Mekong Subregion, three rhytidhysteron-like taxa were collected from dead woody twigs in China and Thailand. These were further investigated based on morphological observations and multi-gene phylogenetic analyses of a combined DNA data matrix containing SSU, LSU, ITS, and tef1-α sequence data. A new species of Rhytidhysteron, R.xiaokongense sp. nov. is introduced with its asexual morph, and it is characterized by semi-immersed, subglobose to ampulliform conidiomata, dark brown, oblong to ellipsoidal, 1-septate, conidia, which are granular in appearance when mature. In addition to the new species, two new records from Thailand are reported viz. Rhytidhysterontectonae on woody litter of Betula sp. (Betulaceae) and Fabaceae sp. and Rhytidhysteronneorufulum on woody litter of Tectonagrandis (Lamiaceae). Morphological descriptions, illustrations, taxonomic notes and phylogenetic analyses are provided for all entries.


Introduction
Hysteriaceae was introduced by Chevallier (1826) with Hysterium as the type genus, which was characterized by hysterothecial or apothecioidal, carbonaceous ascomata with a pronounced, longitudinal slit running the length of the long axis, 8-spored, clavate to cylindric asci with an ocular chamber as well as obovoid, clavate, ellipsoid or fusoid, hyaline to light-or dark brown, one to multi-septate or muriform, smoothwalled ascospores with or without a sheath (Boehm et al. 2009b;Hongsanan et al. 2020;Hyde et al. 2020a). In recent outlines of Dothideomycetes Pem et al. 2020;, 14 genera have been accepted in Hysteriaceae.
Rhytidhysteron species have been documented from a wide range of hosts in various countries such as Australia, Bermuda, Bolivia, Brazil, China, Colombia, Cuba, France, Hawaii, India, New Zealand, Thailand, Ukraine, USA, and Venezuela (Kutorga and Hawksworth 1997;de Silva et al. 2020). Most Rhytidhysteron species are identified as saprobes on woody-based substrates in terrestrial habitats as well as from mangrove wood in marine habitats (Thambugala et al. 2016;Kumar et al. 2019;Hyde et al. 2020a, b;Wanasinghe et al. 2021). However, they have also been reported as endophytes or weak pathogens on woody plants and seldom as human pathogens (Soto and Lucking 2017;de Silva et al. 2020). From a biotechnological perspective, Rhytidhysteron species have great potential for their commercial applications and in industry. In particular, interest in secondary metabolites has rekindled in recent years, for instance with the discovery of palmarumycins. The latter is a potential inhibitor of thioredoxin-thioredoxin reductase cellular redox systems, with potential antimicrobial and antifungal properties (Murillo et al. 2009). Other Rhytidhysteron species discovered from the Southeast Asian region, such as R. bruguierae (MFLUCC 17-1515) and R. chromolaenae (MFLUCC 17-1516) also showed antimicrobial activity against Mucor plumbeus  and hence this demonstrates a potential biotechnological application.
The Greater Mekong Subregion (GMS) is regarded as a global biodiversity hotspot due to its widely varying environmental conditions. Accordingly, the GMS harbors a diverse array of numerous florae, fauna and microorganisms . Woody litter microfungi is an overlooked group of fungi in GMS and based on previous fungal estimates, there is undoubtedly a large number of new species yet to be described from this region. Our ongoing studies into the diversity of microfungi of the GMS are actively contributing towards filling in the knowledge gap in fungal taxonomy, phylogeny, host association and ecological distribution of Rhytidhysteron species in this region (Luo et al. 2018;Bao et al. 2019;Dong et al. 2020;Hyde et al. 2020b;Monkai et al. 2020Monkai et al. , 2021Wanasinghe et al. 2020Wanasinghe et al. , 2021Yasanthika et al. 2020). Our specific objectives of this study are as follows: 1) to describe a novel species of Rhytidhysteron with evidence from morphology and DNA sequence data; 2) to characterize (based on morphology and phylogeny) additional new records of Rhytidhysteron; 3) to investigate the phylogenetic relationships of our Rhytidhysteron samples based on DNA sequence analyses from rDNA and protein coding genes and update the taxonomy of Rhytidhysteron.

Samples collection and morphological analyses
Woody litter samples were collected from China (Kunming, Yunnan Province) during the wet season (August 2019) and during the dry season (December 2019) collections were done in Thailand (Chiang Rai and Tak Provinces). Samples were brought to the laboratory in plastic Ziploc bags. Fungal specimens were then examined using a stereomicroscope (Olympus SZ61, China). Pure cultures were obtained via single spore isolation on potato dextrose agar (PDA) following the methods described in Senanayake et al. (2020). Cultures were incubated at 25 °C for one week in the dark. Digital images of the fruiting structures were captured with a Canon (EOS 600D) digital camera fitted to a Nikon ECLIPSE Ni compound microscope. Squash mount preparations were prepared to determine micro-morphology and free hand sections of sporocarps made to observe the shapes of ascomata/conidiomata and peridium structures. Measurements of morphological structures were taken from the widest part of each structure. When possible, more than 30 measurements were made. Measurements were taken using the Tarosoft (R) Image Frame Work program. Figures were processed using Adobe Photoshop CS6. Field data are presented in 'Material examined'. Other details pertaining to good practices of morphological examinations were done following guidelines by Senanayake et al. (2020). New species are established based on recommendations proposed by Jeewon and Hyde (2016). Type specimens were deposited in the herbarium of the Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS). Ex-type living cultures were deposited at the Culture Collection of Mae Fah Luang University (MFLUCC) and Kunming Institute of Botany Culture Collection (KUMCC).

DNA extraction, amplification and sequencing
Genomic DNA was extracted from the mycelium grown on PDA at 25-30 °C for one week using a Biospin Fungus Genomic DNA Extraction Kit (BioFlux Hangzhou, P. R. China). Three partial rDNA genes and a protein coding gene were processed in our study, including the small ribosomal subunit RNA (SSU) using the primer pair NS1/ NS4 (White et al. 1990), internal transcribed spacer region (ITS) using the primer pair ITS5/ITS4 (White et al. 1990), large nuclear ribosomal subunit (LSU) using primer pair LR0R/LR5 (Vilgalys and Hester 1990), translation elongation factor 1-alpha gene (tef1-α) using primer pair 983F/2218R (Rehner and Buckley 2005). Amplification reactions were performed in a total volume of 25 μL of PCR mixtures containing 8.5 μL ddH 2 O, 12.5 μL 2X PCR MasterMix (TIANGEN Co., China), 2 μL DNA template and 1 μL of each primer. PCR thermal cycle program for SSU, LSU, ITS, and tef1-α were set as described in Wanasinghe et al. (2020). The PCR products were sent to the Qingke Company, Kunming City, Yunnan Province, China, for sequencing. Sequences were deposited in GenBank (Table 1).

Phylogenetic analyses
Representative species used in the phylogenetic analyses were selected based on previous publications (Thambugala et al. 2016;Mapook et al. 2020;. Sequences were downloaded from GenBank (http://www.ncbi.nlm.nih. gov/) and their accession numbers are listed in Table 1. The newly generated sequences in this study were assembled by BioEdit 7.0.9.0 (Hall 1999). Individual gene regions were separately aligned in MAFFT v.7 web server (http://mafft.cbrc.jp/alignment/ server/) (Katoh et al. 2019). The alignments of each gene were improved by manually deleting the ambiguous regions and gaps, and then combined using BioEdit 7.2.3. Final alignments containing SSU, LSU, ITS, and tef1-α were converted to NEXUS format (.nxs) using CLUSTAL X (2.0) and PAUP v. 4.0b10 (Thompson et al. 1997;Swofford 2002) and processed for Bayesian and maximum parsimony analysis. The FASTA format was changed into PHYLIP format via the Alignment Transformation Environment (ALTER) online program (http://www.sing-group.org/ALTER/) and used for maximum likelihood analysis (ML).
ML was carried out in CIPRES Science Gateway v.3.3 (http://www.phylo.org/por-tal2/; Miller et al. 2010) using RAxML-HPC2 on XSEDE (8.2.12) (Stamatakis 2014) with the GTRGAMMA substitution model and 1,000 bootstrap iterations. Maximum parsimony analysis (MP) was performed in PAUP v. 4.0b10 (Swofford 2002) with the heuristic search option and Tree-Bisection-Reconnection (TBR) of branch-swapping algorithm for 1,000 random replicates. Branches with a minimum branch length of zero were collapsed and gaps were treated as missing data (Hillis and Bull 1993). ML and MP bootstrap values (ML) ≥ 75% are given above each node of the phylogenetic tree (Fig. 1).
Bayesian analysis was executed in MrBayes v.3.2.2 (Ronquist et al. 2012). The model of evolution was estimated using MrModeltest v. 2.3 (Nylander et al. 2008) via PAUP v. 4.0b10 (Ronquist and Huelsenbeck 2003). The HKY+I for SSU; GTR+I+G for ITS, LSU and tef1-α were used in the final command. Markov chain Monte Carlo sampling (MCMC) in MrBayes v.3.2.2 (Ronquist et al. 2012) was used to determine posterior probabilities (PP) (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002). Bayesian analyses of six simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 200 generations (resulting in 10,001 total   Figure 1. RAxML tree based on a combined dataset of partial SSU, LSU, ITS, and tef1-α sequence analyses. Bootstrap support values for ML and MP equal to or higher than 75% and Bayesian PP equal to or greater than 0.95 are shown at the nodes. Hyphens (--) represent support values less than 75% / 0.95 BYPP. The ex-type strains are in bold and the new isolate in this study is in blue. The tree is rooted with Gloniopsis calami (MFLUCC 15-0739) and G. praelonga (CBS 112415). trees). The first 25% of sampled trees were discarded as part of the burn-in procedure, the remaining 7,501 trees were used to create the consensus tree, and the average standard deviation of split frequencies was set as 0.01. Branches with Bayesian posterior probabilities (BYPP) ≥ 0.95 are indicated above each node of the phylogenetic tree (Fig. 1). Phylogenetic trees were visualized in FigTree v1.4.0 (http://tree.bio.ed.ac. uk/software/figtree/; Rambaut 2012). The tree was edited using Microsoft PowerPoint before being, then saved in PDF format and finally converted to JPG format using Adobe Illustrator CS6 (Adobe Systems, USA). The finalized alignments and trees were deposited in TreeBASE, submission ID: TB2:S28620 (http://purl.org/phylo/treebase/ phylows/study/TB2:S28620).

Phylogenetic analysis
The phylogenetic analysis was conducted using 38 strains in Rhytidhysteron, and two outgroup taxa viz. Gloniopsis calami (MFLUCC 15-0739) and G. praelonga (CBS 112415) in Pleosporales (Table 1) Topologies of the phylogenetic trees under ML, MP and BI criteria recovered for each gene dataset were visually compared, and the overall tree topology was similar to those obtained from the combined dataset (Figure 1). Our analyzed molecular data generated phylogeny of Rhytidhysteron species was consistent with those of Wanasinghe et al. (2021). The maximum likelihood tree generated based on sequence analysis of the combined (ribosomal DNA: SSU, LSU and ITS; and protein coding gene: tef1-α) dataset recovered three major monophyletic clades within Rhytidhysteron (A-C, Figure  1) and two basal lineages viz. R. hysterinum (EB 0351) and R. opuntiae (GKM 1190). Clade A comprises Rhytidhysteron magnoliae, R. neorufulum, R. rufulum and R. tectonae with 96% ML, 98% MP and 1.00 BYPP support values.
Two Etymology. The species epithet reflects the location where the species was collected.
Key to asexual morphs of Rhytidhysteron species

Discussion
Rhytidhysteron is one of the first genera that trainee mycologists working on microfungi find in nature, as the hysterothecia are conspicuous (Hyde et al. 2020a). Species also easily germinate in culture and can easily be sequenced (Hyde et al. 2020a). Thus, it is even more remarkable that we found a new species in this study, indicating we are far from finding all species in this genus, and that more collections need be done on other continents (Hyde et al. 2020c). Most of Rhytidhysteron species are saprobes, which are essential for ecosystems functioning in terrestrial habitats and are commonly recognized as key biotic agents of wood decomposition, playing a vital role in carbon and nitrogen cycling in arid ecosystems, soil stability, plant biomass decomposition, and endophytic interactions with plants (Lustenhouwer et al. 2020;Dossa et al. 2021). Furthermore, Rhytidhysteron species have numerous antimicrobial and antifungal applications (Murillo et al. 2009;Mapook et al. 2020), and the discovery of new species provides new resources for future applied research in the field of biotechnology and industry.
Since the genus was established in 1881, a total of 24 species have been found to date, and the most commonly encountered species are Rhytidhysteron neorufulum and R. rufulum, so it might be difficult for mycologists to find new species within Rhytidhysteron. Rhytidhysteron is mainly identified via its sexual morph de Silva et al. 2020;Hyde et al. 2020a, b;Mapook et al. 2020;. The asexual morphs of Rhytidhysteron have been reported as aposphaeria-like or diplodia-like, including R. hysterinum and R. rufulum (Samuels and Müller 1979). Thambugala et al. (2016) confirmed the asexual-sexual morph connection for R. thailandicum by aposphaeria-like asexual morphs forming in culture on PDA. Herein, we found a diplodia-like asexual morph of Rhytidhysteron from woody litter of Prunus sp. in China. In comparison to the occurrence of the sexual morph of Rhytidhysteron, asexual morphs seldom form under natural conditions. The discovery of this new species provides an important reference for the study of the asexual morphs of Rhytidhysteron. Moreover, findings from this study further enrich GMS Rhytidhysteron species diversity.
In our phylogenetic analyses, the new species, Rhytidhysteron xiaokongense was basal to R. thailandicum (Fig. 1). Although species in Rhytidhysteron are morphologically similar, our new species is an asexual form of the species found in nature, so it is easy to distinguish from other speices excluding the asexual forms of R. hysterinum, R. rufulum and R. thailandicum. Rhytidhysteron xiaokongense shares similar morphological characters to R. hysterinum and R. rufulum in having black, unilocular, subglobose conidiomata and dark brown, 1-septate conidia but conidial features differ (Samuels and Müller 1979). Rhytidhysteron thailandicum can be differentiated from R. xiaokongense with respects to its globose to subglobose, hyaline conidia (Thambugala et al. 2016). To further support the establishment of the new taxon as proposed by Jeewon and Hyde (2016), we examined the nucleotide differences within the ITS regions (ITS1-5.8S-ITS2) gene region. Comparison of the 507 nucleotides across the ITS regions reveals 39 bp (7.7%) differences between Rhytidhysteron thailandicum and R. xiaokongense.
Rhytidhysteron species are widely distributed throughout the globe (de ; however, they appear to be particularly abundant in Asia, where they are well studied. There is an abundance of species and collections in the Greater Mekong Subregion (China and Thailand), such as R. brasiliense, R. camporesii, R. chromolaenae, R. erioi, R. hongheense, R. hysterinum, R. magnoliae, R. mangrovei, R. neorufulum, R. tectonae and R. thailandicum (Thambugala et al. 2016;Doilom et al. 2017;Soto-Medina et al. 2017;Kumar et al. 2019;Cobos-Villagran et al. 2020;de Silva et al. 2020;Hyde et al. 2020a;Mapook et al. 2020;Wanasinghe et al. 2021). We provide morphological and phylogenetic data for three species of Rhytidhysteron collected from the Greater Mekong Subregion: one new species, Rhytidhysteron xiaokongense, as a geographical record from China, two new host records of R. tectonae from woody litter of Betula sp and Fabaceae sp, and one new host record of R. neorufulum from woody litter of Tectona grandis. Based on our current work and that of past studies (de Hyde et al. 2020a, b;Mapook et al. 2020;Wanasinghe et al. 2021), it is clear that species within Rhytidhysteron are likely cosmopolitan and not host-specific, with evidence of the same species being found on a number of different hosts. Importantly, the morphology of a single species sometimes shows slight variations under different environmental conditions, geographical regions, hosts and different life modes (Senanayake et al. 2020). It is therefore crucial to collect more species of Rhytidhysteron across different geographic regions and hosts, obtain more cultures and sequence data, and describe their morphology to improve knowledge of taxonomy and phylogeny.