Lignicolous freshwater ascomycota from Thailand: Phylogenetic and morphological characterisation of two new freshwater fungi: Tingoldiago hydei sp. nov. and T. clavata sp. nov. from Eastern Thailand

Abstract Lignicolous freshwater fungi represent one of the largest groups of Ascomycota. This taxonomically highly diverse group plays an important role in nutrient and carbon cycling, biological diversity and ecosystem functioning. The diversity of lignicolous freshwater fungi along a north-south latitudinal gradient is currently being studied in Asia. In this paper, we introduce two novel freshwater taxa viz. Tingoldiago hydeisp. nov. and T. clavatasp. nov. which were collected from freshwater substrates in Eastern Thailand. Morphological comparison based on the size of ascomata, asci and ascospores, as well as multi-gene phylogenetic analyses based on LSU, SSU, ITS and TEF1-α DNA sequences, supports their placement in Tingoldiago (Lentitheciaceae). Descriptions and illustrations of these two new species are provided.


Introduction
Freshwater fungi are those which the whole or part of their life cycle is found in a freshwater habitat (Thomas 1996, Wong et al. 1998) and they are an evolutionary important group (Vijaykrishna et al. 2006). The members of freshwater fungi can be saprobes, parasites, endophytes and mutualistic taxa (Vijaykrishna et al. 2005, Zhang et al. 2008, Swe et al. 2009). There is a wide range of organisms that can be freshwater fungi hosts, such as wood, plants, alga, foams, fish etc. (Sparrow 1960, Ellis and Ellis 1985). However, a lot of studies on freshwater fungi have focused on lignicolous freshwater fungi (Tsui et al. 2000, Cai et al. 2002, Luo et al. 2004, which were defined as those fungi that grow on submerged woody debris in freshwater streams, ponds, lakes and tree hollows . They also grow on submerged wood in peat swamps and dams (Pinnoi et al. 2006, Pinruan et al. 2007, 2014, Hu et al. 2010. Lignicolous freshwater fungi are a diverse group comprising species from different phyla (Aphelidiomycota, Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Monoblepharomycota, Mortierellomycota and Rozellomycota) (Shearer et al. 2007, Kagami et al. 2012, Zhang et al. 2012, Wijayawardene et al. 2018. The dominant groups of lignicolous freshwater fungi are Dothideomycetes and Sordarialmycets .
We are studying the diversity of lignicolous freshwater fungi in Thailand, in order to establish the phylogenetic relationships of lignicolous freshwater fungi, understanding the natural classification of this group and contributing to the biogeographical diversity of fungi ). The study on freshwater fungi in Thailand was first investigated by Tubaki et al. (1983) and they reported 40 freshwater fungal species from foam. Subsequently, mycologists started to study lignicolous freshwater fungi in Thailand and several taxa have been reported (Sivichai et al. 1998, 1999, Marvanová et al. 2000, Hu et al. 2010, Luo et al. 2015. Lentitheciaceae was introduced by Zhang et al. (2012) to accommodate Massarina-like species in the order Pleosporales. Presently, 13 genera are accepted in this family . Species in this family are widely distributed in the world (China, Egypt, Hungary, Italy, Japan, Russia, Saudi, Thailand, UK, Uzbekistan) and are commonly saprobic on stems and twigs of herbaceous and woody plants in terrestrial or aquatic habitats (Wanasinghe et al. 2014, Knapp et al. 2015, Luo et al. 2016 In this paper, we introduce two new freshwater species of Tingoldiago (Lentitheciaceae), based on morpho-molecular studies. Detailed descriptions and illustrations of these two new species are provided.

Collection, Isolation and morphological studies
Submerged decaying wood samples were collected from That Phanom, Nakhon Phanom, Thailand and brought to the laboratory in plastic bags. The samples were incubated in plastic boxes lined with moistened tissue paper at room temperature for one week. Specimen observations and morphological studies were conducted, following the protocols provided by Luo et al. (2018).
Pure cultures were obtained by single spore isolation followed by Chomnunti et al. (2014). Germinating ascospores were transferred aseptically to potato dextrose agar (PDA) plates and grown at 16-25 °C in daylight. Colony colour and other characters were observed and measured after three weeks. The specimens were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand. Living cultures are deposited in the Culture Collection of Mae Fah Luang University (MFLUCC). Facesoffungi numbers and Index Fungorum numbers were obtained, following Jayasiri et al. (2015) and Index Fungorum (2019). New species have been established as recommended by Jeewon and Hyde (2016).

DNA extraction, PCR amplification and sequencing
Fungal mycelium was scraped from the surface of colonies grown on a PDA plate or MEA plate at 25 °C for 4 weeks, transferred into a 1.5 ml centrifuge tube and ground using liquid nitrogen. The EZ geneTM fungal gDNA kit (GD2416) was used to extract DNA from the ground mycelium according to the manufacturer's instructions. The gene regions of the large subunit of the nuclear ribosomal DNA (LSU), the internal transcribed spacers (ITS), the small subunit of the nuclear ribosomal DNA (SSU) and the translation elongation factor (TEF1-α) RNA were amplified using the primer pairs LR0R/LR7 (Vilgalys and Hester 1990), ITS5/ITS4, NS1/ NS4 (White et al. 1990) and 983F/2218R (Liu et al. 1999), respectively. The amplification reactions were performed in 25 μl of PCR mixtures containing 9.5 μl ddH 2 O, 12.5 μl 2× PCR MasterMix (Tsingke Co., China), 1 μl DNA sample and 1μl of each primer. The PCR thermal cycle programme for LSU, ITS, SSU and TEF1-α amplification were as follows: 94 °C for 3 minutes, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 56 °C for 50 seconds, elongation at 72 °C for 1 minute and a final extension at 72 °C for 10 minutes and finally kept at 4 °C. PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide. PCR products were sequenced using the same set of primers used in PCR in Beijing Tsingke Biological Engineering Technology and Services Co. Ltd. (Beijing, P.R. China).

Sequencing and sequence alignment
The sequence was assembled by using BioEdit and sequences with high similarity indices were determined from a BLAST search to find the closest matches with taxa in Lentitheciaceae and from recently published data ). All consensus sequences and the reference sequences were aligned using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh and Standley 2013), then checked visually and manually optimised using BioEdit v.7.0.9 (Hall 1999). Ambiguous regions were excluded from the analyses and gaps were treated as missing data. The phylogeny website tool "ALTER" (Glez-Peña et al. 2010) was used to convert the alignment fasta file to Phylip format for RAxML analysis and Clustalx BETA and PAUP 4.0 were used to convert the alignment fasta file to a Nexus file for Bayesian analysis. Phylogenetic analyses were obtained from Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian analysis.

Phylogenetic analyses
Maximum likelihood trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis 2006, Stamatakis et al. 2008 in the CIPRES Science Gateway platform  using GTR+ I + G model of evolution which was estimated by MrModeltest 2.2 (Nylander et al. 2008). Maximum likelihood bootstrap values (ML), equal to or greater than 75%, are given above each node (Figure 1).
MP analyses were performed using the heuristic search option with 1000 random taxa addition and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equal weight and gaps were treated as missing data. Maxtrees were unlimited, branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Clade stability was assessed using a bootstrap (BS) analysis with 1000 replicates, each with ten replicates of random stepwise addition of taxa (Hillis and Bull 1993).
The Bayesian analysis was performed with MrBayes v3.2 (Ronquist et al. 2012), with the best-fit model of sequence evolution estimated with MrModeltest 2.2 (Nylander et al. 2008) to evaluate posterior probabilities (PP) (Rannala andYang 1996, Zhaxybayeva andGogarten 2002) by Markov Chain Monte Carlo (MCMC) sampling. Six simultaneous Markov chains were run for 10,000,000 generations, trees were sampled every 1000 th generation and 1,0000 trees were obtained. 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 cal culate posterior probabilities in the majority rule consensus tree (critical value for the top ological convergence diagnostic set to 0.01).  The phylograms were visualised in FigTree 1.4.2 (Rambaut 2014) and made in Adobe Illustrator CS5 (Adobe Systems Inc., USA). All newly generated sequences of this study have been submitted in GenBank.

Phylogenetic analyses
The aligned sequence matrix comprises LSU, SSU, ITS and TEF1-α sequence data for 69 taxa, with Corynespora smithii and Corynespora cassiicola as out-group taxa. The dataset comprises 3334 characters after alignment including gaps (LSU: 1-897; SSU: 898-1920; ITS: 1921-2522; TEF1-α: 2523-3479). The topologies of RAxML, MP and Bayesian are similar and the bootstrap support values for Maximum Likelihood (ML), Maximum Parsimony (MP) higher than 75% and Bayesian posterior probabilities (PP) greater than 0.95 are given above the nodes. Maximum parsimony analyses indicated that 2,442 characters were constant, 232 variable characters parsimony uninformative and 805 characters are parsimony-informative. The RAxML analysis of the combined dataset yielded the best scoring tree ( Figure 1)  The novel species Tingoldiago hydei and T. clavata, introduced in this paper, are supported by multi-phylogenetic analyses. Four newly generated strains clustered together within Tingoldiago with strong statistical support (100 ML/95 MP/1.00 PP, Figure. 1). Three strains of T. clavata clustered together and sister to T. hydei with strong bootstrap support (99 ML/97 MP/1 PP, Figure 1). Etymology. Referring to Kevin D. Hyde for his contributions in fungal taxonomy.
Culture characteristics. Ascospores germinating on PDA within 24 hours. Colonies on MEA effuse, greyish-white to dark brown from above and below, reaching 3-4 cm diameter within 30 days at room temperature under natural light, composed of subhyaline to pale brown, septate, smooth hyphae.
Culture characteristics. Ascospores germinating on PDA within 24 hours. Colonies on MEA effuse, velvety, greyish-white to dark brown from above and below, reaching 2.5-3 cm diameter within 30 days at room temperature under natural light, composed of subhyaline to brown, septate, smooth hyphae.

Discussion
During the last decade, freshwater fungi in Thailand have been mainly reported from north, south and northeast of Thailand , Marvanová and Hywel-Jones 2000, Sivichai et al. 1998, Sri-indrasutdhi et al. 2010. No freshwater fungi from Eastern Thailand have been reported so far. In this study, two new freshwater species, viz. Tingoldiago hydei and T. clavata from Eastern Thailand, are introduced, based on morphology and phylogeny. Tingoldiago hydei and T. clavata satisfied the generic concept of the genus Tingoldiago (Hirayama et al. 2010). They comprise globose to conical, immersed to erumpent ascomata, cellular pseudoparaphyses, bitunicate, fissitunicate asci and clavate ascospores with a median primary septum and a large fusiform gelatinous sheath around the ascospore (Hirayama et al. 2010). Morphologically, T. hydei and T. clavata are quite similar as they have similar shape of asci and ascospores; however, we can distinguish them by the size of ascomata, asci and ascospores (Table 2). In addition, we also compared the morphological differences of these two species with the type species, T. graminicola. Ascopores of T. hydei and T. clavata are hyaline, uniseptate, with appendages at the septum and the upper cells are broader and shorter than the lower cells, while the ascopsores of T. graminicola are hyaline, uniseptate, but becoming brown and 3-septate with age, lacking appendages at the septum, upper cells and lower cells are similar lengths. Phylogenetic analyses showed that our two new isolates clustered together and are sister to the type species, Tingoldiago graminicola with strong bootstrap support (100 ML/92 MP/1.00 PP). This evidence strongly supports our two isolates to be the new species. Hyde et al. (2020) introduced a new genus, Pseudomurilentithecium in Lentitheciaceae. In their phylogenetic analysis, Pseudomurilentithecium clustered with Poaceascoma and was basal to Lentitheciaceae. However, in our phylogenetic analysis, Pseudomurilentithecium grouped with the members of Massarinaceae, rather than Lentitheciaceae. Therefore, further investigation is required to confirm the placement of the genus.
Tingoldiago is a well-resolved genus in this family with a stable clade within Lentitheciaceae. The genus can be distinguished from other genera in this family by having hyaline, uniseptate, upper cells are broad and basal cells are narrow ascospores with a large fusiform gelatinous sheath. The sheath is considered to be an adaptation by the genus that enables ascospores to attach to the substrates in moving water (Shearer 1993, Hyde and Goh 2003, Jones 2006, Devadatha et al. 2019. It is reported that the genus Tingoldiago is exclusively found in freshwater habitats (Hirayama et al. 2010) and our two new species were collected from lotic habitats of Mekong River.