﻿Morphology and multigene phylogeny reveal three new species of Distoseptispora (Distoseptisporales, Distoseptisporaceae) on palms (Arecaceae) from peatswamp areas in southern Thailand

﻿Abstract Peatswamp forest is a unique habitat that supports high biodiversity, particularly fungal diversity. The current study collected submerged and dead plant parts from Eleiodoxaconferta, Eugeissonatristis and Licualapaludosa from a peatswamp forest in Narathiwat Province, Thailand. Morphological features coupled with multigene phylogenetic analyses of ITS, LSU, rpb2 and tef1-α sequence data identified our isolates as new Distoseptispora species (viz. D.arecacearumsp. nov., D.eleiodoxaesp. nov. and D.narathiwatensissp. nov.). Morphological descriptions, illustrations and notes are provided.


Introduction
Most peatswamp forests can be found in tropical rainforests where peat is submerged for most of the year and characterised by low nutrient contents and high acidity due to lack of fully decomposed plant materials (Page et al. 1999(Page et al. , 2011;;Jackson et al. 2009;Lampela et al. 2016;Ratnayake 2020).Peatswamp forests are unique ecosystems due to their high species diversity and significant role in maintaining a stable global climate.They function as carbon sinks, storing twice as much carbon as all global forest biomass (Hakim et al. 2017;Fujimoto et al. 2019;Shuhada et al. 2020).Beyond carbon storage, peatlands offer valuable benefits.They play vital roles in the water cycle, storing and filtering water and mitigating floods by slowing peak flows.Home to diverse plants and animals, these wetlands support millions of people.Additionally, they hold archaeological relics and provide insights into past environmental conditions MycoKeys 102: 55-81 (2024), DOI: 10.3897/mycokeys.102.112815 Omid Karimi et al.: Novel peatswamp fungi through their peat layers, aiding predictions about the future climate (Parish et al. 2008;Posa et al. 2011;Minayeva and Sirin 2012;UNEP 2022).Asian peatlands are amongst the most diverse and geographically extensive in the world, with over 160 million hectares and the majority of tropical peatlands are found in Southeast Asia (e.g.Brunei, Indonesia, Malaysia, Papua New Guinea and Thailand) (UNEP 2022).
Distoseptispora K.D. Hyde, McKenzie & Maharachch belongs to Distoseptisporaceae, Distoseptisporales, Sordariomycetes, Ascomycota and comprises sporidesmium-like taxa (Wijayawardene et al. 2022).Su et al. (2016) proposed Distoseptisporaceae to accommodate sporidesmium-like taxa with Distoseptispora as the type genus and D. fluminicola McKenzie, Hong Y. Su, Z.L. Luo & K.D. Hyde as the type species.Subsequently, Luo et al. (2019) introduced Distoseptisporales to accommodate Distoseptisporaceae, based on multigene phylogenetic analyses of LSU, SSU, rpb2 and tef1-α sequence data.Distoseptispora is characterised by short, septate, olivaceous to brown conidiophores.The conidiogenous cells are monoblastic and determinate, bearing acrogenous conidia that are brown, euseptate, distoseptate or muriform and cut off by cross walls at the basal cell with a basal scar (Yang et al. 2018).The genus exhibits morphology similar to Sporidesmium, but can be distinguished by having shorter conidiophores and darker conidia with pale round apexes (Su et al. 2016).To date, Distoseptispora comprises 65 species listed in the MycoBank database (https://www.mycobank.org/;Accessed in August 2023), with molecular data available for all reported species in the GenBank.The estimated divergence time for Distoseptisporaceae is approximately 44.21 million years ago (MYA), after the Tertiary-Cretaceous extinction event (Hyde et al. 2020), which could have created conducive conditions for Distoseptispora to thrive as a saprobe on various hosts (Phukhamsakda et al. 2022).
Peatswamp forests are unique, endangered ecosystems and their fungal biodiversity is little known.Therefore, in the current study, we aimed to study fungal species on different palm materials from peatswamp forests in Thailand, based on morphology and phylogeny.This study introduces three new species, Distoseptispora arecacearum, D. eleiodoxae and D. narathiwatensis, associated with Eleiodoxa conferta, Eugeissona tristis and Licuala paludosa from a peatswamp forest in Narathiwat Province, Thailand, based on morphological characteristics coupled with multigene phylogenetic analyses (ITS, LSU, rpb2 and tef1-α).

Sample collection, morphological study and isolation
Decaying leaves of Eleiodoxa conferta, Eugeissona tristis and Licuala paludosa were collected from a peatswamp forest in Narathiwat Province, Thailand, in April 2022.Wet (submerged) and dry (aerial part) palm specimens were placed in plastic bags and brought to the laboratory.The submerged materials were kept moist and examined periodically for fungal fruiting structures and the dry materials were examined immediately or incubated in moisture chambers.Small pieces of the collected specimens were examined under a Leica EZ4 stereomicroscope and isolated into axenic culture using a single spore technique (Choi et al. 1999) in the Difco potato dextrose agar (PDA) media supplemented with Streptomycin 0.5 g/l.Germinating spores were transferred to new PDA and incubated at 25 ± 1 °C in dark conditions for two weeks.The micro-morphological characters were examined and photographed using a digital camera (Canon 600D, Japan) fitted to a compound microscope (Nikon ECLIPSE Ni, Japan) and the measurements were obtained using the Tarosoft (R) Image Frame Work programme version 0.9.7 (Tarosoft, Thailand).The ex-type living cultures were deposited at the Mae Fah Luang University Culture Collection (MFLUCC) and the herbarium specimens at the Mae Fah Luang University Herbarium (MFLU).The Facesoffungi (FoF) and Index Fungorum numbers were obtained, as explained in Jayasiri et al. (2015) and Index Fungorum (http://www.indexfungorum.org),respectively.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fresh fungal mycelia using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, P.R. China), according to the manufacturer's standard protocol.Polymerase chain reactions (PCR) were conducted to amplify the internal transcribed spacer region rDNA (ITS), 28S large subunit rDNA (LSU), RNA polymerase II second largest subunit (rpb2) and translation elongation factor 1-alpha (tef1-α) using primers and conditions listed in Table 1.The PCR products were visualised on 1% agarose gels, stained with 4S Green Stain and sequenced at SolGent Co., Ltd (South Korea).

Sequence alignment and Phylogenetic analyses
The obtained sequences of ITS, LSU, rpb2 and tef1-α were assembled using SeqMan software version 7.1.0(DNASTAR Inc., WI) and subjected to BLASTn search against the GenBank nucleotide database at National Center for Biotechnology Information (NCBI) to identify closely-related sequences.Sequence data of related taxa were obtained from previous publications (Su et al. 2016;Yang et al. 2018Yang et al. , 2021;;Crous et al. 2019;Hyde et al. 2019;Luo et al. 2019;Monkai et al. 2020;Phukhamsakda et al. 2020;Sun et al. 2020;Ma et al. 2022;Zhai et al. 2022;Zhang et al. 2022;Afshari et al. 2023) and downloaded from the GenBank database (Table 2).The sequences were aligned using MAFFT v.7 online web server (http://mafft.cbrc.jp/alignment/server/index.html,Katoh et al. 2019) under default settings and the alignments were trimmed in NGPhylogeny online web server (https://ngphylogeny.fr/workflows/wkmake/3a4ab-1bef8e7ff3c, Lemoine et al. 2019).The sequence datasets were combined using SequenceMatrix software version 1.9 (Vaidya et al. 2011).The Maximum Likelihood (ML) phylogenetic analysis was run in the CIPRES Science Gateway platform (Miller et al. 2010), using RAxMLHPC2 on the XSEDE (v.8.2.10) tool (Stamatakis 2014) under the GTRCAT substitution model and 1,000 non-parametric bootstrap replicates.For Bayesian Inference (BI) analysis, the optimal substitution model of each region was determined using jModelTest2 on the CIPRES Science Gateway under the Akaike Information Criterion (AIC) (Darriba et al. 2012).Bayesian analysis was performed using MrBayes v. 3.2.6 on XSEDE at the CIPRES Science Gateway with four simultaneous Markov Chain runs for 1,000,000 generations.The resulting trees were visualised in FigTree v. 1.4.0 (Rambaut 2012) and edited in Microsoft PowerPoint 2019 (Forethought, Inc., The United States).

Phylogenetic analyses
The   Culture characteristics.Colonies grown on PDA, reaching 50 mm in diameter after 15 days at 25 °C, under dark conditions, circular, fimbriate edge, flat, dull surface, radiating outwards, felted, medium dense, without pigment diffusion and sporulation, brown on the top, reverse dark brown to black.
Culture characteristics.Colonies grown on PDA, reaching 30 mm in diameter after 15 days at 25 °C, under dark conditions, circular, entire to radially with lobate edge, well-defined margin, low convex, dull surface, felted, dense, mycelium superficial to immersed, without pigment diffusion and sporulation, greyish-brown on the top with dark brown margin, reverse brown with dark brown centre and margin.
Culture characteristics.Colonies grown on PDA, reaching 50 mm in diameter after 15 days at 25 °C, under dark conditions, circular, entire margin, well-defined margin, low convex, dull surface, felted, dense, mycelium mostly superficial, without pigment diffusion and sporulation, medium brown to reddish-brown with dark brown edge on the top, reverse-side dark brown to black.

Discussion
Peatswamp forests are unique habitats found in only a few regions worldwide (Jackson et al. 2009).The destruction caused by humans threatens them; hence more extensive studies on fungal identification are needed before the extinction of fungal species.Pinnoi et al. (2006Pinnoi et al. ( , 2009) ) recorded sporidesmium-like taxa on the palm species Eleiodoxa conferta and Calamus sp. in Sirindhorn peatswamp forest, Narathiwat, Thailand, based on morphological data.In this study, three new Distoseptispora species (D. arecacearum, D. eleiodoxae and D. narathiwatensis) from peatswamp forest in Thailand are introduced, based on multilocus phylogenetic analysis (ITS, LSU, rpb2 and, tef1-α) (Fig. 1) and morphology (Figs 2-4).
Except for Distoseptispora hyalina J. Yang & K.D. Hyde and D. licualae Konta & K.D. Hyde, most Distoseptispora species have been recorded as having an asexual morph and their characters, such as size, shape, colour and the number of septa in conidiophores and conidia, are crucial for distinguishing species.Morphologically, Distoseptispora is similar to Ellisembia Subram and Sporidesmium Link; therefore, it is problematic to recognise Distoseptispora species by only morphological signatures (Su et al. 2016;Hyde et al. 2019;Luo et al. 2019;Yang et al. 2021).Different studies have explored the taxonomy of Distoseptispora using various combinations of gene regions, such as combined ITS, LSU (Tibpromma et al. 2018), combined LSU, ITS, rpb2 (Monkai et al. 2020) or combined LSU, ITS, tef1-α and rpb2 (Zhang et al. 2022).In our study, we constructed the phylogenetic tree using concatenated ITS, LSU, rpb2 and tef1-α.In this study, Distoseptispora clematidis (MFLUCC 17-2145) and D. nabanheensis Jing W. Liu, X.G.Zhang & Jian Ma (HJAUP C2003) formed a sister clade, consistent with previous research (Liu et al. 2023).However, D. clematidis (KUN-HKAS:112708) appeared separated from these two taxa, presenting an unresolved relationship.The phylogenetic relationship amongst these three taxa is not comparable with the previous studies due to the lack of all these taxa together in their phylogenetic trees (Afshari et al. 2023;Liu et al. 2023).The unresolved clade's origin may stem from the lack of rpb2 sequence data for D. clematidis (KUN HKAS:112708) in contrast to the other two taxa where this gene region is available.This suggests that different taxon sampling and protein-coding sequences can influence the topology of the tree.However, further studies are essential to validate this hypothesis.
Morphologically, some taxa that share similarities exhibit distinct phylogenies.For instance, D. arecacearum shares a morphological resemblance with D. dehongensis, although they are phylogenetically distinct.Similarly, D. eleiodoxae shows morphological similarities to D. tropica, but resides in a separate clade in the phylogenetic tree.Distoseptispora narathiwatensis forms a sister clade with D. saprophytica despite the differences highlighted by the pairwise comparison of ITS, LSU and other genetic markers.These encompass 22/580 bp (3.8%) and 16/870 bp (1.8%) differences for ITS and LSU, respectively, excluding gaps.Moreover, distinctions in the morphology of conidiophores and the absence of acrogenous conidia further contribute to the differentiation between D. narathiwatensis and D. saprophytica.Our study confirmed the necessity of associating molecular data with morphological characters to distinguish them, as previously demonstrated in other studies (Su et al. 2016;Hyde et al. 2019;Luo et al. 2019;Yang et al. 2021;Ma et al. 2022).
To date, the majority of Distoseptispora species have been reported from China (42 species) and Thailand (23 species), primarily on dead plant materials in freshwater (44 species) and terrestrial (21 species) habitats.In most cases, the hosts are unknown.Although in 19 cases, their hosts have been identified, two of which have been reported from palm, including D. palmarum from Cocos nucifera and D. licualae from dead leaves of Licuala glabra in terrestrial habitats (Hyde et al. 2016(Hyde et al. , 2021;;Su et al. 2016;Xia et al. 2017;Tibpromma et al. 2018;Yang et al. 2018;Luo et al. 2019;Phookamsak et al. 2019;Monkai et al. 2020;Phukhamsakda et al. 2020Phukhamsakda et al. , 2022;;Song et al. 2020;Sun et al. 2020;Dong et al. 2021;Li et al. 2021;Shen et al. 2021;Jayawardena et al. 2022;Ma et al. 2022;Zhai et al. 2022;Zhang et al. 2022;Konta et al. 2023;Liu et al. 2023).Distoseptispora species have been recorded as saprophytes and their reported limited geographic distribution (China and Thailand) is most likely due to increased attention by mycologists in these areas on saprophytic fungi in aquatic and terrestrial habitats.This study shows that there is much to be done in this regard.Ongoing and future investigations will reveal the diversity and functions of these microorganisms in this ecosystem.

Figure 1 .
Figure 1.Maximum Likelihood tree generated from combined ITS, LSU, rpb2 and tef1-α sequence data.Bootstrap support values ≥ 65% and Bayesian posterior probabilities ≥ 0.95 are demonstrated at the nodes.The new taxa are indicated in red bold.Ex-type strains are in black bold.

Table 1 .
Primers and PCR protocols.