Appendage-Bearing Sordariomycetes from Dipterocarpus alatus Leaf Litter in Thailand

Leaf litter is an essential functional aspect of forest ecosystems, acting as a source of organic matter, a protective layer in forest soils, and a nurturing habitat for micro- and macro-organisms. Through their successional occurrence, litter-inhabiting microfungi play a key role in litter decomposition and nutrient recycling. Despite their importance in terrestrial ecosystems and their abundance and diversity, information on the taxonomy, diversity, and host preference of these decomposer taxa is scarce. This study aims to clarify the taxonomy and phylogeny of four saprobic fungal taxa inhabiting Dipterocarpus alatus leaf litter. Leaf litter samples were collected from Doi Inthanon National Park in Chiang Mai, northern Thailand. Fungal isolates were characterized based on morphology and molecular phylogeny of the nuclear ribosomal DNA (ITS, LSU) and protein-coding genes (tub2, tef1-α, rpb2). One novel saprobic species, Ciliochorella dipterocarpi, and two new host records, Pestalotiopsis dracontomelon and Robillarda australiana, are introduced. The newly described taxa are compared with similar species, and comprehensive descriptions, micrographs, and phylogenetic trees are provided.


Phylogenetic Analyses
The obtained sequence chromatograms were checked with Chromas 2.6.6 (Technelysium Pty Ltd., South Brisbane, Australia), and the low-quality regions were trimmed. The sequences were subjected to BLASTn searches against the NCBI nucleotide non-redundant databases, with the option "sequences from type material" selected. Separate data sets were used to perform phylogenetic analyses for Pestalotiopsis, Ciliochorella, and Robillarda. Reference sequences were obtained from recent literature [50,51] and downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank/ (accessed on 18 November 2022)) (Tables 2-4). Taxon sampling for Pestalotiopsis was performed by selecting the type strains and a duplicate strain if available from all the recorded species, excluding unverified sequences, to obtain better topology for the phylogenetic tree. The single locus of each data set was aligned by MUSCLE [52] implemented in MEGA (v. 7.0.26), applying the default settings. The aligned sequences were automatically trimmed using trimAl 1.2 [53] under the default -gappyout option for all loci and concatenated using BioEdit v. 7.0.5.2 [54]. Phylogenetic analyses were based on maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) posterior probability (PP). ML, MP, and BI were performed for Pestalotiopsis, while Ciliochorella, and Robillarda were resolved using model-based methods (ML, BI) following the previous literature [23,29,50]. ML and BI were performed in the CIPRES Science Gate-way platform [55]. ML was executed by RAxML-HPC2 on XSEDE v. 8.2.8 [56,57], with 1000 bootstrap replicates under the GTRGAMMA nucleotide substitution model.        [59,60], under the nucleotide evolutionary models calculated by jModelTest v.2.1.6 (Table 5). Then, 2 parallel independent runs with 6 MCMCs were run for 15,000,000 (Pestalotiopsis) and 3,000,000 generations (Ciliochorella and Robillarda), with trees sampled every 1000th generation. Twenty-five percent of the trees representing the burn-in phase were discarded, and the remaining trees were used to calculate the PP in the majority-rule consensus tree.
Maximum-parsimony trees were generated by PAUP v4.0b10 [59], using the heuristic search option with 1000 random sequence additions, with Maxtrees set to 1000. Branches of zero length were collapsed, and all maximum parsimony trees were saved. Descriptive tree statistics for parsimony-tree length (TL), consistency index (CI), retention index (RI), relative consistency index (RC), and homoplasy index (HI) were calculated following the Kishino-Hasegawa test (KHT) criteria [61].
The phylogenetic trees were visualized and exported using FigTree v.1.4.0 [62], and the phylograms were edited and annotated in Microsoft PowerPoint (2013) and Adobe Photoshop CS6.

Phylogenetic Analyses
The combined alignment (ITS, tef1-α, and tub2) of Pestalotiopsis comprised 146 taxa, including 5 outgroup taxa. The best-scoring ML tree ( Figure 1  5.503206, and GT = 1.000000; gamma distribution shape parameter α = 0.120262; and tree length = 0.748074. In the Bayesian analysis, the average standard deviation of split frequencies at the end of 3,000,000 MCMC generations was calculated with a stop value of 0.009935. The ML and BI trees were similar in topology. The resulting phylogenetic tree from the concatenated alignment resolved C. dipterocarpi (MFLUCC 22-0132) isolate into a well-supported distinct lineage with strong statistical support (94% ML, 0.95 PP) ( Figure  2).  1.040419, CT = 5.807309, and GT = 1.000000; gamma distribution shape parameter α = 0.244419; and tree length = 3.825788. In the Bayesian analysis, the average standard deviation of split frequencies at the end of 3,000,000 MCMC generations was calculated with a stop value of 0.009951. The ML and BI trees were similar in topology. The resulting phylogenetic tree from the concatenated alignment grouped the isolate MFLUCC 22-0121 with the ex-type strain R. australiana (CBS 143882) and formed a well-supported clade with strong statistical support (84% ML, 0.99 PP) (Figure 3).    Known host: Pathogenic on leaves of Dracontomelon mangifera (Anacardiaceae) [63], leaf spots of Podocarpus sp. (Podocarpaceae) [27], saprobic on D. alatus leaf litter (this study).

Discussion
Appendages are informative morphological characteristics in delineating fungal species [28]. Additionally, they support the adherence of spores to their substrate and dispersal, characterizing important ecological functions [28]. In this study, we used a polyphasic approach for the species boundaries of novel collections of leaf-litter-inhabiting fungi [42,[66][67][68]. With this approach, out of three appendage-bearing Sordariomycetes collected from Doi Inthanon National Park, northern Thailand, one was placed as Ciliochorella dipterocarpi sp. nov, and two (P. dracontomelon and R. australiana) were placed as new records on D. alatus.
Ciliochorella is characterized by its distinct morphological characteristics [64,65] and molecular phylogeny based on the ITS, LSU, and tub2 sequence data [50]. Nevertheless, many species and isolates lack the sequence data for one or more loci, which may cause biases in the tree topology and phylogenetic placement [69][70][71][72] [e.g., 71,72,73,74]. Still, our newly introduced taxon C. dipterocarpi is clustered sister to C. mangiferae (MFLUCC 12-0310) (Figure 2), which has all the loci with available sequence data (Table 3). Like C. dipterocarpi, Ciliochorella species have been mainly reported as saprobes on various plant litter. In the present study, using a taxonomic approach (Figure 2), we expand the diversity of Ciliochorella with the introduction of C. dipterocarpi, isolated from the dead leaves of D. alatus.
Regarding the new litter host association of P. dracontomelon (MFLUCC 22-0119) and D. alatus, Pestalotiopsis species have been reported as endophytes producing important bioactive secondary metabolites, pathogens on several economically important crops, and saprobes on different plant litter [36,[73][74][75][76]. Since the last detailed morphology and molecular phylogeny update [23,24], several species have been introduced, and around 100 Pestalotiopsis species are currently accepted [29,30,[77][78][79]. However, the taxonomic placement is doubtful, mainly due to the lack of strong morphological characteristics [36] and the poor resolution of the employed molecular markers (ITS, tub2, and tef1-α), which presumably identify species rather than populations within the genus [80]. In our analysis, as taxon sampling is fundamental for the taxonomic placement and delimitation [81,82], to obtain better phylogenetic resolution (Figure 1), we excluded distantly related species and those with inconsistent sequence data and included only representative type strains (Table 2). Thus, a taxonomic revision of this genus is urgently needed, especially using the polyphasic approach with additional genomic regions [24,42,80,83,84].
Unlike Pestalotiopsis, Robillarda is characterized by its distinct morphological features [85,86], coupled whenever possible with the multilocus phylogeny of the ITS, rpb2, tub2, and tef1-α barcodes [29]. Nevertheless, out of its 41 species listed in the Index Fungorum database [40], only 8 have molecular data available [29,67,85,87,88]. Robillarda species are found as saprobic on decaying leaves in terrestrial and aquatic habitats, dust particles, and soil [85]. Here, we contribute with the expansion of the host and geographical record of R. australiana, which was found as a saprobe on decaying leaves of D. alatus ( Figure 6) at Doi Inthanon National Park, Thailand. This species was only reported in Australia on unidentified plant litter [29].
Many fungal species have been discovered in Thailand, using polyphasic approaches [89]. As evident from the present study, much work remains to be done in this regard. This includes examining unstudied areas, such as protected national parks and hosts, to address whether fungi are host-specific and at which level [13]. Tennakoon et al. [90] contributed significantly to this matter. This is important in predicting the number of species [19] and in assessing the diversity in a given area or ecosystem [91], which have the same significance [92]. Currently, only approximately 10% of the 2.2-3.8 million species estimated by Hawksworth and Lucking [93] have been described [13,16,94], highlighting the potential for unraveling novel fungal taxa in largely untapped ecosystems. In this regard, this study contributes to the above topics, and the number of teleomorph species of Ascomycota estimated by Senanayake et al. [95] was between 1.37 and 2.56 million. Finally, the findings of the present study provide additional insights into fungal diversity in pristine tropical environments, as predicted by Hyde et al. [89]. However, given the different lifestyles of the species, elucidating their evolution, host and lifestyle shifting, and environmental adaptations is needed for a better understanding of their roles [13].

Conclusions
Ecological preference and evolutionary relationships can support the emergence of novel species in contrasting life modes [4,13]. This hypothesis expands the scope of saprobic fungal habitats and their host preferences [4,13]. This scenario is supported by identifying saprobic lifestyles of different genera from the present study. Moreover, we also identified fungal species associated with novel hosts and geographical locations (P. dracontomelon and R. australiana), along with the novel species C. dipterocarpi. Data Availability Statement: All sequences generated in this study were submitted to GenBank (https://www.ncbi.nlm.nih.gov (accessed on 4 April 2023)).