Three novel species and a new record of Daldinia (Hypoxylaceae) from Thailand

In an investigation of stromatic Xylariales in Thailand, several specimens of Daldinia were discovered. Three novel species (D. flavogranulata, D. phadaengensis, and D. chiangdaoensis) were recognized from a molecular phylogeny based on concatenated ITS, LSU, RPB2, and TUB2 sequence data, combined with morphological characters and secondary metabolite profiles based on high performance liquid chromatography coupled to diode array detection and mass spectrometry (HPLC-MS). The major components detected were cytochalasins (in D. flavogranulata and D. chiangdaoensis) and daldinin type azaphilones (in D. phadaengensis). In addition, D. brachysperma, which had hitherto only been reported from America, was found for the first time in Asia. Its phylogenetic affinities were studied, confirming previous suspicions from morphological comparisons that the species is closely related to D. eschscholtzii and D. bambusicola, both common in Thailand. Daldinia flavogranulata, one of the new taxa, was found to be closely related to the same taxa. The other two novel species, D. phadaengensis and D. chiangdaoensis, share characters with D. korfii and D. kretzschmarioides, respectively.


Introduction
The genus Daldinia was erected by Cesati and De Notaris (1863) in honor of the Swiss monk, Agostino Daldini.
Today, it is one of largest genera in the Hypoxylaceae (Ascomycota, Xylariales). Traditionally, Daldinia species were recognized by the internal concentric zones below the perithecial layer in their stroma and by the presence of KOH-

Section Editor: Hans-Josef Schroers
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11557-020-01621-4) contains supplementary material, which is available to authorized users. extractable pigments on and below their stromatal surface (Ju et al. 1997). The latest world monograph of the genus compiled morphological, ultra-structural, and chemotaxonomic data for more than a thousand specimens and cultures, and included a preliminary phylogeny based on ITS sequence data . Daldinia species are extremely prolific secondary metabolite producers, and the metabolites of their stromata and cultures can be used as taxonomic markers, while others exert selective and prominent activities in biological systems (Helaly et al. 2018).
While the majority of Daldinia species are associated with dicots, some of them like D. bambusicola are associated with bamboo (monocot) in Thailand (Ju et al. 1997). Hsieh et al. (2005) reported that D. bambusicola is closely related to D. caldariorum based on TUB2 and ACTA1 sequences. In India, Daldinia graminis and D. sacchari are found on sugarcane (Dargan and Thind 1985). Narmani et al. (2018) revealed that D. sacchari is phylogenetically related to D. eschscholtzii, and even isolated two new cytochalasins, which are the characteristic stromatal metabolites of the D. eschscholtzii complex. Furthermore, several species of Daldinia produce stromata on fire-damaged woods, including D. vernicosa, D. loculata, D. cal dariorum, D. gelatinoides, and D. loculatoides .
S t r o m a t a o f s o m e s p e c i e s o f D a l d i n i a ( i . e . , D. placentiformis, D. korfii, and D. kretzschmarioides) appear morphologically similar to Hypoxylon as they are lacking internal concentric zones. However, the affinities of these species to Daldinia were confirmed by ITS and TUB2 sequences, and by the fact that stromata of D. korfii contain cytochalasins and concentricol B (Sir et al. 2016b). These compounds can be used as molecular markers for D. concentrica, D. eschscholtzii, and some members of the D. eschscholtzii group (Quang et al. 2002;Stadler et al. 2014). Morphologically, D. kretzschmarioides is very closely linked to Hypoxylon, while multiple loci analyses and metabolomics profiles indicate a closer relationship with Daldinia . The phylogenetic affinities of Daldinia and allied genera were also recently confirmed using a multi-locus phylogeny in two independent studies by Wendt et al. (2018) and Daranagama et al. (2018). They used many type and authentic strains of the stromatic Xylariales, which led to a rearrangement of the genera, and provided a phylogenetic backbone tree of these pyrenomycetes for the first time. Recently, some strains representing important lineages of the Hypoxylaceae have been selected for a phylogenomic study relying on high quality genomes and the first papers on comparative functional genomics ) and on the occurrence of ITS polymorphisms ) have been published. Nevertheless, numerous species of the Hypoxylaceae remain to be recollected and cultured, and new taxa are steadily being discovered in particular from tropical countries.
In the course of taxonomic studies on stromatic Xylariales in Thailand, involving extensive field work, we have recently encountered three new species and a new record for the country. The present study is dedicated to their description and illustration, and we also provide evidence on their phylogenetic position and their chemotaxonomy.

Survey and sample collection
Stromatic Xylariales were collected in selected forests, i.e., community forests, national parks, and reforestation areas (Pha Daeng Zinc Mine area) in Thailand. Macrophotographs were taken using a Canon 60D digital camera (Canon Inc. Tokyo, Japan). Fungal cultures were obtained using a multiple spore isolation method (Sir et al. 2016a). Germinated ascospores were transferred to new agar plates. Axenic cultures and vouchers were deposited in Thailand Bioresource Research Center (TBRC, BCC) and BIOTEC Bangkok Herbarium (BBH), respectively. Scanning electron microscopy (SEM) was carried out using a conventional procedure as described by Kuhnert et al. (2017).

Morphological characterizations and HPLC profiling
Morphological characters, such as stromatal size and shapes, perithecia, asci, and ascospores were examined in accordance with Stadler et al. (2014) using an Olympus ZX31 (Olympus Corporation, Tokyo, Japan) and a dissecting microscope Olympus SZ61 (Olympus). Fungal cultures were obtained on several media, i.e., oatmeal agar (Difco OA), potato dextrose agar (Difco PDA), and yeast malt glucose agar (1% malt extract, 0.4% glucose, and 0.4% yeast extract; agar 1%; YMGA). The morphological studies were carried out on 9 cm Petri dishes. Conidiogenous cells and conidiophore branching patterns of the anamorph were investigated as proposed by Ju and Rogers (1996). Furthermore, stromatal color, KOH-extractable pigments, and cultures are recorded using the color chart of Rayner (1970). For chemotaxonomic studies, stromatal secondary metabolites were extracted with acetone and analyzed using high performance liquid chromatography coupled with diode array and high resolution electrospray mass spectrometric detection (HPLC/DAD-HR-ESIMS) in a similar manner as described by Yuyama et al. (2018) and Kretz et al. (2019). Instrumental settings and conditions were the same as described in Kuhnert et al. (2017).

Phylogenetic analyses
All sequences were aligned in MUSCLE (Edgar 2004) and refined by direct examination. Multiple sequence alignments were analyzed with closely matched sequences and other reference taxa obtained from GenBank as shown in Table 1. Sequences were analyzed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian algorithm (MB). The MP analysis was performed in PAUP*4.0b10 (Swofford 2002), and all characters were equally weighted and gaps were treated as missing data. The most parsimonious trees were obtained from heuristic searches: 100 replicates of stepwise random addition and tree-bisection-reconnection (TBR) as branch swapping algorithm. Maximum parsimony bootstrap supports (MPBS) were estimated by 1000 replicates (10 replicates of stepwise random sequence addition). Tree length, consistency index (CI), retention index (RI), relative consistency index (RC), and homoplasy index (HI) were estimated. The ML tree and bootstrap analyses (MLBS) were conducted through the CIPRES Science Gateway V. 3.3 (Miller et al. 2010) using RAxML 8.2.4 (Stamatakis 2014 with the BFGS method to optimize GTR rate parameters. Bayesian posterior probabilities (BPP) of the branches were computed using MrBayes 3.0B4 (Huelsenbeck and Ronquist 2001) with the best-fit model (GTR + I + G) selected by AIC in Mr Modeltest 2.2 (Nylander 2004), tested with hierarchical likelihood ratios (hLRTs). Three million generations were run in four Markov chains and sampled every 100 generations with a burn-in value set at 3000 sampled trees. Sequence alignments were deposited at TreeBase (submission ID 25485; www.treebase.org). Sequences of Graphostoma platystomum CBS 270.87 and Xylaria hypoxylon CBS12260 obtained from GenBank were used as outgroups. The RAxML based phylogenetic tree is shown in Fig. 6.

Molecular phylogeny
Sixty-one new sequences were generated and included into a combined ITS, LSU, RPB2, and TUB2 dataset to clarify the phylogenetic relationships of newly collected Thai specimens of Daldinia and distinguish them from other species and genera in the Hypoxylaceae (Table 1). PCR amplifications yielded approximately 840 bp, 1213 bp, 829 bp, and 1583 bp of ITS, LSU, RPB2, and TUB2 sequences. The dataset of the multi-locus DNA sequences included 67 taxa from the Hypoxylaceae based on Annulohypoxylon (5), Daldinia (35), Hypoxylon (12), H y p o m o n t a g n e l l a ( 4 ), J ac k r o g e r s e l l a ( 3 ) , a n d Pyrenopolyporus (6). The combined dataset consisted of 4465 characters, of which 2600 were constant, 1434 parsimony informative, and 431 uninformative. In MP analysis, a CI of 0.357, a RI of 0.638, and a HI of 0.643 yielded three equally most parsimony trees. The phylogenetic tree included 5 major clades: a Daldinia clade subdivided into five branches (D I-D V) and one clade e a c h r e p r e s e n t i n g P y r e n o p o l y p o r u s ( P y ) , H y p o m o n t a g ne l l a ( H y ) , A n n u l o h yp o x y l o n , and Jackrogersella (AJ) and Hypoxylon (H) (Fig. 6). Clade D I, accommodating D. flavogranulata (BCC 89363, BCC 89365, and BCC 89376) and D. caldariorum appeared monophyletic and was supported with high bootstrap values. These data are in agreement with the morphological characters. Clade D II also group with a strong bootstrap support and comprised D. bambusicola and D brachysperma. Clade D III included the D. eschscholtzii complex, where D. placentiformis and D. theissenii were grouping as a strongly supported monophyletic clade. The strongly supported clade D IV grouped with clades D II and D III as sister clades and consisted of D. korfii, D. kretzschmarioides, D. phadaengensis (BCC 89349, BCC 89350), and D. chiangdaoensis (BCC 88220, BCC 88221). In agreement with the morphological evidence, the four taxa were separated in a highly supported clade (100% BSMP, 100% BSML, and 1.00 BPP). Clade D V also formed a fully statistically supported, monophyletic clade (100% BSMP, 100% BSML, 1.00 BPP) appearing as sister clade to clades D II and D III.   Hypoxylon, which is in agreement with data of Wendt et al. (2018). In summary, the phylogeny allowed for a clear separation of the taxa that are described below as new, even though the topology of the phylogenetic tree was not in accordance with the grouping of Daldinia as proposed by Stadler et al. (2014) based on ITS sequences, chemotaxonomy, and morphology. This may be due to different modes of taxon selection and the variability of ITS.
Anamorph on YMGA. Conidiophores with the same branching pattern and dimensions of conidiogeneous cells and conidia as on OA.

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Etymology. "phadaengensis" referring to the locality where the type specimen was collected.
Anamorph on YMGA similar to that on OA.
Cultures on PDA not producing anamorphic structures in 3 months.
Notes. Daldinia phadaengensis is morphologically similar to D. chiangdaoensis, D. korfii, and D. kretzschmariodes in lacking internal concentric zones below the perithecial layer. The new species is distinguishable from the aforementioned species by morphology as well as by comparison of the molecular phylogenetic data. S trikingly, D. phadaengensis also differs from the other species by having yellowish orange KOH-extractable stromatal pigments and the tissue below the perithecial layer, and has the thinnest tissue below the perithecial layer (1.4-2 mm) of all known Daldinia species. Table 2 provides a synopsis of the morphological characters and secondary metabolites of this group of Daldinia species and the related genus Pyrenopolyporus. Daldinia placentiformis, another morphologically similar species, which has so far not been found in Thailand, has olivaceous pigments, owing to the presence of daldinone A (Bitzer et al. 2008). Daldinin A derivatives were originally isolated from a species referred to as "D. concentrica" by Hashimoto (1994), which was revised as D. childiae by Stadler et al. (2014). They are chemically similar to the lenormandins and fragirubrins that are known from Hypoxylon species (Kuhnert et al. 2015;Surup et al. 2018). However, this is the first time they have been identified as a major metabolites in a species that does not belong to the D. childiae group as defined by Stadler et al. (2014). Several peaks corresponding to cytochalasans were also observed but could not be further elucidated without preparative isolation, which was not possible due to scarcity of material. A major unknown compound (UCP) was also detected, whose molecular formula could not yet be identified.
Cultures on YMGA and PDA not producing anamorphic structures in 3 months.
Anamorph on YMGA and PDA similar to that on OA. Secondary metabolites. BNT (1) in traces and a multitude of peaks corresponding to cytochalasans that could not further elucidated without preparative isolation, which was not possible due to scarcity of material. Additionally, two unidentifiable peaks (UCB1, UCB2) not corresponding to cytochalasans were detected.
Notes. The Thai specimen of D. brachysperma corresponds well with the descriptions made in Ju et al. (1997) and Stadler et al. (2014). This species is distinctive for its stromatal morphology and the characteristic short ascospores. The HPLC profile matched the data reported by Stadler et al. (2014). The phylogenetic position and the characteristics of the anamorph are reported here for the first time, and this confirmed the affinities of this species to the D. eschscholtzii group as postulated by Stadler et al. (2014) (Figs. 6, 7, and 8).

Conclusion
The present study focused on the taxonomy of Daldinia in Thailand, from which only four species (D. bambusicola, D. eschscholtzii, D. kretzschmarioides, D. subvernicosa) had been recorded. Here, we describe three additional novel taxa and a new  Phylogeny of the Hypoxylaceae. The RAxML tree was generated based on multiple loci alignment of concatenated ribosomal (ITS and LSU) and proteinogenic (TUB2 and RPB2) sequence data. Support values were calculated via MP, ML, and Bayesian analysis and are indicated above (MPBS/MLBS) and below (BPP) the respective branches. Branches of significant support (BS ≥ 95% and PP ≥ 0.98) are thickened record using a polyphasic approach. Several potentially new secondary metabolites have been detected in the stromata of these species by chemotaxonomic methodology, but these metabolites remain to be isolated and identified, which was not possible from the scarce stromatal material representing the type specimens. Therefore, either artificial stromata production or re-collection of the fungi in the field will be necessary in the future to accomplish this task. Daldinia as well as other genera of the stromatic Xylariales in Thailand (e.g., Pyrenopolyporus and in particular the large genus Hypoxylon) need further studies. Apart from molecular systematics and chemotaxonomy, this also concerns the generation of data based on innovative technologies such as genomics, proteomics, and metabolomic data in order to explore the full biotechnological potential of these fungi. 4a Stromata with short stout stipe; ascospores dark brown to blackish brown, unicellular, ellipsoid-inequilateral, with narrowly rounded to almost acute ends, 6-7 × 3-4 μm

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
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