Multigene Phylogeny Reveals Endophytic Xylariales Novelties from Dendrobium Species from Southwestern China and Northern Thailand

Xylariales are common endophytes of Dendrobium. However, xylarialean species resolution remains difficult without sequence data and poor sporulation on artificial media and asexual descriptions for only several species and old type material. The surface-sterilized and morph-molecular methods were used for fungal isolation and identification. A total of forty-seven strains were identified as twenty-three species belonging to Apiosporaceae, Hypoxylaceae, Induratiaceae, and Xylariaceae. Five new species—Annulohypoxylon moniliformis, Apiospora dendrobii, Hypoxylon endophyticum, H. officinalis and Nemania dendrobii were discovered. Three tentative new species were speculated in Xylaria. Thirteen known fungal species from Hypoxylon, Nemania, Nigrospora, and Xylaria were also identified. Another two strains were only identified at the genus and family level (Induratia sp., Hypoxylaceae sp.). This study recorded 12 new hosts for xylarialean endophytes. This is the first report of Xylariales species as endophytes from Dendrobium aurantiacum var. denneanum, D. cariniferum, D. harveyanum, D. hercoglossum, D. moniliforme, and D. moschatum. Dendrobium is associated with abundant xylarialean taxa, especially species of Hypoxylon and Xylaria. We recommend the use of oat agar with low concentrations to induce sporulation of Xylaria strains.


Introduction
Dendrobium Sw. is one of the three largest genera in Orchidaceae [1]. Many Dendrobium orchids have important medicinal and ornamental values [2,3]. However, the majority of Dendrobium species are endangered due to low germination rates, habitat destruction, and over-exploitation as reported in the IUCN (International Union for Conservation of Nature) Red List of Threatened Species. Fungal endophytes play an important role in orchid development and defense against stress [4][5][6][7]. The symbiotic germination of eleven orchid species can be enhanced by some fungal endophytes [8,9]. Extracts of both Dendrobium and fungal endophytes have been found to possess various bioactivities such as angiogenesis inhibitory, anti-cancer, anti-inflammatory, anti-mutagenic, and anti-oxidative bioactive properties [10][11][12][13][14][15][16].

Phylogenetic Analysis
The original DNA sequence data were manually trimmed and assembled into contigs using ContigExpress (Vector NTI suite 6.0, Informax). The consensus sequences were subjected to BLASTn in the NCBI GenBank database for the initial screening of the most similar sequences, particularly for those of ex-type/ex-epitype strains (www.ncbi.nlm.nih. gov/BLAST, 3 July 2021). All new sequences in this study were deposited in GenBank (Supplementary Tables S1 and S2). The selected sequences were aligned using MAFFT version 6 [56] (http://mafft.cbrc.jp/ alignment/server/, 3 July 2021). Aligned datasets were visually inspected and misaligned regions were trimmed by TrimAL v. 1.2 in PhyloSuite v. 1.2.2 [57,58] and AliView [59]. All base pair differences, excluding gaps, were calculated by MEGA 7.0 [60]. Gaps were treated as missing data in maximum likelihood and Bayesian inference trees. For the Apiosporaceae tree, the general time-reversible (GTR) model with the gamma distribution option was implemented for the TEF-1α and TUB2 datasets. The GTR model with Inv-Gamma distribution was selected for the ITS and LSU datasets. For another tree, the GTR model with the Inv-Gamma distribution option of nucleotide substitution was selected for the ITS, LSU, and RPB2 datasets. The Hasegawa, Kishino & Yano (HKY) model with Inv-Gamma distribution was used for the TUB2 datasets. The ML tree was constructed using RAxML-HPC2 on XSEDE in CIPRES Science Gateway V. 3.3 (http://www.phylo. org/index.php/, 4 July 2021). The BI tree was achieved by MrBayes version 3.2.7 via the public resource platform CIPRES Science Gateway V3.3 (Ronquist & Huelsenbeck 2003, https://www.phylo.org/, 4 July 2021). The concatenated dataset was partitioned, and the ultrafast bootstrap [61] implemented in the IQ-TREE software [62] was used to estimate the best fitting models according to the Bayesian information criterion (BIC).
Two sets of six simultaneous independent chains of Markov Chains Monte Carlo (MCMC) simulations were run for 5,000,000 (For Apiosporaceae phylogenetic tree) and 400,000,000 (For Xylariaceae related taxa phylogenetic tree) generations, 25% of trees were discarded, and the remaining trees were used to calculate the posterior probabilities. Convergence was assumed when the standard deviation of split sequences was less than 0.01. The best scoring RAxML trees with bootstrap values of maximum likelihood (ML) (equal to or above 70%) and Bayesian posterior possibilities (BPP) from MCMC analysis trees (equal to or above 90%) are shown in phylogenetic trees. The two phylogenetic trees were submitted to TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2: S28326; http://purl.org/phylo/treebase/phylows/study/TB2:S28327, 5 July 2021). All trees were viewed in FigTree v. 1

Morphological Study
Pure strains were cultivated on 2% malt extract agar (MEA). Fresh mycelia were transferred to potato dextrose agar (PDA), 4% oat agar (OA), and water agar (WA) using sterilized toothpicks for sporulation [45]. Cultures were kept in a dark cabinet at room temperature (28 • C) and observed every seven days or less. The growth rate was evaluated when fungal mycelia nearly covered the whole plate. Fungal structures were observed and captured with a stereomicroscope (SteREO Discovery. V8, Carl Zeiss Microscopy GmBH, Jena, Germany). Cross-sections of conidiomata, or ascomata-like, structures were mounted in 10% hydrogen peroxide and observed using a Nikon ECLIPSE 80i compound microscope with a Cannon 600D digital camera (Japan). The size of the morphological characteristics was measured with Tarosoft (R) Image Frame Work (IFW) v.0.9.7 and the photo plates were made by Adobe Photoshop CS 22.2.0 (Adobe Inc., USA).

Isolated Fungi
Among the 47 fungal isolates from 23 Dendrobium orchid samples (13 from leaves, 22 from roots, 12 from stems), a total of 23 species (including 3 speculated novel Xylaria species and 2 undetermined species-Induratia sp. and Xylariaceae sp.) were identified based on phylogenetic analysis and morphology (Table 3). Xylaria species account for 51% of the isolated fungi. Nine new species from Annulohypoxylon, Hypoxylon, Nemania, and Xylaria were introduced. The newly introduced species are listed in Table 4.

Taxonomy
Xylariales [63]. Xylariales are commonly found as fungal endophytes in herbaceous plants, saprobes, or pathogens on rotting wood with a preference for humid, shady habitats and have a worldwide distribution [23,64]. Hyde et al. (2020) accepted 15 families in this order. Stroma is an important characteristic for distinguishing most of the genera in Xylariales. However, some genera are astromatic, and there are asexual Xylariales [19]. Many novel structures of interesting secondary metabolites have been discovered in species of this order, which indicates a great potential for investigating bioactive compounds [33].
Apiospora dendrobii XY Ma & JC Kang, sp. nov. Figure 3. indicates that the most similar species is Sordariomycetes sp. (Sequence similarity 99%). Both LSU and TUB2 blast searches indicated that MFLUCC 14-0152 was the most similar species to A. xenocordella with sequence similarity of 99% and 94%, respectively. Although the morphological characteristics were not sufficient to designate the novel species, the base pair differences support it as a different species from Apiospora xenocordella. Nigrospora Zimm. [74] Nigrospora are ubiquitous endophytes and saprobes, and some species are human and plant pathogens, with global distribution [55]. Nigrospora species are characterized by spherical to subspherical conidiogenous cells, black and globose to subglobose conidia [55,75]. Numerous secondary metabolites isolated from this genus have promising bioactive potentials, such as plant growth-inhibiting nigrosporolide and anti-mosquitoes phomalactone [76,77]. Index Fungorum number: IF551811; Facesoffungi number: FoF 10263. Etymology-Name after its host species-genus name. Culture characteristics-Colonies on PDA superficial, white to light yellow hypha, circular, lightly undulate, radiant from the middle; reverse yellow. Vegetative hyphae septate, branched, hyaline, and thick-walled. Growth rate: 5 mm/day.
On 4% OA, vegetative hyphae hyaline was 1-4 µm diam., septate, smooth, branched, and thick-walled. Conidiophores were reduced to conidiogenous cells, hyaline to brown, thick-walled. Conidiogenous cells were erected from mature mycelia, aggregated, hyaline to brown, and straight to curved. Conidiomata 20 µm diam., superficial or immersed in aerial mycelia, globose to irregular, dark brown to black, aggregated. Conidia 7-11.5 × 9-12.5 µm (x = 9.5 × 10.3 µm, n = 50), hyaline to light brown, solitary, in surface view, ellipsoid to globose or sub-globose, lenticular from side view. Notes-The conidiogenous cell of Apiospora dendrobii is straight, which differs from that of A. xenocordella (verruculose, globose to clavate to doliiform). Apiospora dendrobii forms an individual branch clustered with A. xenocordella sustained by 100% ML/1.0BPP. The ex-type MFLUCC 14-0152 differs from the ex-type of CBS 478.86 of A. xenocordella by 1.95% (13/666 bp) of ITS and 3.24% (7/216 bp) of TUB2 sequences. The ITS blast search indicates that the most similar species is Sordariomycetes sp. (Sequence similarity 99%). Both LSU and TUB2 blast searches indicated that MFLUCC 14-0152 was the most similar species to A. xenocordella with sequence similarity of 99% and 94%, respectively. Although the morphological characteristics were not sufficient to designate the novel species, the base pair differences support it as a different species from Apiospora xenocordella. Nigrospora Zimm. [74] Nigrospora are ubiquitous endophytes and saprobes, and some species are human and plant pathogens, with global distribution [55]. Nigrospora species are characterized by spherical to subspherical conidiogenous cells, black and globose to subglobose conidia [55,75]. Numerous secondary metabolites isolated from this genus have promising bioactive potentials, such as plant growth-inhibiting nigrosporolide and anti-mosquitoes phomalactone [76,77].  Notes-Nigrospora chinensis (MFLUCC 14-0109 and MFLUCC 18-1215) was isolated from leaves of Dendrobium cariniferum collected from northern Thailand and southwestern China. The two strains clustered with ex-type of Nigrospora chinensis (CGMCC 3.18127) and another strain, LC4593, that were from Camellia sinensis in Guang Dong, China with 100% ML/1.0BPP support. Compared with the ex-type CGMCC 3.18127, MFLUCC 14-0109 and MFLUCC 18-1215 have very similar sequences and asexual morphological characteristics. The blast search also indicates that their most similar species is Nigrospora Culture characteristics-colonies on PDA were superficial, white, and cottony on the entire edge. Reverse white. Notes-Nigrospora chinensis (MFLUCC 14-0109 and MFLUCC 18-1215) was isolated from leaves of Dendrobium cariniferum collected from northern Thailand and southwestern China. The two strains clustered with ex-type of Nigrospora chinensis (CGMCC 3.18127) and another strain, LC4593, that were from Camellia sinensis in Guang Dong, China with 100% ML/1.0BPP support. Compared with the ex-type CGMCC 3.18127, MFLUCC 14-0109 and MFLUCC 18-1215 have very similar sequences and asexual morphological characteristics. The blast search also indicates that their most similar species is Nigrospora chinensis (Sequence similarity 99%).
Notes-Nigrospora sphaerica (GZAC O37S13) was isolated from Dendrobium hercoglossum from Xingyi, Guizhou in southwestern China. It failed to sporulate on PDA after one month. From the phylogeny, the isolate GZAC O37S13 and two Nigrospora sphaerica strains, including the ex-type, were isolated from Musa paradisiaca in Hai Nan, China. Compared with the ex-type of LC 13523 of Nigrospora sphaerica, the sequences of GZAC O37S13 are similar to that of LC 13523. The robust support value 100% ML/1.0BPP exhibited their intimate relationship.
Notes-Annulohypoxylon moniliformis and An. annulatum clustered with 98% ML/1.0BPP support. It is difficult to judge their morphology because our characteristics are insufficient and unclear, although they differ by color and size of ascospores (hyaline 7-8.5 × 4-6 μm in 1 month (An. moniformis) vs. brown to dark brown, unicellular, 7.5-11 × 3.6-6 μm in 2-3 weeks (An. annulatum)) on different media (WA of slide culture vs. OA). The blast search of all sequences indicated that An. annulatum was the most similar species to An. moniliformis (Sequence similarity 99%). However, a pairwise nucleotide comparison between the MFLUCC 18-1214 and An. annulatum (ex-type CBS 140775) revealed 1.53% (13/847 bp) of ITS and 4.51% (42/932 bp) of TUB2 sequence differences. Therefore, based on the principal that more than 1.5% nucleotide differences in the ITS gene perhaps indicate a new species [83], we introduce MFLUCC 18-1214 as a new species based on the base pair differences.
Hypoxylon Bull. [84]  Notes-Annulohypoxylon moniliformis and An. annulatum clustered with 98% ML/1.0BPP support. It is difficult to judge their morphology because our characteristics are insufficient and unclear, although they differ by color and size of ascospores (hyaline 7-8.5 × 4-6 µm in 1 month (An. moniformis) vs. brown to dark brown, unicellular, 7.5-11 × 3.6-6 µm in 2-3 weeks (An. annulatum)) on different media (WA of slide culture vs. OA). The blast search of all sequences indicated that An. annulatum was the most similar species to An. moniliformis (Sequence similarity 99%). However, a pairwise nucleotide comparison between the MFLUCC 18-1214 and An. annulatum (ex-type CBS 140775) revealed 1.53% (13/847 bp) of ITS and 4.51% (42/932 bp) of TUB2 sequence differences. Therefore, based on the principal that more than 1.5% nucleotide differences in the ITS gene perhaps indicate a new species [83], we introduce MFLUCC 18-1214 as a new species based on the base pair differences.
Hypoxylon Bull. [84] Hypoxylon is the type of genus of Hypoxylaceae, which mainly inhabit dead wood as saprobes, and some occur as endophytes of a wide range of hosts or facultative parasites on diseased hosts. They have Nodulisporium-like asexual morphs [20,80]. Stomatal (including pigment) and ascospore morphology were often used for morphological delimitation of the Hypoxylon species [20]. Multigene genealogy revealed that they are a polyphyletic group [19,67]. Many bioactive secondary metabolites have been identified from Hypoxylon species, especially from endophytic strains [33,85,86].
saprobes, and some occur as endophytes of a wide range of hosts or facultative parasites on diseased hosts. They have Nodulisporium-like asexual morphs [20,80]. Stomatal (including pigment) and ascospore morphology were often used for morphological delimitation of the Hypoxylon species [20]. Multigene genealogy revealed that they are a polyphyletic group [19,67]. Many bioactive secondary metabolites have been identified from Hypoxylon species, especially from endophytic strains [33,85,86]. Hypoxylon endophyticum XY Ma & JC Kang, sp. nov. Figure 6. Etymology-Name after its life strategy as a fungal endophyte. Culture characteristics-Colony on PDA superficial, greyish to white, velvety, loose, and on entire edge; reverse brown to black. Growth rate: 5.6 mm/day.
On WA (slide culture), vegetative hyphae 2-5 µm, hyaline to brown, smooth to rough, and branched. Conidiomata 200-500 µm wide, light brown, and irregular. Conidiophores mononematous or macronematous, Periconiella-like, cylindrical, hyaline to brown, integrated, septate, branched, and finely rough. Conidia 3.5-6.5 × 2-5.5 µm (x = 5.5 × 3 µm, n = 20), hyaline, globose to ellipsoid, sympodially, and smooth. Notes-Hypoxylon endophyticum formed an independent clade close to H. investiens. The sequence variation among the six strains is less than 1% except for the MFLUCC 18-1207 that may be caused by the crossing contamination or intraspecific variation. Hypoxylon endophyticum failed to be discriminated with H. investiens although the size of conidia in this study are larger than that of Hypoxylon investiens in Ju & Rogers (1996) considering different media (3.5-6.5 × 2-5.5 µm on WA in 1 month vs. 2.5-3.5 × 2.2-3.5 µm on OA in 2 weeks) [78]. The distinction based on base pairs between Hypoxylon endophyticum and H. investiens have been listed in Table 4. The large difference exists in the sequences of LSU and RPB2. Blast searches for the six isolates showed that Hypoxylon investiens is the most similar species with sequence similarity from 96-97%.
Hypoxylon officinalis XY Ma & JC Kang, sp. nov. Figure 7. Notes-Hypoxylon endophyticum formed an independent clade close to H. investiens. The sequence variation among the six strains is less than 1% except for the MFLUCC 18-1207 that may be caused by the crossing contamination or intraspecific variation. Hypoxylon endophyticum failed to be discriminated with H. investiens although the size of conidia in this study are larger than that of Hypoxylon investiens in Ju & Rogers (1996) considering different media (3.5-6.5 × 2-5.5 μm on WA in 1 month vs. 2.5-3.5 × 2.2-3.5 μm on OA in 2 weeks) [78]. The distinction based on base pairs between Hypoxylon endophyticum and H. investiens have been listed in Table 4. The large difference exists in the sequences of LSU and RPB2. Blast searches for the six isolates showed that Hypoxylon investiens is the most similar species with sequence similarity from 96-97%.
Material Notes-Hypoxylon officinalis, represented by MFLUCC 14-0075, MFLUCC 14-0078, and MFLUCC 21-0060, formed an independent clade adjacent to Hypoxylon lateripigmentum (MUCL 53304). The three strains were all from Dendrobium roots. Compared with the ex-type MUCL 53304, Hypoxolon officinalis was not observed having the periconiella-like conidiogenous structure, and the shape of conidia is a bit more changeable. Their sequence base pair discrepancy is listed in Table 4.
Hypoxylon investiens (Schwein.) M.A. Curtis [87] Figure 8. Notes-The strain MFLUCC 15-1155 was isolated from the stems of Dendrobium moschatum collected from northern Thailand. It clustered with three Hypoxylon investiens strains sustained by low support. Type specimens with sequence data of Hypoxylon investiens is absent. Contrasted to the morphological descriptions of Hypoxylon investiens in Ju and Rogers (1996), MFLUCC 15-1155 is the same as Hypoxylon investiens although periconiella-like conidiogenous structure was not observed on MFLUCC 15-1155, and the latter has smaller conidia perhaps resulting from different media. However, the ITS base pair discrepancy among the four Hypoxylon investiens strains-YMJ 89062905, CBS 118185, CBS 118183, and MFLUCC 15-1155 can be up to 3.5%. We speculate that a complex exists which needs further verification and comparison with confirmed type species and molecular data. The blast search shows that Hypoxylon investiens has the highest 96% sequence similarity with strain MFLUCC 15-1155. Culture characteristics-Colony on PDA superficial, white to brown, velvety, mycelia gather unevenly, with an entire edge, reverse brown to black, and radial. Growth rate: 7 mm/day.
On MEA and WA (slide culture), vegetative hyphae 1.2-3.2 µm diam., hyaline to brown, smooth to rough, and branched. On MEA, conidiomata was superficial or immersed, scattered, black, and globose to irregular. Ascospores was brown to dark brown, sub-globose, ellipsoid and clavate, nearly equilateral, with one or two rounded ends, faint, and straight germ slit shorter than spore-length. Conidia on WA 2-4.5 × 1. Notes-The strain MFLUCC 15-1155 was isolated from the stems of Dendrobium moschatum collected from northern Thailand. It clustered with three Hypoxylon investiens strains sustained by low support. Type specimens with sequence data of Hypoxylon investiens is absent. Contrasted to the morphological descriptions of Hypoxylon investiens in Ju and Rogers (1996), MFLUCC 15-1155 is the same as Hypoxylon investiens although periconiellalike conidiogenous structure was not observed on MFLUCC 15-1155, and the latter has smaller conidia perhaps resulting from different media. However, the ITS base pair discrepancy among the four Hypoxylon investiens strains-YMJ 89062905, CBS 118185, CBS 118183, and MFLUCC 15-1155 can be up to 3.5%. We speculate that a complex exists which needs further verification and comparison with confirmed type species and molecular data. The blast search shows that Hypoxylon investiens has the highest 96% sequence similarity with strain MFLUCC 15-1155.
Hypoxylaceae sp. (Unresolved species) Figure 10. Culture characteristics-Colony on PDA was superficial, white to amber, velvety, entire edge, and reverse brown to black. Growth rate: 6 mm/day.
Hypoxylaceae sp. (Unresolved species) Figure 10. Culture characteristics-Colonies on PDA were superficial white, irregular, with lobbed concentric ring and undulate margin, flossy, velvety, reverse white, with the ununiform concentric ring.  [89]. Induratiaceae was introduced by Smarakoon et al., (2020) to accommodate Emarcea and Induratia (=Muscodor) with apiospores and independent phylogenetic clade trees. This group is usually saprobic on dead wood and leaves and endophytic on leaves, stems, and bark [89]. The hyphae of Induratiaceae are rope-like with cauliflower-like hyphal bodies [89]. Moscodor is an endophyte genus without morphological record and established only based on molecular data, which can produce volatile antibiotics and be distinguished from each other by their chemical profiles [23]. However, the genus was not accepted by  Culture characteristics-Colonies on PDA were superficial white, irregular, with lobbed concentric ring and undulate margin, flossy, velvety, reverse white, with the ununiform concentric ring. Moscodor is an endophyte genus without morphological record and established only based on molecular data, which can produce volatile antibiotics and be distinguished from each other by their chemical profiles [23]. However, the genus was not accepted by Muscodor were thought to be the same genus corresponding to the sexual and asexual states in phylogenetic studies [88]. Induratia was adopted as the genus name according to the One-Fungus-One-Name proposal [89]. This group is saprobic on dead wood and endophytic on bark, leaves, roots, and stems [89]. The asexual morph is characterized by terminal conidiogenous cells bearing inconspicuous denticles, conidia narrowly ellipsoidal to subglobose, hyaline, smooth-walled, with a flat, wide, basal scar [89].
Notes-Induratia rarely produces morphological structures on artificial media. Induratia sp. was isolated as a fungal endophyte from the root of Dendrobium nobile [90]. The endophytic strain MFLUCC 15-1218 was isolated from the roots of Dendrobium sp. and collected from a steep forest in northern Thailand. It clustered with Induratia brasiliensis and I. ziziphi. However, it is hard to discern its taxonomic placement based on the ITS sequences with less than 1% base pairs difference.
Nemania Gray [91] Nemania is a large genus of Xylariaceae and characterized by stromata without bark rupturing appearance, a lack KOH-extractable pigments, and finely papillate ostioles [20]. phylogenetic studies [88]. Induratia was adopted as the genus name according to the One-Fungus-One-Name proposal [89]. This group is saprobic on dead wood and endophytic on bark, leaves, roots, and stems [89]. The asexual morph is characterized by terminal conidiogenous cells bearing inconspicuous denticles, conidia narrowly ellipsoidal to subglobose, hyaline, smooth-walled, with a flat, wide, basal scar [89]. Induratia sp. (Unresolved species) Figure 9B. Culture characteristics-Colony on PDA was white, cottony, undulate edge, and reverse light brown. Growth rate: 3.6 mm/day.
Notes-Induratia rarely produces morphological structures on artificial media. Induratia sp. was isolated as a fungal endophyte from the root of Dendrobium nobile [90]. The endophytic strain MFLUCC 15-1218 was isolated from the roots of Dendrobium sp. and collected from a steep forest in northern Thailand. It clustered with Induratia brasiliensis and I. ziziphi. However, it is hard to discern its taxonomic placement based on the ITS sequences with less than 1% base pairs difference.
Xylariaceae Tul. & C. Tul. [19] Xylariaceae is one of the largest families of Xylariales and can be a saprobe, pathogen, or endophyte on a wide range of hosts, substrates, or associated insect vectors [20,23]. Many xylariaceous species have been founded as endophytes and involved in the study for natural products [11,18,23,33]. The stromata and ascomata of Xylariaceae are variable in size [19,20]. Their sexual morph is hyphomycetous, which are mostly geniculosporiumlike [20,23]
Notes-Nemania bipapillata comprising three isolates MFLUCC 14-0105, MFLUCC 14-0138, 90080610 (HAST) were well separated with N. dendrobii. The two strains, MFLUCC 14-0105 and MFLUCC 14-0138, were isolated from stems of Dendrobium cariniferum and roots of Dendrobium sp. collected from northern Thailand. The asexual morphological descriptions and type molecular data are absent. The blast search showed that Nemania bipapillata matched each gene with a high sequence similarity of 97-100%.
Notes-Nemania bipapillata comprising three isolates MFLUCC 14-0105, MFLUCC 14-0138, 90080610 (HAST) were well separated with N. dendrobii. The two strains, MFLUCC 14-0105 and MFLUCC 14-0138, were isolated from stems of Dendrobium cariniferum and roots of Dendrobium sp. collected from northern Thailand. The asexual morphological descriptions and type molecular data are absent. The blast search showed that Nemania bipapillata matched each gene with a high sequence similarity of 97-100%.
Note-The strain MFLUCC 14-0139 was isolated from the root of Dendrobium sp. collected from Thailand. Nemania diffusa represented by three strains 91020401 (HAST), JZB3370003, and MFLUCC 14-0139 formed a single clade close to Hypoxylon argillaceum Culture characteristics-colonies on PDA were superficial, white, cottony, radial, entire edge, exudate colorless, and reverse white. Growth rate: 7 mm/day.
Note-The strain MFLUCC 14-0139 was isolated from the root of Dendrobium sp. collected from Thailand. Nemania diffusa represented by three strains 91020401 (HAST), JZB3370003, and MFLUCC 14-0139 formed a single clade close to Hypoxylon argillaceum (CBS 527.63) with robust support (100% ML/1.0BPP). Hypoxylon argillaceum (CBS 527.63) is the only hypoxylaceous species in the 'Nemania + Rosellinia' clade. The same situation also occurred in U'ren et al. 2016 and re-evaluation was recommended. We speculate that CBS 527.63 should be 'Nemania argillaceum.' Although MFLUCC 14-0139 holds 3.8% of the different TUB2 sequences compared with 91020401 (HAST), considering their identical ITS sequences and scanty morphological information, incorporating the blast search for each gene, we identified it as Nemania diffusa.
Xylaria Hill ex Schrank [93] Xylaria is a genus type of Xylariaceae and is characterized by large stromata, long asci with stipes, dark ascospores, and geniculosporium-like asexual morph [20,85,90]. The highly diversified nature of the genus may be the result of highly convergent evolution within the genus [19,20]. Most Xylaria are saprobic on deciduous dead wood and endophytes of numerous plants, especially pantropical areas, some associated with termites [23].
Xylaria sp.1 Figure 14. Xylaria is a genus type of Xylariaceae and is characterized by large stromata, long asci with stipes, dark ascospores, and geniculosporium-like asexual morph [20,85,90]. The highly diversified nature of the genus may be the result of highly convergent evolution within the genus [19,20]. Most Xylaria are saprobic on deciduous dead wood and endophytes of numerous plants, especially pantropical areas, some associated with termites [23].
Material Notes-Xylaria sp.1 (MFLUCC 14-0137 and GZAC O6LA2) is adjacent to the isolate GENT 159 of X. cubensis, which forms a sister group to another two X. cubensis isolates. No type of herbarium specimens for X. cubensis was assigned. Compared with the asexual morph of Xylaria cubensis recorded by Rodrigues et al. (1993), Xylaria. sp. 1 has much bigger conidia with no obvious denticulate secession scars on the conidiogenous cell and a flat basal abscission scar on conidia. In gene sequence comparison, as listed in Table 4, Xylaria sp.1 and X. cubensis mainly differed by the ITS and TUB2 gene sequences. Here we introduce both MFLUCC 14-0137 and GZAC O6LA2 as a new species separated with Xylaria cubensis. X. cubensis isolate GENT 159, and Xylaria sp.1 MFLUCC 14-0137 has only six distinguished base pairs in the TUB2 sequence. However, GENT 159 is more different from the other two X. cubensis isolates. The taxonomic placement of GENT 159 perhaps needs re-evaluation with other gene sequence data.
Material Notes-Xylaria sp.1 (MFLUCC 14-0137 and GZAC O6LA2) is adjacent to the isolate GENT 159 of X. cubensis, which forms a sister group to another two X. cubensis isolates. No type of herbarium specimens for X. cubensis was assigned. Compared with the asexual morph of Xylaria cubensis recorded by Rodrigues et al. (1993), Xylaria. sp. 1 has much bigger conidia with no obvious denticulate secession scars on the conidiogenous cell and a flat basal abscission scar on conidia. In gene sequence comparison, as listed in Table 4, Xylaria sp.1 and X. cubensis mainly differed by the ITS and TUB2 gene sequences. Here we introduce both MFLUCC 14-0137 and GZAC O6LA2 as a new species separated with Xylaria cubensis. X. cubensis isolate GENT 159, and Xylaria sp.1 MFLUCC 14-0137 has only six distinguished base pairs in the TUB2 sequence. However, GENT 159 is more different from the other two X. cubensis isolates. The taxonomic placement of GENT 159 perhaps needs re-evaluation with other gene sequence data.
Notes-The conidia and conidiophores of Xylaria sp.2 sporulated on 4% OA are similar to the asexual morph of Xylaria [45]. In view that only two conidia were observed in this culture, the conidia size was not measured because the sample was too small to be representative. Xylaria sp. 2 clusters with X. phyllocharis. However, both type specimens of Xylaria phyllocharis and its asexual morph are absent. The strong evidence for the unique taxonomic placement of Xylaria sp. 2 is from the phylogenetic analysis (Table 4). Xylaria cubensis, X. digitata, and X. enteroleuca are the best match in blast search for MFLUCC 21-0014 with sequence similarities from 85.5-98% (ITS could not match a known species).
Notes-The conidia and conidiophores of Xylaria sp.2 sporulated on 4% OA are similar to the asexual morph of Xylaria [45]. In view that only two conidia were observed in this culture, the conidia size was not measured because the sample was too small to be representative. Xylaria sp. 2 clusters with X. phyllocharis. However, both type specimens of Xylaria phyllocharis and its asexual morph are absent. The strong evidence for the unique taxonomic placement of Xylaria sp. 2 is from the phylogenetic analysis (Table 4). Xylaria cubensis, X. digitata, and X. enteroleuca are the best match in blast search for MFLUCC 21-0014 with sequence similarities from 85.5%-98% (ITS could not match a known species).
Notes-The blast search for each gene of the strain MFLUCC 21-0059 indicated that Xylaria bambusicola is the best match with sequence similarities from 89-98%. Despite few available asexual morph records about X. bambusicola, the observed conidial characteristics resemble the asexual descriptions for endophytic Xylaria [45]. Erroneous or low-quality sequencing was excluded by checking sequence chromatogram and consensus blast results for each gene. We speculated that MFLUCC 21-0059 represents a tentative new species.
Notes-The blast search for each gene of the strain MFLUCC 21-0059 indicated that Xylaria bambusicola is the best match with sequence similarities from 89-98%. Despite few available asexual morph records about X. bambusicola, the observed conidial characteristics resemble the asexual descriptions for endophytic Xylaria [45]. Erroneous or low-quality sequencing was excluded by checking sequence chromatogram and consensus blast results for each gene. We speculated that MFLUCC 21-0059 represents a tentative new species.   Culture characteristics-colonies on PDA were superficial, white, velvety or cottony, stacked, lobbed or entire or undulate edge, and reverse white to brown. Growth rate: 4 mm/day.
On PDA, vegetative hyphae 2.7-4.8 µm, hyaline to brown, septate, smooth, branched, and thick-walled. Conidiomata were superficial, erect through the media, globose, with flattened apex, and white to light brown. Conidiophores had a 4.4 µm diam., were cylindrical to clavate, hyaline to brown, branched or unbranched, smooth, and sometimes laterally compressed into a tight layer. Conidia were hyaline to brown, ovoid to ellipsoid, with granular contents, smooth-walled, thick-walled, with a flattened basal scar.
Xylaria curta Fr. [95] Figure 9G. Culture characteristics-colonies on PDA were superficial, white, cottony aerial hyphae and brown concentric rings in the center, leathery outside the center, radial with bunched mycelia, denticulate edge, and reverse light brown. Growth rate: 5 mm/day.
Notes-As a fungal endophyte, Xylaria curta was isolated from the roots of Dendrobium aphyllum and D. chrysanthum [37]. Although GZAC O36L23 and 92092022 (HAST) have 7.1% of distinct ITS base pairs with 1.3% and 1.1% of sequence differences in TUB2 and RPB2, respectively, we tend to identify GZAC O36L23 as Xylaria curta due to the phylogenetic placement and scanty morphology. The ITS blast results showed that Xylaria cf. curta (K.-L. Chen L148) was the best match species with 94.2% sequence similarity. Xylaria cf. curta (K.-L. Chen L148) represents an endophyte isolated from leaves of lotus [96]. However, the classification of Xylaria cf. curta is unclear. The TUB2 and RPB2 gene sequence blast search indicated that X. curta is the most similar species to GZAC O36L23 (Sequence similarity 93-99%). We speculate that GZAC O36L23 might be an intraspecific variant of Xylaria curta.
Xylaria laevis Lloyd [97] Figure 9F. Culture characteristic-colonies on PDA were superficial, white, velvety, radial, zonate with several concentric circles, entire edge, and reverse white and brown around some marginal parts. Vegetative hyphae were septate, branched, hyaline, and thick-walled. Growth rate: 4.5 mm/day. Notes-Two target strains, GZAC O33L12 and GZAC O6LA2, were isolated from the leaves of Dendrobium aurantiacum and D. officinale sampled in Xingyi City, Guizhou, China. The gene blast search showed that Xylaria laevis was their best match species. (Sequence similarity 95-98%). Despite gene sequence disparity between the two isolates, due to a lack in further evidence, both are identified as Xylaria laevis.
Notes-Xylaria grammica has been isolated as endophyte from the root of Dendrobium aphyllum, D. nobile, D. chrysanthum, D. chrysotoxum, D. crystallinum, and D. fimbriatum [37,84]. In this study, Xylaria grammica (MFLUCC 14-0093 and MFLUCC 14-0146) were isolated from leaves of Dendrobium sp. collected from northern Thailand. The mononematous conidiophores and conidia with abscission scar resembled the asexual morph of Xylaria, but the conidia are much larger with more variant shapes [45]. Although lacking type specimens, they have identical sequences as Xylaria grammica (Strains BCC 1002 and 5228) and all cluster with well-supported values (100% ML/1.0BPP). The blast results for the two strains showed that Xylaria grammica has the highest sequence similarity of 99-100%.
Notes-Xylaria populis GZAC O32S24 was isolated from the stems of Dendrobium chrysotoxum collected from Luodian County, Guizhou Province, southwestern China. Xylaria papulis 5118 as an endophyte was isolated from the roots of Dendrobium aphyllum, D. chrysotoxum and D. fimbriatum [37]. In view that the isolate GZAC O32S24 is identified as Xylaria papulis with only ITS sequence in the polyphasic analysis, it may not be a stable result. The ex-type of Xylaria hongkongensis, GDGM 40058, has less than 0.5% ITS sequence discrepancy with Xylaria papulis 5118. Therefore, Xylaria hongkongensis GDGM 40058 perhaps need to be re-evaluated with further gene sequences.
Notes-Xylaria populis GZAC O32S24 was isolated from the stems of Dendrobium chrysotoxum collected from Luodian County, Guizhou Province, southwestern China. Xylaria papulis 5118 as an endophyte was isolated from the roots of Dendrobium aphyllum, D. chrysotoxum and D. fimbriatum [37]. In view that the isolate GZAC O32S24 is identified as Xylaria papulis with only ITS sequence in the polyphasic analysis, it may not be a stable result. The ex-type of Xylaria hongkongensis, GDGM 40058, has less than 0.5% ITS sequence discrepancy with Xylaria papulis 5118. Therefore, Xylaria hongkongensis GDGM 40058 perhaps need to be re-evaluated with further gene sequences.
Xylaria venosula Speg. [100]. Figure 19. =Xylaria arbuscula CBS 126415, CBS 126416 =Xylaria apiculata EF6 Culture characteristics-colonies on PDA were superficial, white to light yellow, velvety, had an entirely or slightly undulated edge, and were reverse light brown to dark brown. Vegetative hyphae were septate, branched, hyaline, and thick-walled. Growth rate: 4.3 mm/day. Colonies on MEA were white, velvety, on the entire edge, with 1-2 cm high charcoal stroma, stroma cylindrical with a white top, and reverse white with black spots. Growth rate: 4.5 mm/day. Stroma formed after 2 months.
Notes-Xylaria venosula were isolated as endophytes from the root of Dendrobium nobile [87]. The type specimens of Xylaria venosula is absent. Our asexual morph coincides with the description for Xylaria [45]. In the present study, it was isolated from roots and stems of Dendrobium fimbriatum, D. primulinum, and Dendrobium sp. Xylaria venosula, X. arbuscula and X. apiculate formed a monophyletic group with robust support (100% ML/1.0BPP). mboxXylaria arbuscula (CBS 126415 and CBS 126416) were isolated from the branch of Quercus and Robinia in a fern greenhouse (Brandenburg, Germany), respectively [81]. Xylaria apiculata EF6 as an endolichenic strain was isolated and identified with the ITS sequence from the lichen Pyxine petricola in India [94]. All nine strains in this group have very similar sequences of each gene with only 0.35-2.5% base pair differences. Few morphological information is available for comparison. Therefore, we regard all of them as Xylaria venosula.

Xylariaceous Endophyte Associated with Dendrobium
From this study, each Dendrobium species is associated with two different Xylarialean species on average (including the unidentified taxa). The actual numbers would be higher considering the unidentified hosts. Xylaria is the most frequently isolated Xylariaceous genera associated with Dendrobium, followed by Hypoxylon and Nemania. Hypoxylon and Xylaria species account for the majority (29% and 51%) of all xylariaceous species. Hypoxylon and Xylaria endophytes mostly occurred on angiosperms, and a few gymnosperms were mainly distributed in pantropical areas [23,[101][102][103][104]. Hypoxylon investiens, Nemania diffusa, Nigrospora species, Xylaria cubensis, Xylaria curta, X. feejeensis, X. grammica, and X. venosula are common fungal endophytes in orchids, and most of them were identified by molecular analysis [35][36][37][105][106][107][108]. Hypoxylon officinalis and Xylaria venosula were isolated from both China and Thailand. Three new hosts were recorded for Xylaria venosula in this study. It is worth noting that five novel taxa with one cryptic species (Hypoxylaceae sp.) were introduced from forty-seven strains, which implied that fungal endophyte perhaps contains many unrecognized or cryptic species. Many xylarialean endophytes from various hosts in the USA have been inferred as novel species with multi-locus [109]. Due to all novel taxa being far away from the species of Xylaria sensu stricto (X. hypoxylon aggregate, Figure 2), we defined them as Xylaria sensu lato.
Despite the limited sample size and scope, we speculate that Dendrobium species are associated with highly diverse Xylariales, especially the Hypoxylon and Xylaria species. The result coincides with that of Chen et al. (2013) who investigated 217 xylariaceous strains from seven medical Dendrobium species with multi-gene phylogenetic analysis in China. Xylaria grammica is the most isolated taxon from the roots of Dendrobium in southwestern China [37]. Our results showed that the penzigioid species Xylaria berteri was repeatedly isolated from various Dendrobium organs in northern Thailand, while most Hypoxylaceae strains were from Chinese Dendrobium. As an endophyte, Xylaria berteri has often been recorded in tropical areas such as Brazil, Hawaiian Islands, Mexico, and Panama [110][111][112]. Hypoxylon officinalis was introduced based on the traits of three strains from roots of different Dendrobium, so we speculated Hypoxylon officinalis could be an organ specificity species. However, the organ, climatic or geographic preference, and host specificity in Xylariales endophyte needs further investigation in a wide range [48].

The Dilemma for Xylaria Endophyte Verification
Xylariaceae has been revised several times [19,23]. The best investigated Xylaria combines the traditional morphological concepts based on the type of material with multi-gene sequence analysis [16,23]. However, many old but well-defined Xylaria species lack type material and sequence data [23,48]. Few reports relate to the Xylaria asexual morph. In addition, a considerable number of Xylaria species produce sterile mycelia only, and reproduction rarely occurs on artificial media [37,78]. Altogether these make the Xylaria endophyte species resolution very difficult [23,45,109,113]. Therefore, a multi-locus phylogenetic analysis is the most reliable approach to know the taxonomic placement of the Xylaria endophyte [37]. Some cryptic Xylaria species have been found [37,102,109]. In this study, most Xylaria species identification mainly depends on molecular phylogenetic analysis. We could only present limited morphological characterization with asexual morph sporulated on 4% OA and little available sexual morph record for comparison. However, the discrimination based on base pairs could result from pseudogene or over-evaluation of genes [114]. With the type specimen re-inspection, assignment, and phylogenomic deciphering, we may approach a well-supported taxonomy for Xylariales [114][115][116]. Therefore, all novelties in this study could be regarded as tentative new taxa. Besides type (and ex-type) assignment, sporulation for endophytes such as Xylaria is always encouraged [42]. More attention should be paid to Xylaria asexual morph, which links endophytes with the sexual morph of known species and contributes to deciphering its genealogy with more novel, even cryptic species [54]. A breakthrough is needed for Xylaria asexual morph study, and any pertaining characteristics should be recorded for recognition, comparison, and discussion even though they would be criticized and potentially wrong. We recommend using OA with low concentrations under alternative fluorescent light to induce sporulation of Xylaria endophytes as more and more studies successfully sporulate Xylaria species with this medium [45,111]. The three new species, Xylaria insolita, X. necrophora, and X. subescharoidea, have been introduced recently with asexual morph sporulated on OA [117,118]. Due to a lack of full information for type species, we did not designate any novel Xylaria species in this study.

HTS Application for Fungal Endophyte
Compared with our results, the high-throughput amplicon sequencing (HTS) implemented by ITS-rRNA metagenomics analysis for the fungal endophyte composition of Dendrobium officinale showed a very low Xylaria frequency, which perhaps is due to the designed primers not being enough for all endophytes [119]. Although multi-gene markers have been employed for further resolving many taxonomic groups, including fungal endophytes, the traditional DNA-sequencing method only allows individual specimen sequencing [120]. The next generation sequencing (NGS) makes it possible to sequence mixed environmental bulk samples [121]. The collection of the resulting set of tens of thousands of genetic reads in parallel contributes to understanding historical, functional, and ecological biodiversity [2,122]. HTS application to fungal endophytes reveals some sympatric cryptic species, which discovered taxa that could not grow on artificial media [118,123,124]. Meanwhile, the HTS often produces false negative or erroneous results, and its application normally requires multiple barcoding reads into operational taxonomic units (OTUs), which is feasible to discern environmental DNA to generic levels but loses information on intraspecific diversity [122]. Therefore, applying the culture-dependent method or HTS only maybe not reveal the real composition of fungal endophytes. It is likely that combining both methods or improving the HTS primers can contribute to discovering the real composition, taxonomic placements, and ecological strategies [125][126][127]. Several advanced metabarcoding strategies and platforms have been introduced to achieve species identification in dietary, gut microbiome, and wildlife forensic species detection, which is likely to be used for more effective and exact fungal endophytes identification [128].

Potential Roles of Xylariaceous Endophyte
In this study, several species were initially identified as pathogens reported on other plants. Most grasses and reeds perhaps harbor species of Apiospora endophyte [65]. Apiospora (=Arthrinium) sp. was also associated with Dendrobium candidum and D. nobile [36,90]. Nigrospora sphaerica is an opportunistic pathogen causing onychomycosis in humans and can cause leaf blight on Camellia sinensis [55,129,130]. Nigrospora chinensis is a common pathogen that has been reported from Ginkgo biloba [90]. Nigrospora as ubiquitous endophyte, plant, and human pathogens, have been found in several orchids including Bulbophyllum neilgherense, Dendrobium candidum, and Vanda testacea [90,131]. The occurrence of the appressoria-like structure found in the 4% of OA culture of Xylaria venosula suggests this species could be invasive in the germination period. These fungal endophytes probably become potential pathogens or saprobes involving senescence during later life [25,[132][133][134]. It is reported that some volatile organic compounds isolated from Xylariales species can cause pest larval mortality [88,130,135]. For further understanding of xylarialean endophyte, an optimized methodology is needed on the taxonomy, physiology, and functional roles.

Conclusions
Dendrobium are associated with various Xylariales species. Hypoxylon and Xylaria take the majority of culturable xylarialean fungal endophytes in Dendrobium species. Hypoxylon endophyticum, Xylaria berteri and X. grammica are the most frequently culturedependent isolates. Novel endophytic species need robust backbone phylogenetic analysis with complete type material information. The low concentration of OA is recommended for inducing sporulation of Xylaria endophytes.