Multi-Gene Phylogeny and Taxonomy of Hypoxylon (Hypoxylaceae, Ascomycota) from China

: The Hypoxylon species play an important ecological role in tropical rainforest as wood-decomposers, and some might have beneﬁcal effects on their hosts as endophytes. The present work concerns a survey of the genus Hypoxylon from Hainan Tropical Rainforest National Park of China. Four new species: H. wuzhishanense , H. hainanense , H. chrysalidosporum , and H. cyclobalanopsidis , were discovered based on a combination of morphological characteristics and molecular data. Hypoxylon wuzhishanense is characterized by Rust pulvinate stromata, amyloid apical apparatus and brown ascospores, with most of the perispore being indehiscent in 10% KOH. Hypoxylon hainanense has effused–pulvinate and Violet stromata, amyloid apical apparatus, light-brown to brown ascospores with straight germ slit and dehiscent perispore. Hypoxylon chrysalidosporum is distinguished by glomerate to pulvinate stromata, highly reduced or absent inamyloid apical apparatus, and light-brown to brown ascospores with very conspicuous coil-like ornamentation. Hypoxylon cyclobalanopsidis has Livid Purple pulvinate stromata, highly reduced amyloid apical apparatus, faint bluing, brown ascospores and dehiscent perispore, and it grows on dead branches of Cyclobalanopsis . Detailed descriptions, illustrations, and contrasts with morphologically similar species are provided. Phylogenetic analyses inferred from ITS, RPB2, LSU, and β -tubulin sequences conﬁrmed that the four new species are distinct within the genus Hypoxylon


Introduction
Hypoxylon Bull., described by Bulliard in 1791 [1], is a genus that contains primarily saprotrophs and endophytes of angiospermous plants [2,3]. The genus Hypoxylon, together with Annulohypoxylon Y.M. Ju, J.D. Rogers, H.M. Hsieh and Daldinia Ces., De Not., are all closely associated with both dicots and, infrequently, monocots in forest ecosystems [4]. Most hypoxylaceous fungi have a strong capacity to degrade cellulose and lignin and are important elements in forest ecosystems, playing a key ecological role in carbon circulation [5]. In addition, the endophytic stages of these fungi may even benefit their host plants by protecting them from pathogens [6,7].
The type genus Hypoxylon is the largest genus in the Hypoxylaceae, with more than 200 species [8] and 1173 epithets in the Index Fungorum (http://www.indexfungorum.org/ names/names.asp, accessed on 1 November 2021). Members of the genus have a worldwide distribution, but they display a higher diversity in the tropics and subtropics [4,6,9,10].
In the 20th century, the generic concept of Hypoxylon was based only on morphological characteristics [1,4,[11][12][13][14][15]. Currently, morphological, phylogenetic, and chemotaxonomic evidence, has also been used to infer species limits in inter-and intra-genera in Hypoxylaceae [3,6,10] and to segregate some new genera such as Annulohypoxylon [16], Hypomontagnella [17], Jackrogersella, and Pyrenopolyporus [18] from the genus Hypoxylon. The genus Hypoxylon is quite common in China; however, the occurrence of the species in China has not been confirmed by molecular phylogenetic analyses, and the species diversity and distribution of the genus in China are unclear [19][20][21][22].
Hainan Tropical Rainforest National Park is located in south-central Hainan province, between 18 • 33 16 -19 • 14 16 N and 108 • 44 32 -111 • 04 43 E and has a tropical monsoon climate. More than 3577 plant species, 1142 genera, and 220 families have been reported in the rainforest park (http://www.hntrnp.com, accessed on 15 November 2021), including abundant hypoxylaceous fungi. During investigations on Xylariales from Hainan Province, China, some specimens of Hypoxylaceae were collected. These collections were carefully studied using both morphological and phylogenetic methods, and four undescribed species of Hypoxylon were identified. The aims of this study were to confirm the taxonomic status of the new species, explore the species diversity of Hypoxylon in Hainan Tropical Rainforest National Park, and infer the evolutionary relationships of the genus Hypoxylon.

Sample Sources
The studied specimens were collected from Hainan Tropical Rainforest National Park, China, in 2020. These specimens were deposited at the Fungarium of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS).

Morphological Characterization
The micromorphological observations, micrographs, and measurements were obtained using an Olympus IX73 inverted fluorescence microscope (Tokyo, Japan) with the laser capture microdissection system of model MMI CellCut Plus (Zurich, Switzerland), while the same processes for observing the morphological characteristics of stromatal sufaces and perithecia were performed using a VHX-600E microscope from the Keyence Corporation. The photographs of ascospores were examined by scanning electron microscope (SEM) (Hitachi Corporation, Tokyo, Japan). Sexual structures were microscopically observed in water, 10% KOH, and Melzer's reagent, as determined by Ju and Rogers [4]. The color codes appearing in this article refer to Rayner [23]. In the text, the following abbreviations are used: KOH = 10% potassium hydroxide, n = number of ascospores measured from a given number of specimens, M = arithmetical average of sizes of all ascospores.

DNA Extraction, PCR Amplification, and Sequencing
Following the instructions of the manufacturer, total genomic DNA of studied samples was extracted using an improved cetyltrimethylammonium bromide (CTAB) rapid extraction kit for plant genomes (Aidlab Biotechnologies, Beijing, China) and a Thermo Scientific Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA). Four DNA loci of ITS (internal transcribed spacer regions), nLSU (nuclear large subunit ribosomal DNA), RPB2 (RNA polymerase II second largest subunit), and β-tubulin (beta-tubulin) were amplified by polymerase chain reaction (PCR) using HS Taq Mix (Dongsheng Biotech, Guangzhou, China). The 40 µL PCR mixtures contained 16 µL of ddH2O, 20 µL of 2 × HS™ Mix, 2 µL of DNA template, and 1 µL of each forward and reverse primer. The primer pairs ITS5/ITS4, LR0R/LR5, fRPB2-7CR/fRPB2-5F, and T1/T22 were used to amplify ITS, LSU, RPB2, and β-tubulin, respectively [24][25][26][27][28]. The PCR thermal cycling program for ITS was set as initial denaturation at 95 • C for 3 min, followed by 30 cycles of denaturation at 94 • C for 40 s, annealing at 55.8 • C for 45 s, extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min. For generation of LSU sequence data, the following program was used: initial denaturation at 94 • C for 3 min, 36 cycles of 1 min at 94 • C, 50 s at 52 • C and 1 min at 72 • C, with a final extension period of 10 min at 72 • C. The PCR amplification of RPB2 and β-tubulin was set up as follows: initial denaturation at 95 • C for 3 min, followed by 35 cycles at 94 • C for 1 min, 52 • C for 50 s, 72 • C for 1 min, with a final extension of 72 • C for 10 min [29]. PCR sample purification and DNA sequencing were carried out by BGI, Guangzhou, China. The generated sequences were submitted to GenBank to acquire accession numbers.

Phylogenetic Analysis
All newly generated sequences and relevant sequences of closely related species within the genus Hypoxylon and among genera of the family Hypoxylaceae and some related genera of the Xylariales based on ITS, LSU, RPB2, and β-tubulin were obtained in the phylogenetic analysis ( Table 1). The other genera included Annulohypoxylon, Daldinia, Hypomontagnella, Jackrogersella, Pyrenopolyporus, Rhopalostroma, and Thamnomyces. The phylogenetic trees were rooted with Xylaria hypoxylon (L.) Grev. and Biscogniauxia nummularia (Bull.) Kuntze as outgroups.  The sequence alignment was conducted using fast Fourier transformation (MAFFT) online (http://mafft.cbrc.jp/alignment/server/, accessed on 22 November 2021) [48]. Further sequence processing was conducted using BioEdit 7.0.5, and the concatenation of four DNA loci of ITS, LSU, RPB2, and β-tubulin was completed using MEGA 6.0 [29,49,50].
After aligned by MAFFT online, the sequence datasets contained 2046 character positions for ITS alignment, 3320 character positions for LSU alignment, 1285 character positions for RPB2 alignment, and 2298 character positions for β-tubulin alignment. With less informative positions trimmed, and four DNA loci connected, the generated multi-gene alignment (MGA) had an aligned length of 3836 characters, of which 1977 characters were parsimony-informative. Phylogenetic trees generated from BI and ML analyses of the combined dataset of ITS-LSU-RPB2-β-tubulin were highly similar in topology. Only the ML tree is shown in Figure 1, with ML bootstrap values ≥ 50% and Bayesian posterior probabilities ≥ 0.95 labelled along the branches. The phylogenies reveal a paraphyly of Hypoxylon, with the genera Annulohypoxylon, Daldinia, Hypomontagnella, Jackrogersella, Pyrenopolyporus, and Thamnomyces embedded within the former. The phylogeny inferred from the ITS-LSU-RPB2-β-tubulin sequences demonstrated that the four new species, i.e., H. wuzhishanense, H. hainanense, H. chrysalidosporum, and H. cyclobalanopsidis, formed distinct well-supported lineages (Figure 1).
Most species of Hypoxylon play an important ecological role in tropical rainforests as wood-decomposers [3], and some might have benefical effects on their hosts during their endophytic life stage [65]. In addition, many species have been found to produce highly bioactive secondary metabolites [41,43,[66][67][68][69][70]. Although approximately 33 species of Hypoxylon have been recorded in China [4,[19][20][21]29,71], species diversity, evolution, population dynamics, and the host-fungus interactions of this genus are still obscure. Therefore, comprehensive studies on the diversity, phylogeny, evolution, host-fungus interactions, and secondary metabolites of the genus Hypoxylon are needed in the future.

Conclusions
The current study revealed four new taxa of Hypoxylon from Hainan Tropical Rainforest National Park based on morphological characteristics, ecological distributions, and a combined ITS-LSU-RPB2-β-tubulin phylogeny.