Multi-Locus Phylogeny and Morphology Reveal Two New Species of Hypoxylon (Hypoxylaceae, Xylariales) from Motuo, China

Hypoxylaceous fungi are abundant in China, but their discovery and report are uneven in various provinces, with more fungi in Yunnan and Hainan and fewer fungi in Tibet. During the investigation of macro-fungi in Motuo county, Tibet Autonomous Region, we collected a number of xylarialean specimens. Six hypoxylaceous specimens growing on dead angiosperm were collected from the forests of Motuo county, and they were described and illustrated as two new species in Hypoxylon based on a combination of morphological characters and molecular evidence. Hypoxylon diperithecium was characterized by its bistratal perithecia, purple-brown stromatal granules, citrine to rust KOH-extractable pigments, and light brown to brown ascospores ellipsoid-inequilateral with conspicuous coil-like ornamentation. Hypoxylon tibeticum was distinct from other species by having pulvinate and applanate stromata, surface vinaceous, with orange granules, orange KOH-extractable pigments, and brown ascospores with inconspicuous ornamentation. The multi-gene phylogenetic analyses (ITS-LSU-RPB2-TUB) supported the two new taxa as separate lineages in the genus Hypoxylon. A key to all known Hypoxylon taxa from China is provided.


Introduction
Motuo county, between 27 • 33 -29 • 55 N and 93 • 45 -96 • 05 E, is located in the southeastern Tibet Autonomous Region of southwestern China, and it covers an area of 34,000 square kilometers [1][2][3].The area enjoys the tropical monsoon rainforest and subtropical humid monsoon climate and is one of the most abundant regions of light, heat and water [4,5].Its complex topography and diverse habitat abound with different kinds of biological resources, and the area has long been reputed as the "world's biological gene bank".There are extremely abundant animal and plant resources, and more than 3000 plant species, 850 genera and 230 families have been reported in the county (http://www.motuo.gov.cn/,accessed on 18 September 2023) [6][7][8][9][10].Due to severe climatic conditions and inconvenient transportation, few investigations and studies of macro-fungi diversity have been carried out in Motuo county.In the past, about 200 species of macro-fungi have been reported in Motuo county [11][12][13][14][15][16][17][18], among which four species are pyrenomycetous fungi [12].In recent years, some new species and new records of pyrenomycetous fungi have been discovered in the area, e.g., Eutypella motuoensis Hai X. Ma [19][20][21].
In order to further understand the diversity of macro-fungi in Motuo county, we carried out a field survey focusing on xylarialean fungi in September 2021.We collected a number of xylarialean specimens, including Annulohypoxylon, Daldinia, Diatrype, Eutypella, Neoeutypella, Hypoxylon, Jackrogersella, and Xylaria.Hypoxylon Bull.(Hypoxylaceae, Ascomycota) was established by Bulliard in 1791 and typified with H. fragiforme (Pers.)J. Kickx f. [22,23].The type genus is the largest genera in the family Hypoxylaceae, with more than 200 species accepted [24][25][26] and 1188 epithets in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp,accessed on 22 September 2023).Most taxa of the genus are mainly associated with angiosperm wood as saprotrophs and endophytes, and degrade cellulose and lignin, which play a key role in the substance circulation of a forest ecosystem [24,[27][28][29][30][31].Currently, the placement of Hypoxylon and related genera in Hypoxylaceae is confusing because many are polyphyletic [32][33][34][35][36].In order to further understand the species diversity and phylogeny of Hypoxylaceae, we carried out complete morphological and multi-gene phylogenetic studies on these specimens from Motuo county.In this study, two new species are introduced based on morphological and phylogenetic evidence.

Morphological Studies
The studied specimens were deposited at the Fungarium of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS).Morphological observations and measurements in this study followed Ma et al. (2018) [24] and Song et al. (2022) [37].The morphology of stromata and perithecia were observed and measured by a VHX-6000 microscope (Osaka, Japan).Microscopic characteristics, measurements and photographs of the teleomorph were made from slide preparations of fresh stromata mounted in water, 10% KOH and Melzer's reagent.Sections were observed at a magnification up to ×1000 by using an Olympus IX73 inverted fluorescence microscope (Olympus, Tokyo, Japan).The ornamentation of ascospores were observed with a scanning electron microscope (SEM) (Phenom Corporation, Rotterdam, The Netherlands).The colors were described based on the color-codes by Rayner (1970) [38].The following abbreviations were used: KOH = 10% potassium hydroxide, n = number of measuring objects, M = average of sizes of all measuring objects.

Phylogenetic Analysis
The phylogeny of Hypoxylon and related genera based on a combined ITS-nrLSU-RPB2-TUB dataset included 98 ITS, 64 nrLSU, 65 RPB2 and 95 TUB sequences from 97 specimens representing 93 taxa.There were 2852 character positions for ITS alignment, 3462 character positions for LSU alignment, 1288 character positions for RPB2 alignment, and 2225 character positions for TUB alignment.The dataset of four DNA loci had an aligned length of 3538 characters, of which 1520 characters were parsimony informative.
Additional specimens examined.China: Tibet Autonomous Region, Motuo County, Damu Township, Kabu Village, 29 •  Note.Some stromata of Hypoxylon diperithecium have two layers perithecia visible, and the upper and the lower may be same species according to morphology of ascospore and perithecia; this feature is similar to H. subgilvum Berk.& Broome.Hypoxylon subgilvum has three stromatal layers with the basal layer an effete Biscogniauxia, and other two layers are considered the same species [23,68].Morphologically, H. subgilvum can be distinguished from H. diperithecium by its orange red stromatal granules, KOH-extractable pigments orange, and brown to dark brown ascospores [23].Moreover, molecular evidence supported H. diperithecium as a distinct species from H. subgilvum (Figure 1).
Etymology.The epithet tibeticum (Lat.)refers to the locality (Tibet Autonomous Region) of the type specimens.

Figure 1 .
Figure 1.BI phylogenetic tree of the genus Hypoxylon inferred from multi-gene alignment of ITS-LSU-RPB2-TUB.ML bootstrap support (BS) ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.95 are given at the nodes in this order.New species in this study are indicated in bold.

Figure 1 .
Figure 1.BI phylogenetic tree of the genus Hypoxylon inferred from multi-gene alignment of ITS-LSU-RPB2-TUB.ML bootstrap support (BS) ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.95 are given at the nodes in this order.New species in this study are indicated in bold.

Table 1 .
GenBank accession numbers of sequences used in phylogenetic analyses are presented.