Three New Species of Hypoxylon (Xylariales, Ascomycota) on a Multigene Phylogeny from Medog in Southwest China

During a survey of hypoxylaceous fungi in Medog county (Tibet Autonomous Region, China), three new species, including Hypoxylon damuense, Hypoxylon medogense, and Hypoxylon zangii, were described and illustrated based on morphological and multi-gene phylogenetic analyses. Hypoxylon damuense is characterized by its yellow-brown stromatal granules, light-brown to brown ascospores, and frequently indehiscent perispore. Hypoxylon medogense is morphologically and phylogenetically related to H. erythrostroma but differs in having larger ascospores with straight spore-length germ slit and conspicuously coil-like perispore ornamentation. Hypoxylon zangii shows morphological similarities to H. texense but differs in having Amber (47), Fulvous (43) and Sienna (8) KOH-extractable pigments and larger ascospores with straight spore-length germ slit. The multi-gene phylogenetic analyses inferred from the datasets of ITS-RPB2-LSU-TUB2 supported the three new taxa as separate lineages within Hypoxylon. A key to all known Hypoxylon species from China and related species worldwide is provided.


Introduction
Polyphasic taxonomic studies based on phylogenetic, chemotaxonomic, and morphological data were extensively applied to identify species and reflect evolutionary relationships of hypoxylaceous fungi in recent years [1][2][3]. Since resurrected and emended by Wendt et al. [2], 15 genera were rearranged and recognized to Hypoxylaceae by having stromatal pigments and a nodulisporium-like anamorph. According to the arrangement of the families in Sordariomycetes by Hyde et al. [4], 19 genera were accepted in Hypoxylaceae as saprobes and endophytes. Interesting, Hypoxylon species in endophytic stages may play an important ecological role in protecting their host plants from pathogens [4], and some species are related to insect vectors [2,[5][6][7]. As the main family of Xylariales, Hypoxylaceae exhibits high diversity in tropical and subtropical areas [8][9][10][11]. In the classification system of Ju and Rogers [12], the genus Hypoxylon Bull. contains two subclades, the Annulata and Hypoxylon sections. Then they were segregated and the Annulata section was accepted as a new genus, Annulohypoxylon, based on molecular phylogenetic data inferred from ACT and TUB2 sequences [13]. Hypoxylon species are mainly saprobic on dead and decaying wood of angiospermous plants [14]. In this genus, more than 200 species with 1189 epithets included in the Index Fungorum have been reported so far [4,15,16]. Despite species of The alignment, trimming, and concatenation of sequences followed Song et al. [21]. The multi-gene phylogenetic analyses were performed by using two methods of maximum likelihood (ML) and Bayesian analyses (BA) based on ITS-LSU-RPB2-β-tubulin datasets and ITS-β-tubulin datasets. The latter was used for an added validation to the former. Maximum likelihood analyses used raxmlGUI 2.0 with 1000 bootstrap replicates and GTRGAMMA+G as a substitution model [20,31,32]. Bayesian analyses used MrBayes 3.2.6 with jModelTest 2 conducting model discrimination and Markov chain Monte Carlo (MCMC) sampling. Every 100th generation was sampled as a tree with 1,000,000 generations running for six MCMC chains [20,33]. Phylogenetic trees were viewed and edited by FigTree version 1.4.3 and Photoshop CS6. Table 1. GenBank accession numbers of sequences used in the multi-gene phylogenetic analyses. T and ET represent holotype and epitype specimens, respectively. Species in bold were derived from this study. N/A: not available.  This study selected 89 taxa from 10 genera to perform phylogenetic analysis, including 3 Annulohypoxylon spp., 2 Daldinia spp., 3 Hypomontagnella spp., 72 Hypoxylon spp., 2 Jackrogersella spp., 3 Pyrenopolyporus spp., 1 Rhopalostroma sp., and 1 Thamnomyces sp. with X. hypoxylon and B. nummularia added as the outgroups. The sequence datasets comprised 306 sequences with 91 ITS, 62 LSU, 62 RPB2, and 91 β-tubulin sequences. After being aligned and trimmed, the combined dataset contained 3530 characters including gaps with 587 characters for ITS, 867 characters for LSU, 729 characters for RPB2, and 1347 characters for β-tubulin alignment, of which 1537 characters were parsimony-informative.

Phylogenetic Analysis
The best-scoring ML tree was built with a final ML optimization likelihood value of −77,579.198447. Bayesian posterior probabilities were calculated with a final average standard deviation of split frequencies of less than 0.01. Phylogenetic trees of BA and ML analyses were found to be highly similar in topology, and the ML tree is represented in Figure 1. ML bootstrap support (BS) ≥ 50% and Bayesian posterior probabilities (PP) ≥ 0.95 were labelled along the branches, while branches with BS ≥ 70% and PP ≥ 0.98 were considered to be significant.  Mill. with full support in clade H3. The phylogenetic tree shows that Hypoxylon is a paraphyletic group with other genera embedded (e.g., Annulohypoxylon, Daldinia, and Hypomontagnella).

Taxonomy
Hypoxylon damuense Hai X. Ma, Z.K. Song and Y. Li, sp. nov., Figure 2. MycoBank: MB 843581 Diagnosis. Differs from H. rubiginosum in its larger asci, light-brown to brown ascospores with conspicuous coil-like ornamentation and most of the perispore indehiscent. Differs from H. hypomiltum in its smaller perithecia, larger asci and apical apparatus. Differs from H. wujiangense in its larger stromata and stromatal KOH-extractable pigments.
Etymology Teleomorph. Stromata pulvinate to effused-pulvinate, 1-9 cm long × 0.4-2 cm broad × 0.6-0.9 mm thick; with inconspicuous to conspicuous perithecial mounds; surface Bay (6), Rust (39) and Livid Purple (81), exposing black subsurface layer when colored coating worn off; with yellow-brown granules immediately beneath the surface and between perithecia; yielding luteous (12) and ochreous (44)  Note. Hypoxylon damuense was found in the subtropics, and characterized by large pulvinate stromata, long asci stipes, amyloid apical apparatus, light-brown to brown ascospores with straight germ slit, most of the perispore indehiscent in 10% KOH, with conspicuous coil-like ornamentation. The new species is quite similar to H. rubiginosum in ascospore dimensions and KOH-extractable pigments, but the latter has darker colored ascospores, smaller asci (100-170 µm total length), dehiscent perispores and smooth or with inconspicuous coil-like ornamentation. Hypoxylon rubiginosum sensu stricto was always discovered in the temperate northern hemisphere except for samples reported in Florida [12,15,48]. Moreover, the status of H. damuense as a new species is also supported in the phylogenetic trees, where it appears distant from H. rubiginosum.
Hypoxylon zangii Hai X. Ma, Z.K. Song and Y. Li, sp. nov., Figure 4. MycoBank: MB 843580 Diagnosis. Differs from H. fendleri and H. retpela in its smaller ascospores. Differs from H. rubiginosum in its stromatal granules and a subtropical distribution. Differs from H. texense in its stromatal KOH-extractable pigments and larger ascospores. Differs from H. guilanense in its stromatal morphology.

Discussion
In the present study, three species of Hypoxylon from Medog in China, H. damuense, H. medogense, and H. zangii, are described as new species based on molecular analyses and morphological features. Phylogenetic analyses on the species of Hypoxylon presented confirmed that Hypoxylon is a polyphyletic genus. The species analyzed appeared mainly distributed in six separate clades (except H. papillatum Ellis, Everh. and H. dieckmannii Theiss.). Hypoxylon damuense and H. zangii were clearly separated from other sampled species of Hypoxylon and from each other in the clade H2, and H. medogense was included in clade H3 containing H. fragiforme (Pers.) J. Kickx f., the type species of the genus. The phylogenetic tree shows that the classification of Hypoxylon is confusing. It did not suggest any apparent correlation in morphological features with the distribution of species in the phylogenetic trees. Therefore, more collections, more gene sequences and new taxonomic features, as well as the application of polyphasic taxonomic approaches based on morphological (sexual and asexual), chemotaxonomic, and phylogenetic data of this genus are needed in the further studies. Previously numerous new species have been found in Southwest China [49,50], and present paper confirmed that more known fungal species in the area.