Four New Pale-Spored Species of Xylaria (Xylariaceae, Xylariales) with a Key to Worldwide Species on Fallen Fruits and Seeds

Simple Summary Xylaria, a large, complex, and cosmopolitan genus of Ascomycota, are known as a source of bioactive secondary metabolites with antibacterial, antioxidative, anti-carcinogenic, and other properties. The species of this genus usually grow on decayed wood, fallen fruits or seeds, fallen leaves or petioles, and termite nests. The present paper describes species of Xylaria associated with fruits and seeds using morphological and multigene phylogenetic analyses based on specimens collected in Southwest China. There are few detailed reports on Xylaria taxonomy from China, especially on the species associated with fallen fruits and seeds. In this study, we describe four new species from the genus Xylaria with pale-colored ascospores on fallen fruits. They are described, illustrated, and compared with morphologically similar species, and their nucleotide sequences of ITS, RPB2, and β-tubulin were obtained and analysed. Our study reports new species of Xylaria with pale-colored ascospores associated with fallen fruits and seeds in China for the first time. Abstract Xylaria, a large and cosmopolitan genus of Ascomycota, plays an important ecological role in forest ecology as wood-decomposers, and serve as a source of bioactive secondary metabolites. The present work concerns a survey of Xylaria from Southwest China. Four new species of Xylaria with pale-colored ascospores associated with fallen fruits and seeds are described and illustrated based on morphological and phylogenetic evidences. The phylogeny inferred from a combined dataset of ITS-RPB2-β-tubulin sequences supports these four species as distinct species. The four new taxa, namely Xylaria rogersii, X. schimicola, X. theaceicola, and X. wallichii, are compared and contrasted against morphologically similar species. A dichotomous identification key to all the accepted species of Xylaria associated with fallen fruits and seeds is given.


Introduction
The genus Xylaria Hill ex Schrank is one of the most complex and difficult genera in the Xylariaceae. Stromata morphology of many species often vary greatly in color, size, and even in general shape with stages of development. The genus is widely distributed in tropical, subtropical, and temperate regions. More than 300 Xylaria species have been reported in the world [1], and more than 800 epithets are listed in Index Fungorum (http://www.indexfungorum.org/, accessed on 1 March 2022) [2]. Xylaria species are

Sample Collection and Morphological Study
Field sampling trips in nature reserves and forest parks in tropical and subtropical regions of Southwest China were carried out by the authors. The photos of the materials were taken with a Canon camera G15 (Canon Corporation, Tokyo, Japan). Fresh specimens were dried with a portable drier (manufactured in China). Dried specimens were labeled and stored in an ultra-low freezer at −80 • C for 1 week to kill insects and their eggs, and then they were ready for morphological and molecular studies. Voucher specimens are deposited in the Fungarium of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS).
Microscopic features and measurements were made from slide preparations mounted in water, Melzer's iodine reagent, 5% KOH, 1% SDS, and Indian ink. The average range of ascospore size refers to more than 95% of spores, and the extreme values are given in parentheses. In the text, the following abbreviations are used: L = mean ascospore length (arithmetical average of all ascospores); W = mean ascospore width (arithmetical average of all ascospores); M = L × W; Q = L/W ratio; n (a/b) = number of ascospores (a) measured from number of specimens (b). The photographs of asci, ascus apical apparatus, and ascospores were examined by differential interference microscopy (DIC) and bright field microscopy (BF) with a Zeiss Axio Scope A1 (Zeiss Corporation, Oberkochen, Germany) and a scanning electron microscope (SEM) (Hitachi Corporation, Tokyo, Japan), respectively. Stromatal surface and perithecia were observed and photographed using a VHX-600E microscope of the Keyence Corporation (Osaka, Japan). Color codes and names followed Rayner [25].

Molecular Procedures and Phylogenetic Analyses
Total DNA from herbarium specimens was extracted using a cetyltrimethylammonium bromide (CTAB) rapid extraction kit for plant genomes (Aidlab Biotechnologies, Beijing, China) according to the manufacturer's instructions. Target regions of the ITS rDNA, RPB2, and β-tubulin, were amplified by polymerase chain reaction (PCR) using TaKaRa Taq (TaKaRa Bio, Kusatsu, Japan) and fungal specific primers. Approximately 500 base pairs of the ITS region were amplified with primers ITS5 and ITS4 [26], using the following procedure: initial denaturation at 98 • C for 5 min, followed by 30 cycles of 95 • C for 1 min, 55 • C for 1 min, and 72 • C for 2 min, and a final extension of 72 • C for 10 min. For the RPB2 gene, about 1200 base pairs were amplified with primers fRPB2-5F and fRPB2-7cR [27], using the following procedure: initial denaturation at 95 • C for 5 min, followed by 30 cycles of 95 • C for 1 min, 55 • C for 2 min, and 72 • C for 2 min, and a final extension of 72 • C for 10 min. For the β-tubulin gene, about 1500 base pairs were amplified with primers T1 and T22 [28], using the following procedure: initial denaturation at 95 • C for 2 min, followed by 30 cycles of 95 • C for 1 min, 54-45 • C for 1.5 min, and 72 • C for 2 min, and a final extension of 72 • C for 10 min [29]. DNA sequencing was performed at BGI tech (Guangzhou, China), and all the newly generated sequences were submitted to GenBank (Table 1).
Two separate datasets, the concatenated ITS-RPB2-β-tubulin sequences of Xylaria and related genera in the family Xylariaceae, and ITS-only sequences of Xylaria from GenBank, were analyzed. Poronia pileiformis (Berk.) Fr. was selected as an outgroup [30]. The sequences of ITS, RPB2, and β-tubulin were aligned separately using the MAFFT V.7 online server (https://mafft.cbrc.jp/alignment/server/, accessed on 12 March 2022) [31] with the G-INS-i iterative refinement algorithm, and rechecked and improved manually using BioEdit v. 7.0.5.2 [32]. Phylogenetic analyses were carried out with maximum likelihood (ML) and Bayesian inference (BI) analysis, respectively. The ML analysis was performed using RaxML v.8.2.10 [33] with the bootstrap values obtained from 1000 replicates and the GTRGAMMA model of nucleotide evolution. The BI was performed using MrBayes 3.2.6 [34]. ITS sequences were inferred and used to confirm the Xylaria species identification carried out in the study. Phylogenetic trees were viewed in FigTree version 1.4.2 [35].

Molecular Phylogenetic Analysis
Ten ITS, six RPB2, and seven β-tubulin sequences were generated from this study. The concatenated ITS-RPB2-β-tubulin dataset contained 82 sequences from each gene obtained from 82 samples representing 80 xylariacean taxa and the outgroup ( Table 1). The concatenated dataset had an aligned length of 2807 characters, of which 1718 were parsimony-informative. Phylogenetic trees generated from BI and ML analyses of the combined dataset of ITS-RPB2-β-tubulin were highly similar in topology. Only the ML tree is shown in Figure 1 with Bayesian posterior probabilities ≥0.95 and ML bootstrap values ≥ 50% labeled along the branches, while the tree generated by BI analysis is provided in supplementary materials ( Figure S1). The ITS dataset contained 68 ITS sequences, representing 62 Xylaria taxa with 382 characters, of which 238 were parsimony-informative, and the ML tree is shown in Figure 2.

Discussion and Conclusions
Previous investigations have discovered several new species in Southwest China [43,44], and the current study confirmed the unexplored species diversity of the area. Here, four pale-spored Xylaria species from Southwest China were introduced as new taxa based on morphological characteristics, host association, and phylogenetic analyses. Combined ITS, RPB2, and β-tubulin sequence data of a representative sample of the entire genus showed that the four new species are distributed in two distinct lineages of the phylogenetic tree. Considering all known species associated with fallen fruits and seeds, fructicolous taxa formed clusters in three different clades. This suggests that the fructicolous life style of Xylaria species has evolved independently several times within the genus Xylaria. Moreover, the texture of the fruits or seeds may have promoted or influenced speciation, as reflected by the phylogenetic relationships of Xylaria species associated with fallen fruits and seeds. To further test this hypothesis, it is crucial to carry out additional studies and confirm the phylogenetic position of all Xylaria species associated with fallen fruits and seeds.
Many xylariacean endophytes are a source of bioactive secondary metabolites with antibacterial, antioxidative, anti-carcinogenic, and other properties [45,46]. Unfortunately, we could not obtain cultures from these isolates, and thus, they were not accessible for phylogenetic studies. Future research should include additional specimens of Xylaria from different hosts and substrates using an integrative approach including morphological, chemotaxonomic, and phylogenetic data.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biology11060885/s1, Figure S1: Phylogenetic tree of Xylaria and related genera based on the multigene alignment of ITS-RPB2-β-tubulin in the Bayesian tree. Bayesian posterior probabilities (PP) ≥ 0.95 are labeled above or below the respective branches. Species in bold were sequenced in this study.  Data Availability Statement: All newly generated sequences were deposited in GenBank (https: //www.ncbi.nlm.nih.gov/genbank/, accessed on 7 March 2022; Table 1). Data for all new taxa were deposited in MycoBank (https://www.mycobank.org/, accessed on 5 March 2022; MycoBank identifiers follow new taxa).