﻿Morphology and multigene phylogeny reveal four new Xylaria (Xylariales, Xylariaceae) species from karst region in China

﻿Abstract This study presents the identification of four novel Xylaria species, discovered in the karst region of China. The discovery was facilitated by a rigorous analysis that encompassed both morpho-anatomical features and multi-locus phylogenetics utilizing sequences from the ITS, rpb2, and TUB2 loci. The newly identified species are designated as Xylariajichuaniisp. nov., X.nanningensissp. nov., X.orientalissp. nov., and X.taiyangheensissp. nov. The distinction of these species from their known counterparts was verified through comparison of morphological features and phylogenetic analysis. The study further provides detailed morphological descriptions, illustrative representations, and a phylogenetic tree, all of which contribute to the taxonomic positioning of these novel species.


Introduction
Karst is a geological formation characterized by surface and subsurface features that result from the dissolution of soluble rocks by water.This includes morphological, hydrological, and hydrogeological elements (Marčić et al. 2022).Karst landscapes exhibit distinctive shapes, arising from a unique combination of morphological, hydrological, and hydrogeological features of the surface and subsurface, all rooted in water-soluble rocks.These karst regions and their caves constitute invaluable natural resources, harbouring a broad spectrum of ecological niches, many of which are often singular in nature (Pipan and Culver 2013;Forti 2015).Karst regions boast remarkable biodiversity, characterized by endemic species that are uniquely endemic to their respective locales.The diverse MycoKeys 108: 169-196 (2024), DOI: 10.3897/mycokeys.108.130565 Wenyu Zeng et al.: Four new Xylaria species in China habitats, unique microclimates, and isolation of karst regions contribute to this exceptional biological diversity (Jakob et al. 2022;Marčić et al. 2022;Melekhina et al. 2022;Dong et al. 2023;Mičetić 2023;Zhang et al. 2023).Within karst regions, speciation and the existence of discontinuous populations of organisms with phylogenetically distinct origins are commonplace.Being inaccessible, karst landscapes often serve as natural refuges for species that have vanished elsewhere due to hunting and habitat destruction (Li et al. 2021;Marasinghe et al. 2022;Hyde et al. 2023).These ecosystems, despite being biodiversity hotspots, remain largely unmapped.Consequently, identifying biodiversity and understanding the ecology of karst habitats is paramount, given their sensitivity to disturbance and the challenges associated with restoration efforts (Mičetić 2023).
The genus Xylaria, a member of the Xylariaceae family, comprises a highly diverse group of fungi that possess significant ecological significance.Extensive research has underscored the genus's key role as wood decomposers (Rogers 1986;Fournier et al. 2020;Ma et al. 2022a), a valuable source of bioactive secondary metabolites (Healy et al. 2004), and as endophytes residing within diverse plant species (Davis et al. 2003;Promputtha et al. 2007).Notably, Xylaria has been proven to synthesize a range of bioactive compounds exhibiting promising potential as antibiotic agents (Elias et al. 2018), thereby establishing its pharmaceutical relevance.These fungi are commonly encountered in temperate, subtropical, and tropical regions across the globe, frequently associated with wood, fallen fruits or seeds, leaves or petioles, and termite nests (Dennis 1956;Rogers and Samuels 1986;San and Rogers 1989;Ju and Hsieh 2007;Fournier 2014).Species within the genus are distinguished by their upright, stipitate, and charred stromata, characterized by perithecia that are entirely immersed (San and Rogers 1989;Stadler et al. 2013;Konta et al. 2020;Samarakoon et al. 2022).Globally, there have been reports of more than 300 species belonging to the genus Xylaria (Kirk et al. 2008), while Index Fungorum lists an even more extensive catalog of more than 800 epithets (Ma et al. 2022a).Only in China, including the karst areas of south-western China, about 115 species of woody plants have been recorded (Ma et al. 2022a;Li et al. 2024a), indicating that this genus has rich diversity and universality.
In this study, we collected Xylaria specimens from fallen leaves and twigs of plants in the Karst regions of two neighboring provinces Yunnan and Guangxi Zhuang Autonomous Region, China.We performed comprehensive morphological examinations and phylogenetic analyses to ascertain species identification and their positions within the phylogenetic tree.Our phylogenetic analysis utilized sequences from the ITS, rpb2, and TUB2 loci, employing both maximum likelihood and Bayesian frameworks.The results distinguished these specimens from other known species within the genus, leading us to propose them as new species.

Sample collection
The specimens were collected during surveys conducted in the Karst regions including Yunnan province and Guangxi Zhuang Autonomous Region of China during 2022.All related collection information, including collection time, collector, altitude, latitude and longitude, etc, were recorded.The photos of the collected materials were taken using a Canon G15 camera (Canon Corporation, Tokyo, Japan).Materials were placed in paper bags and taken to the lab for examination.They were dried using a portable fan drier.The specimens were ready for both morphological and molecular studies.The dried specimens were carefully labelled and stored in an ultra-low freezer at -80 °C for one week to eliminate any insects and their eggs.The cultures were obtained before the -80 °C treatment.All specimens were deposited at the herbarium of Guizhou Medical University (GMB) and the Herbarium of Cryptogams, Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS).Living cultures were deposited at the Guizhou Medical University Culture Collection (GMBC).

Morphological characterization and isolation
Morphological characteristics of specimens were examined, and photomicrographs were taken as described in Wu et al. (2022), Senanayake and Calabon (2020).Macroscopic characteristics were observed under an Olympus SZ61 stereomicroscope and photographed with a Canon 700D digital camera fitted to a light microscope (Nikon Ni).Samples for microscopic examination were mounted in distilled water and Melzer's reagent.More than 30 ascospores and 30 asci were measured for each sample using the Tarosoft image framework (v.0.9.0.7).The images were arranged using Adobe Photoshop CS6 (Adobe Systems, USA).Cultures were obtained from single spores method described in Senanayake and Calabon (2020).Germinating spores were observed with a Stereo Zoom microscope and transferred to potato dextrose agar (PDA; 39 g/l distilled water, Difco potato dextrose).The cultures were incubated at 25-30 °C for 4-6 weeks, with frequent observations.

DNA extraction, Polymerase Chain Reaction (PCR) amplification and sequencing
Mycelium was scraped from pure culture plates using a sterilized scalpel and was used for DNA extraction with the methods of the BIOMIGA fungus genomic DNA extraction kit.For some specimens where the ascospores did not germinate, we used a method of directly extracting DNA from the contents of the perithecium.The DNA samples were kept at -20 °C.Internal transcribed spacers (ITS), TUB2 (β-tubulin), and RNA polymerase II second largest subunit (rpb2) were amplified by PCR with primers ITS1/ITS4 (White et al. 1990;Gardes and Bruns 1993), Bt2a/ Bt2b or T1/T22 (Glass and Donaldson 1995;O'Donnell and Cigelnik 1997), and RPB2-5F/RPB2-7cR (Liu et al. 1999) respectively.The components of a 25 μL volume PCR mixture were: 9.5 μL of double distilled water, 12.5 μL of PCR Master Mix, 1 μL of each primer, and 1 μL of template DNA.Qualified PCR products were checked through 1.5% agarose gel electrophoresis stained with GoldenView, and were sent to Sangon Co., China, for sequencing (Xie et al. 2020).

Sequence alignments and phylogenetic analyses
All the obtained sequences were deposited in the GenBank (Table 1).The molecular phylogeny was inferred from a combined dataset of ITS, TUB2 and rpb2 sequences.The reference sequences retrieved from open databases originated from latest Xylaria articles (Hsieh et al. 2010;Stadler et al. 2013;Li et al. 2024a;etc) and the Blastn results of close matches and additional Xylariaceae representatives.Sequences were aligned using the MAFFT v.7.110 online program (Katoh et al. 2019) with the default settings, respectively.The alignment was adjusted manually using BioEdit v.7.0.5.3 (Hall 1999) were necessary, and used trimAl to select available DNA sequences.The maximum likelihood (ML) analysis was implemented in RAxML v.8.2.12 using the GTRGAMMA substitution model with 1,000 bootstrap replicates (Stamatakis 2014).Phylogenetic analyses were conducted using Bayesian inference in MrBayes v. 3.2.1 (Ronquist et al. 2012) online, with Markov chain Monte Carlo (MCMC) sampling in MrBayes v.3.2.2 (Ronquist et al. 2012) used to calculate posterior probabilities (PP).Six simultaneous Markov chains were run for 1,000,000 generations, and trees were sampled every 1,000 th generation.The convergence of the MCMC procedure was assessed from the effective sample size scores (all > 100) using MrBayes.The first 25% of the trees were discarded as burn-ins.The remainder was used to calculate the posterior probabilities (PPs) for individual branches.The phylogenetic tree was visualized in FIGTREE v.1.4.3 (Rambaut 2012).All analyses were run on the CIPRES Science Gateway v 3.3 webportal (Miller et al. 2010).The alignments are available in TreeBASE (www.treebase.org/treebase-web/home.html) under ID 31594 for ITS, TUB2 and rpb2 sequences.
Culture characteristics.Colonies on OA reaching 1.5-2 mm diam.after 2 weeks at 25 °C, white at first, with irregular margins, then extension spreading toward the edge of the Petri dish; the overall color is light white.

Xylaria nanningensis
Culture characteristics.Colonies on OA reaching 3-4 cm diam.after 2 weeks at 25 °C, white at first, with irregular margins, then extension spreading toward the edge of the Petri dish; the overall color is light white.

Xylaria orientalis
Culture characteristics.Colonies on OA reaching 12-15 mm diam.after 2 weeks at 25 °C, white at first, with irregular margins, then extension spreading toward the edge of the Petri dish; the overall color is light white.

Xylaria taiyangheensis
Culture characteristics.Colonies on OA reaching 1-1.5 cm diam.after 2 weeks at 25 °C white at first, with irregular margins, then extension spreading toward the edge of the petri dish; the overall color is light white.

Discussion
The phylogenetic tree (Fig. 1) reveals that Xylaria does not share a common ancestor and evolved from different ancestral lineages.The Xylaria species positions in the phylogram (Fig. 1) are consistent with previous studies (Hsieh et al. 2010;U'Ren et al. 2016;Ma et al. 2022a;Li et al. 2024a).
Phylogenetic analysis in this study reveals that Xylaria jichuanii, X. nanningensis, X. orientalis and X. taiyangheensis form a distinct cluster within the phylogenetic tree.The three folicolous species of this study Xylaria jichuanii, X. nanningensis, and X. orientalis clustered with X. amphithele, X. crinalis, X. filiformis, X. ficicola, X. diaoluoshanensis, X. fulvotomentosa, X. hedyosmicola, X. petchii, X. polysporicola and Entalbostroma erumpens, all of which are related to fallen leaves, except for X. crinalis which is wood-inhabiting (Pan et al. 2024).The wood-inhabiting species of this study X. taiyangheensis clustered with the leaf-inhabiting X. phyllocharis, forming a distinct clade in the phylogram, highlighting the possible influence of substrate on the evolution of these taxa.These findings corroborate the conclusions of Ju and Hsieh (2023) and Pan et al. (2024), affirming consistency in systematic analysis results.This may indicate that species of Xylaria, which associated on plant leaves and petioles, may have a similar evolutionary process.
The fallen leaves and petioles serve as a growth substrate for some Xylaria species.However, these species are often overlooked because of their small stromata.They typically have a small number of stromata, and multiple species may grow on the same leaf, making collection challenging (Ju and Hsieh 2023;Pan et al. 2024).Xylaria petchii C. G. Lloyd, X. diaoluoshanensis Xiao Y. Pan and X. fulvotomentosa Xiao Y. Pan, on the fallen leaves were introduced within Hainan tropical rainforest national park (Pan et al. 2024).Ju and Hsieh (2023) provided a compilation of Xylaria species associated with fallen leaves and petioles worldwide, as well as new species.A total of 44 Xylaria species found on fallen leaves and petioles have been officially documented globally.To date, fourteen taxa, including X. betulicola Hai X. Ma f. major Ciccarone, X. minuscula Y.M. Ju & H.M. Hsieh, X. diaoluoshanensis, X. fulvotomentosa and X. petchii have been discovered on fallen leaves in China (Hsieh et al. 2010;Ma et al. 2011;Zhu and Guo 2011;Huang et al. 2014Huang et al. , 2015;;Ma et al. 2022a;Ju and Hsieh 2023;Pan et al. 2024).Here three new Xylaria species associated on fallen leaves and petioles were introduced.Recent publications have shown that a high number of Xylaria species associated with fallen leaves in tropical and subtropical regions, with most findings confirming new species (Ju and Hsieh 2023;Pan et al. 2024).A more thorough examination of Xylaria species growing on leaves and petioles is needed.
The southwestern region of China boasts the world's largest karst habitat area, harbouring abundant and distinctive fungal species.Recent reports of new Xylaria species from China, particularly from Karst regions, underscore the significance of these environments as focal points of fungal diversity (Li et al. 2024a;Zhu et al. 2024).Pan et al. (2022Pan et al. ( , 2024)), Li et al. (2024aLi et al. ( , 2024b) ) and Zhu et al. (2024) reported more than 80 new species of Xylariales include 45 Xylaria species from the karst region of China.Four new pale-spored species of Xylaria were introduced from Southwest China (Ma et al. 2022a).Zhu et al. (2024) reported two new species, X. aleuriticola and X. microcarpa on fallen fruits, and one new record from south China.Through examination of fallen leaves and twigs of plants in these unique habitats, we identified four new Xylaria species from the Karst regions of Yunnan and Guangxi provinces, China.This extensive documentation underscores the remarkable diversity and ecological significance of the Xylariales in karst environments.These findings not only broaden our comprehension of Xylaria diversity in the region but also underscores the importance of continuous exploration and documentation efforts, particularly in ecologically distinctive areas such as the Karst Region in South China.

Figure 1 .
Figure 1.RAxML tree based on a combined ITS, TUB2 and rpb2 gene sequences data set.Bootstrap support values for maximum likelihood (ML) >75% and Bayesian posterior probabilities (BPP) > 0.95 are displayed above or below the respective branches (ML/BI).The newly described species are marked and red.Type materials were marked bold.

Figure 2 .
Figure 2. Xylaria jichuanii (GMB4703) A type material B stroma C surface of stroma D transverse section of stroma E longitudinal section of stroma F-H asci with ascospores I a J+, ascus apical apparatus (stained in Melzer's Reagent) J-L ascospores M colonies of Xylaria jichuanii on OA.Scale bars: 0.5 mm (C-E); 20 µm (F-L).

Figure 3 .
Figure 3. Xylaria nanningensis (GMB4702) A type material B fertile part of stroma C surface of stroma D transverse section of stroma E longitudinal section of stroma F-H asci with ascospores I a J+, ascus apical apparatus (stained in Melzer's Reagent) J-L ascospores M colonies of Xylaria nanningensis on OA.Scale bars: 0.5 mm (C-E); 10 µm (F-L).

Figure 4 .
Figure 4. Xylaria orientalis (GMB4701) A type material B stroma C surface of stroma D transverse section of stroma E longitudinal section of stroma F-H asci with ascospores I a J+, ascus apical apparatus (stained in Melzer's Reagent) J-L ascospores M colonies of Xylaria orientalis on OA.Scale bars: 0.5 mm (C-E); 15 µm (F-L).

Figure 5 .
Figure 5. Xylaria taiyangheensis (GMB4704) A type material B stroma C surface of stroma D transverse section of stroma E longitudinal section of stroma F-H asci with ascospores I a J+, ascus apical apparatus (stained in Melzer's Reagent) J-L ascospores M colonies of Xylaria taiyangheensis on OA.Scale bars: 0.5 mm (C-E); 15 µm (F-L).

Table 1 .
List of taxa used for the phylogenetic tree.GenBank accession numbers, specimen numbers, country and reference are given.Holotype specimens are labelled with HT.Ex-type cultures are labelled with ET.Species highlighted in bold were derived from this study.N/A: not available.