Multi-gene phylogenetic and taxonomic contributions to Xylaria (Ascomycota) associated with fallen fruits from China
Morphological and phylogenetic analyses on samples of Xylaria species associated with fallen fruits from China were carried out, and two new species were described, namely X. aleuriticola and X. microcarpa. Xylaria aleuriticola is found on fallen fruits of Aleurites moluccana, and characterized by stromata dichotomously branched several times with long acute sterile apices, fertile parts roughened with perithecia and tomentose, and ellipsoid to fusiform ascospores. Xylaria microcarpa differs in its very small stromata with dark brown tomentum, light brown ascospores with an inconspicuous straight germ slit, and grows on leguminous pods. The differences between the new species and morphologically similar species are discussed. Phylogenetic analyses on ITS-RPB2-TUB sequences confirmed that the two species are clearly separated from other species of the genus Xylaria. Xylaria liquidambaris is reported as a new record from China. A key to the Xylaria species associated with fallen fruits and seeds reported from China is provided to facilitate future studies of the genus.
Introduction
Xylaria Hill ex Schrank, with over 878 epithets listed in Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, accessed on 22 November 2023), was currently the largest genus in the family Xylariaceae (Hsieh et al. 2010; Fournier et al. 2018a). The members of Xylaria have a worldwide distribution, but they are highly diverse in the tropics and subtropics (Dennis 1956; Ju and Rogers 1999; Ju and Hsieh 2007; Lodge et al. 2008; Fournier et al. 2011; Wangsawat et al. 2021). Species of Xylaria are saprobic, pathogenic, or endophytic and associated with a wide range of host (Rogers 1979a; Vannini et al. 1996; Whalley 1996; Crozier et al. 2006; Thomas et al. 2008; U’Ren et al. 2009; Vega et al. 2010). According to the substrate that these fungi grow, the taxa of the genus can be divided into four different ecological types, viz., wood-inhabiting type, termite nests inhabiting type, foliicolous type, and fructicolous/seminicolous type. The Xylaria species associated with fallen fruits and seeds preferred to somewhat substrate-specific (Rogers 1979b; Læssøe and Lodge 1994; Ju et al. 2018; Perera et al. 2020).
The generic concept of Xylaria was traditionally based on morphological studies (Dennis 1957, 1958; Rogers et al. 1987, 1988; San Martín and Rogers 1989; Fournier 2014; Fournier et al. 2020). In the past two to three decades, molecular phylogenetic analysis was carried out on the family Xylariaceae by using a single-gene to multi-gene (Lee et al. 2000; Bahl et al. 2005; Ju et al. 2007; Peláez et al. 2008; Hsieh et al. 2010; Læssøe et al. 2013; Wangsawat et al. 2021). Nuclear ribosomal DNA, ITS-5.8S, and protein-coding gene are commonly used for inferring phylogenetic relationships (Tang et al. 2009; Visser et al. 2009). The new genus Neoxylaria was segregated from Xylaria based on morphological and phylogenetic evidence (Konta et al. 2020). The genus Xylaria is quite common in China, however, molecular studies on the Xylaria are still poorly used (Teng 1963; Tai 1979; Li and Li 1994; Xu 1999; Zhu and Guo 2011; Ma et al. 2011, 2013). Especially, the phylogenetic relationships inferring from multi-gene between Xylaria species associated with fruits and other Xylaria species as well as other genera in the Xylariaceae remain unsolved, and the species diversity and geographical distribution in China are unclear.
During the investigation of xylariaceous taxa from China, 18 samples belonging to 3 species of Xylaria associated with fruits were collected. Based on morphological and multi-gene phylogenetic evidences, two new species and one new Chinese record are introduced in this study.
Materials and methods
Sample collection and morphological studies
The studied samples were collected from south China during 2013–2020. The fallen fruits bearing xylariaceous stromata were dried with a SX-770 portable drier of Foshan Taomeihui Electric Appliance Co., Ltd (Guangdong, China), and deposited in the Fungarium of Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS). Macro- and micro-morphological studies were carried out in this study and followed Ma et al. (2022). Stromatal surface and perithecia were observed and measured by using a VHX-600E 3D microscope of the Keyence Corporation (Osaka, Japan). Microscopic observations and examinations were performed by using an Olympus IX73 inverted fluorescence microscope (Tokyo, Japan) and the CellSens Dimensions Software (Olympus, Tokyo, Japan). In presenting ascospore size variation, 5% of measurements were excluded from each end of the range and given in parentheses. The following abbreviations were used: KOH = 5% potassium hydroxide, SDS = 1% sodium dodecyl sulfate, M = mean ascospore length × mean ascospore width, Q = the ration of mean ascospore length / mean ascospore width, n (a/b) = number of ascospores (a) measured from number of specimens (b). Colour terms followed Rayner (1970).
DNA extraction, amplification, and sequencing
Total genomic DNA was extracted from dried specimens using a cetyltrimethylammonium bromide (CTAB) rapid extraction kit (Aidlab Biotechnologies, Beijing) following its instruction with some modifications as in Song et al. (2022). Three DNA gene fragments, the internal transcribed spacer (ITS) region, RNA polymerase II subunit (RPB2) gene, and β-tubulin (TUB) were amplified using the primer pairs ITS5/ITS4 (White et al. 1990), fRPB2-5F/fRPB2-7cR (Liu et al. 1999), and T1/T22 (O' Donnell et al. 1996), respectively. The PCR procedures for the three sequences followed Pan et al. (2022). Newly generated sequences were uploaded on GenBank and listed in Table 1.
Phylogenetic analyses
Xylaria species associated with fallen fruits and seeds were subjected to phylogenetic analyses in other various species of Xylaria and closely related genera including Amphirosellinia, Astrocystis, Kretzschmaria, Nemania, Podosordaria, and Rosellinia (Table 1). Poronia pileiformis (Berk.) Fr. was selected as an outgroup (Wangsawat et al. 2021; Ma et al. 2022).
The sequences of ITS, RPB2 and TUB2 were aligned individually using the online MAFFT tool (http://mafft.cbrc.jp/alignment/server/index.html), and improved manually using BioEdit 7.0.5.3 (Hall 1999) and ClustalX 1.83 (Thompson et al. 1997). The individual gene data sets were concatenated using the MEGA 6.0 (Tamura et al. 2011). The concatenated data set of ITS, RPB2 and TUB (ITS-RPB2-TUB) data set of studied species were carried out using Bayesian inference (BI) and maximum likelihood (ML) analyses. Maximum likelihood (ML) analysis was conducted by raxmlGUI 2.0 using rapid bootstrapping with 1000 replicates, and GTRGAMMA+G as a substitution model (Felsenstein 1981). Bayesian inference (BI) analysis was performed in MrBayes 3.2.6 with jModelTest 2 conducting model discrimination (Huelsenbeck and Ronquist 2001). Six simultaneous Markov chains were run from random starting trees for 1 million generations, and trees were sampled every 1000th generations. The first 25% of sampled trees were discarded as burn-in, and the remaining were used to calculate the posterior probability (PP) of each branch (Larget and Simon 1999).
Results
Molecular phylogeny
The eighteen Xylaria species associated with fallen fruits and seeds that were subjected to phylogenetic analyses based on ITS-RPB2-TUB dataset in Xylariaceae. The BI and ML analyses generated highly similar topologies, the ML tree is presented with bootstrap values ≥ 75% and Bayesian posterior probabilities ≥ 0.90 respectively (Figure 1).
In the phylogenetic tree (Figure 1), the genus Podosordaria separated from other genera, Amphirosellinia, Astrocystis, Kretzschmaria, Nemania, and Rosellinia were nested within Xylaria clade. All Xylaria species associated with fallen fruits and seeds were distributed within clade HY or clade PO as shown in Hsieh et al. (2010) and Ma et al. (2022). In HY clade, a Xylaria species on fruits of Fagus longipetiolata and four known Xylaria species associated with pericarps of fruits, including X. schimicola Hai X. Ma & Yu Li, X. theaceicola Hai X. Ma & Yu Li, X. wallichii Hai X. Ma & Yu Li and X. liquidambaris J.D. Rogers, Y.M. Ju & F. San Martín, formed a subclade with high support values (BS=88, PP=1.00). In the PO clade, the new species X. aleuriticola on fruits of Aleurites moluccana and X. microcarpa on pods grouped with six fructicolous Xylaria species including X. aethiopica J. Fourn., Y.M. Ju, H.M. Hsieh & U. Lindem., X. ianthinovelutina (Mont.) Fr., X. culleniae Berk. & Broome, X. fabaceicola R.H. Perera, E.B.G. Jones & K.D. Hyde, X. vivantii Y.M. Ju, J.D. Rogers, J. Fourn. & H.M. Hsieh, X. rogersii Hai X. Ma & Yu Li, and X. juruensis Henn. on Arenga engleri in a subclade with high support values (BS=100, PP=1.00).
