﻿Three new Xylaria species (Xylariaceae, Xylariales) on fallen leaves from Hainan Tropical Rainforest National Park

﻿Abstract Three new species of Xylaria on fallen leaves in Hainan Province of China are described and illustrated, based on morphological and molecular evidence. Xylariahedyosmicola is found on fallen leaves of Hedyosmumorientale and featured by thread-like stromata with a long sterile filiform apex. Phylogenetically, X.hedyosmicola is closely related to an undescribed Xylaria sp. from Hawaii Island, USA and morphologically similar to X.vagans. Xylarialindericola is found on fallen leaves of Linderarobusta and characterised by its subglobose stromata and a long filiform stipe. It is phylogenetically closely related to X.siculaf.major. Xylariapolysporicola is found on fallen leaves of Polysporahainanensis, it is distinguished by upright or prostrate stromata and ascospores sometimes with a slimy sheath or non-cellular appendages. Xylariapolysporicola is phylogenetically closely related to X.amphithele and X.ficicola. An identification key to the ten species on fallen leaves in China is given.


Introduction
Species of Xylaria Hill ex Schrank are commonly found throughout the temperate, subtropical and tropical regions of the world, associated with wood, fallen fruits or seeds, fallen leaves or petioles and termite nests (Dennis 1956;Rogers 1986;Rogers and Samuels 1986;San Martin and Rogers 1989;Ju and Rogers 1999;Ju and Hsieh 2007;Fournier 2014). Previous studies on Xylaria have dealt primarily with species growing on wood and termite nests Ju and Hsieh 2007;Fournier et al. 2020), but the species diversity and distribution of the genus on other substrates, such as fallen fruits or seeds and fallen leaves or petioles, are still poorly studied (Hsieh et al. 2010;Ju et al. 2018). Especially, the study of Xylaria species growing on fallen leaves or petioles is far behind those mentioned taxa associated with other substrates and only seven species have been reported on those substrates in China (Dennis 1956;Rogers et al. 1988;Zhu and Guo 2011;Huang et al. 2014Huang et al. , 2015Ma and Li 2018).
Hainan Province (20°01.04'N, 110°20.95'E) is located in southern China and enjoys a tropical monsoon climate. More than 6036 plant species, 1895 genera and 243 families have been reported in the province (Yang 2015). Different kinds of tropical vegetations (e.g. Moraceae, Euphorbiaceae and Arecaceae) and rainforests are distributed over the vast territory of the province, in which abundant fungi occur (Dai et al. 2009;Dai 2012;Gao and Yang 2016;Cui et al. 2019). Two intensive surveys of xylariaceous fungi were carried out in Hainan province in 2019 and 2020 and about 400 specimens of Xylariaceae were collected. These materials have been carefully studied through both morphological and phylogenetic methods and three new species on fallen leaves were identified. The new taxa are described and illustrated, and an identification key is provided for the 10 known species of Xylaria on fallen leaves in China.

Morphological studies
Voucher specimens are deposited in the Fungarium of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS), Hainan Province, China. Samples for microscopic examination were mounted in distilled water, Melzer's reagent, India ink or 1% SDS. Microscopic features observation, measurements and photographing were performed by using a Zeiss Axio Imager A2 microscope (Göttingen, Germany) by differential interference contrast microscopy (DIG) and brightfield microscopy (BF). The photographs of stromata, perithecia and ostioles were taken with a VHX-600E stereomicroscope Keyence Corporation (Osaka Japan). The methods of collecting, preservation and identification of the specimens follow Ma and Li (2018).

DNA extraction and sequencing
A modified cetyltrimethylammonium bromide (CTAB) extraction kit (Aidlab Biotechnologies, Beijing, China) was employed for total DNA extraction from dried specimens. The ITS region was amplified with the primer pair ITS4 and ITS5 (White et al. 1990) using the following procedure: initial denaturation at 95 °C for 3 min, followed by 30 cycles of 94 °C for 40 s, 55.8 °C for 45 s and 72 °C for 1 min and a final extension of 72 °C for 10 min. The TUB and RPB2 gene region were amplified with primers T1/T22 (O'Donnell and Cigelnik 1997) and fRPB2-5F/fRPB2-7CR (Liu et al. 1999), respectively, using the following procedure: initial denaturation at 95 °C for 3 min, followed by 35 °C cycles of 94 °C for 1 min, 52 °C for 1 min and 72 °C for 1.5 min and a final extension of 72 °C for 10 min (Hsieh et al. 2005). DNA sequencing was performed at BGI tech (Guangzhou, China) and sequences were deposited in GenBank (Table 1).

Phylogenetic analyses
The molecular phylogeny was inferred from a combined dataset of ITS, TUB and RPB2 sequences.  Table 1).
A combined matrix of ITS-RPB2-TUB and ITS-exons of TUB and RPB2 were used to construct phylogenetic analysis by two methods including maximum likelihood (ML) and Bayesian Inference (BI) analysis, respectively. ML tree generation and bootstrap analyses were performed via the programme RAxML7.2.6 (Stamatakis 2006) running 1000 replicates combined with a ML search. Bayesian analysis was performed with MrBayes 3.1 (Huelsenbeck and Ronquist 2005) implementing the Markov Chain Monte Carlo (MCMC) technique and parameters predetermined by MrModeltest 2.3 (Nylander 2004).

Molecular phylogeny
This study used genetic sequences of 57 species, including 69 ITS sequences, 57 TUB sequences and 54 RPB2 sequences. We applied two tree construction methods to improve the reliability of the results.
After the alignment sequence was adjusted using MAFFT, the ITS alignment, shown in BioEdit 7.0.5, consisted of 778 character positions, 2219 in the TUB alignment and 1241 in the RPB2 alignment. After curing, the constructed multigene alignment (MGA) consisted of 3138 characters (523 of which were derived from the ITS alignment, 1550 from TUB alignment, 1065 from RPB2 alignment). Of the MGA, 1354 characters were considered parsimony-informative.
The analysis results show that the phylogenetic tree, generated by ML in RAxML7.2.6, is basically the same as that generated by BI in MrBayes 3.1. Topology of the phylogenetic analyses, based on ITS-RPB2-TUB and ITS-exons of TUB and RPB2, have no significant conflicts. Only the BI tree is shown in Figure 1 with Bayesian posterior probabilities ≥ 0.95 and ML bootstrap values ≥ 50% labelled along the branches. The phylogenetic tree showed that X. hedyosmicola is clustered with Xylaria sp. 6, X. polysporicola is clustered with X. amphithele Diagnosis. Differs from X. vagans by its stromata without a black rhizomorphoid mycelium connecting dead leaves, larger ascospores and tubular to slightly urn-shaped apical apparatus. Differs from X. betulicola by its smaller stromta and larger ascospores.
Remarks. Xylaria hedyosmicola closely resembles X. vagans Petch by sharing threadlike or long hair-like stromata bearing closely packed or scattered perithecia with a long sterile filiform apex. Xylaria vagans was originally described and illustrated by Petch (1915) from Sri Lanka. However, based on comparisons of the descriptions and illustrations, there were some differences between the two species. Xylaria hedyosmicola has larger sporiferous part of asci (70-100 µm × 8-12 µm) with tubular to slightly urnshaped apical apparatus bluing in Melzer's reagent, brown and larger ascospores with straight ( Fig. 2n and p) to slightly sigmoid germ slit (Fig. 2o), with narrowly rounded ends and a slimy sheath on ventral side swollen at both ends to form rounded noncellular appendages, while X. vagans has a black rhizomorphoid mycelium connecting dead leaves, smaller sporiferous part 68-72 µm × 6 µm and black-brown, cymbiform, smaller ascospores 9-12 × 5-6 µm, with broadly rounded ends and is without apical apparatus, germ slit and sheath or appendages (Petch 1915). Unfortunately, the molecular sequences of X. vagans from Sri Lanka were not available.
Xylaria betulicola Hai X. Ma, Lar.N. Vassiljeva & Yu Li is similar to X. hedyosmicola in stromatal morphology, but differs in having larger stromata 3-7 cm, slightly smaller  ascospores (11.5)12-14(15) × 5-6 µm, without sheath or appendages (Ma and Li 2018). In the phylogenetic tree, X. hedyosmicola formed a fully supported clade with Xylaria sp. 6 from Hawaiian Islands, USA (Hsieh et al. 2010). Although there are no descriptions on Xylaria sp. 6 in the study of Hsieh et al. (2010), we suspected that it is conspecific with X. hedyosmicola. The sequences comparison showed that there are 98.7%, 99% and 99.9% maximal percentage identities, respectively in ITS, TUB and RPB2 between X. hedyosmicola (FCATAS 856) and Xylaria sp. 6 from USA (JDR 258). Diagnosis. Differs from X. sicula f. major by its subglobose stromata without a long sterile apex, larger ascospores and host plant. Differs from X. hypsipoda by its black stromata, glabrous stipes and smaller apical apparatus.
Xylaria polysporicola is somewhat similar to X. amphithele F. San Martín & J.D. Rogers in shape and size of apical apparatus and ascospores. However, X. amphithele has globose to conical stromata with 3-4 to 20 naked perithecia (San Martín and Rogers 1989). In the phylogenetic tree, X. polysporicola formed a lineage close to X. amphithele and X. ficicola, but is distant from X. phyllocharis.

Discussion
We included ten Xylaria species on fallen leaves in the phylogenetic analyses of the present study. Except for X. phyllocharis, the other nine studied species formed a monophyletic clade with two wood-inhabiting species, X. muscula Lloyd and X. crinalis Hai X. Ma, Lar. N. Vassiljeva & Yu Li, in our phylogenetic tree (Figure 1). In China, only three species have been previously reported with molecular evidence: X. ficicola from tropical Yunnan, X. sicula f. major from tropical Taiwan and X. betulicola from temperate Jilin (Ma and Li 2018). Within the clade, X. meliacearum, associated with petioles and infructescence of Guarea guidonia, formed a separate branch from other Xylaria species on other leaves. In Hsieh et al. (2010), X. phyllocharis grouped with the wood-inhabiting Xylaria species, which did not reveal any contradictions in our tree. Three species, X. polysporicola, X. amphithele and X. ficicola formed a highly supported clade. Morphologically, these species have some similar features, such as ascospores with slimy sheath or non-cellular appendages, inverted hat-shaped or urnshaped apical apparatus (San Martín and Rogers 1989;Ma et al. 2020). As Xylaria hedyosmicola formed a fully supported clade with Xylaria sp. 6, the two species should be the same, based on the ITS-TUB-RPB2 (Hsieh et al. 2010). Xylaria lindericola, on leaves of Lindera robusta formed a sister lineage to X. sicula f. major on unknown fallen leaves with high bootstrap value 100%. Xylaria muscula, growing on dead branches, formed a weakly supported branch with X. lindericola and X. sicula f. major associated with fallen leaves in our tree. This may be because our phylogenetic analysis did not include more taxa related to X. muscula.
Until now, ten taxa, X. betulicola, X. diminuta F. San Martín & J.D. Rogers, X. ficicola, X. foliicola G. Huang & L. Guo, X. hainanensis Y.F. Zhu & L. Guo, X. hedyosmicola, X. jiangsuensis G. Huang & L. Guo, X. lindericola, X. polysporicola and X. sicula f. major have been found on fallen leaves in China (Hsieh et al. 2010;Ma et al. 2011;Zhu and Guo 2011;Huang et al. 2014Huang et al. , 2015Ma and Li 2018). Amongst these species, X. diminuta, originally reported from Mexico, was found in Yunnan province of China in 2013 (Huang et al. 2014). Xylaria sicula f. major was first described from Sicily in 1878 and then found in Spain, Kenya, Sardinia, and Taiwan province of China (Hsieh et al. 2010;Fournier 2014). Unfortunately, except for the three species in this study, the molecular data of the other Xylaria species from China were not available. We anticipate that additional species of Xylaria on fallen leaves will be discovered as more studies are conducted.