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Research Article
Three new Xylaria species (Xylariaceae, Xylariales) on fallen leaves from Hainan Tropical Rainforest National Park
expand article infoXiao-Yan Pan‡§, Zi-Kun Song‡|, Zhi Qu‡, Tie-Dong Liu§, Hai-Xia Ma‡
‡ Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China
§ Hainan University, Haikou, China
| Jilin Agricultural University, Jilin, China
¶ Hainan Institute for Tropical Agricultural Resources, Chinese Academy of Tropical Agricultural Sciences, Haikou, China

Corresponding author: Tie-Dong Liu ( liu@hainanu.edu.cn )

Corresponding author: Hai-Xia Ma ( mahaixia@itbb.org.cn )

Academic editor: Thorsten Lumbsch
© 2022 Xiao-Yan Pan, Zi-Kun Song, Zhi Qu, Tie-Dong Liu, Hai-Xia Ma.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Pan X-Y, Song Z-K, Qu Z, Liu T-D, Ma H-X (2022) Three new Xylaria species (Xylariaceae, Xylariales) on fallen leaves from Hainan Tropical Rainforest National Park. MycoKeys 86: 47-63. https://doi.org/10.3897/mycokeys.86.71623
Open Access

Abstract

Three new species of Xylaria on fallen leaves in Hainan Province of China are described and illustrated, based on morphological and molecular evidence. Xylaria hedyosmicola is found on fallen leaves of Hedyosmum orientale 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. Xylaria lindericola is found on fallen leaves of Lindera robusta and characterised by its subglobose stromata and a long filiform stipe. It is phylogenetically closely related to X. sicula f. major. Xylaria polysporicola is found on fallen leaves of Polyspora hainanensis, it is distinguished by upright or prostrate stromata and ascospores sometimes with a slimy sheath or non-cellular appendages. Xylaria polysporicola is phylogenetically closely related to X. amphithele and X. ficicola. An identification key to the ten species on fallen leaves in China is given.

Keywords

Folicolous fungi, Phylogeny, Pyrenomycetes, Taxonomy

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 (Rogers et al. 2005; 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. 2014, 2015; Ma 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.

Materials and methods

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).

Table 1.
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Species, specimens and GenBank accession number of sequences used in this study. New species and sequences are set in bold.

Taxon Substrate / Origin Specimen No. GenBank No. Reference
ITS TUB RPB2
Xylaria acuminatilongissima termite nests / China Taiwan HAST 623 EU178738 GQ502711 GQ853028 Hsieh et al. (2010)
X. adscendens wood / Guadeloupe HAST 570 GU300101 GQ487708 GQ844817 Hsieh et al. (2010)
X. allantoidea trunk / China Taiwan HAST 94042903 GU324743 GQ502692 GQ848356 Hsieh et al. (2010)
X. amphithele dead leaves / Guadeloupe HAST 529 GU300083 GQ478218 GQ844796 Hsieh et al. (2010)
X. apoda bark / China Taiwan HAST 90080804 GU322437 GQ495930 GQ844823 Hsieh et al. (2010)
X. arbuscula bark / China Taiwan HAST 89041211 GU300090 GQ478226 GQ844805 Hsieh et al. (2010)
X. arbuscula var. plenofissura. wood / China Taiwan HAST 93082814 GU339495 GQ478225 GQ844804 Hsieh et al. (2010)
X. atrodivaricata termite nests / China Taiwan HAST 95052001 EU178739 GQ502713 GQ853030 Hsieh et al. (2010)
X. badia bamboo culm / China Taiwan HAST 95070101 GU322446 GQ495939 GQ844833 Hsieh et al. (2010)
X. bambusicola bamboo culm / Thailand JDR 162 GU300088 GQ478223 GQ844801 Hsieh et al. (2010)
X. berteri bark / USA JDR 256 GU324750 GQ502698 GQ848363 Hsieh et al. (2010)
X. berteri bark / China Taiwan HAST 90112623 GU324749 AY951763 GQ848362 Hsieh et al. (2010)
X. betulicola leaves of Betula / China FCATAS 750 MF774332 – – Ma and Li (2018)
X. brunneovinosa termite nests / China Taiwan HAST 720 EU179862 GQ502706 GQ853023 Hsieh et al. (2010)
X. castorea wood / New Zealand PDD 600 GU324751 GQ502703 GQ853018 Hsieh et al. (2010)
X. cirrata termite nests / China Taiwan HAST 664 EU179863 GQ502707 GQ853024 Hsieh et al. (2010)
X. coccophora wood / French HAST 786 GU300093 GQ487701 GQ844809 Hsieh et al. (2010)
X. crinalis wood / China FCATAS 751 MF774330 – – Ma and Li (2018)
X. crozonensis bark / France HAST 398 GU324748 GQ502697 GQ848361 Hsieh et al. (2010)
X. cubensis log / Russian Far East HAST 477 – GQ502699 GQ848364 Hsieh et al. (2010)
X. culleniae pod / Thailand JDR 189 GU322442 GQ495935 GQ844829 Hsieh et al. (2010)
X. escharoidea termite nests / China Taiwan HAST 658 EU179864 GQ502709 GQ853026 Hsieh et al. (2010)
X. feejeensis bark / China Taiwan HAST 92092013 GU322454 GQ495947 GQ848336 Hsieh et al. (2010)
X. ficicola fallen leaves and petioles of Ficus auriculata / China HMJAU 22818 MZ351258 – – This study
X. filiformis herbaceous stem / Iran GUM 1052 KP218907 – – Hashemi et al. (2015)
X. fimbriata termite nests / French West Indies HAST 491 GU324753 GQ502705 GQ853022 Hsieh et al. (2010)
X. cf. glebulosa fruit / French West Indies HAST 431 GU322462 GQ495956 GQ848345 Hsieh et al. (2010)
X. grammica wood / China Taiwan HAST 479 GU300097 GQ487704 GQ844813 Hsieh et al. (2010)
X. griseosepiacea termite nests / China Taiwan HAST 641 EU179865 GQ502714 GQ853031 Hsieh et al. (2010)
X. hedyosmicola fallen leaves of Hedyosmum orientale / China Hainan FCATAS 856 (HT) MZ227121 MZ221183 MZ683407 This study
X. hedyosmicola fallen leaves of Hedyosmum orientale / China Hainan FCATAS 857 MZ227023 MZ221184 MZ851780 This study
X. hypoxylon wood / Belgium HAST 152 GU300096 GQ260187 GQ844812 Hsieh et al. (2010)
X. hypoxylon wood / China Taiwan HAST 95082001 GU300095 GQ487703 GQ844811 Hsieh et al. (2010)
X. hypoxylon leaf debris / Sweden CBS 122617 AM993146 – – Persoh et al. (2009)
X. ianthinovelutina fruit of Swietenia / Martinique HAST 553 GU322441 GQ495934 GQ844828 Hsieh et al. (2010)
X. intraflava termite nests / China Taiwan HAST 725 EU179866 GQ502718 GQ853035 Hsieh et al. (2010)
X. juruensis Arenga engleri / China Taiwan HAST 92042501 GU322439 GQ495932 GQ844825 Hsieh et al. (2010)
X. laevis wood / Martinique HAST 419 GU324746 GQ502695 GQ848359 Hsieh et al. (2010)
X. leavis bark / China Taiwan HAST 95072910 GU324747 GQ502696 GQ848360 Hsieh et al. (2010)
X. lindericola fallen leaves of Lindera robusta / China Hainan FCATAS 852 (HT) MZ005635 MZ031978 MZ031982 This study
X. lindericola fallen leaves of Lindera robusta / China Hainan FCATAS 853 MZ005636 MZ031979 MZ048749 This study
X. liquidambar fruits of Liquidambar formosana / China Taiwan HAST 93090701 GU300094 GQ487702 GQ844810 Hsieh et al. (2010)
X. longissima wood / China FCATAS 749 MF774331 – – Ma and Li (2018)
X. longissima wood / Iran IRAN 16582 F KP218906 – – Hashemi et al. (2015)
X. meliacearum petioles and infructescence of Guarea guidonia / Puerto Rico JDR 148 GU300084 GQ478219 GQ844797 Hsieh et al. (2010)
X. multiplex wood / USA JDR 259 GU300099 GQ487706 GQ844815 Hsieh et al. (2010)
X. muscula dead branch / French West HAST 520 GU300087 GQ478222 GQ844800 Hsieh et al. (2010)
X. nigripes termite nests / China Taiwan HAST 653 GU324755 GQ502710 GQ853027 Hsieh et al. (2010)
X. oxyacanthae fallen seeds / USA JDR 859 GU322434 GQ495927 GQ844820 Hsieh et al. (2010)
X. oxyacanthae fruits / Germany LZ 2010-502 HQ414587 – – Roensch et al. (2010)
X. palmicola fruits / New Zealand PDD 604 GU322436 GQ495929 GQ844822 Hsieh et al. (2010)
X. phyllocharis> dead leaves / French West HAST 528 GU322445 GQ495938 GQ844832 Hsieh et al. (2010)
X. plebeja trunk / China Taiwan HAST 91122401 GU324740 GQ502689 GQ848353 Hsieh et al. (2010)
X. polymorpha wood / USA JDR 1012 GU322460 GQ495954 GQ848343 Hsieh et al. (2010)
X. polymorpha Stump / Germany M:M-0125909 FM164944 – – Persoh et al. (2009)
X. polysporicola fallen leaves of Polyspora hainanensis / China Hainan FCATAS 848
(HT) 		MZ005592 MZ031976 MZ031980 This study
X. polysporicola fallen leaves of Polyspora hainanensis / China Hainan FCATAS 849 MZ005591 MZ031977 MZ031981 This study
X. regalis log of Ficus racemose / India HAST 920 GU324745 GQ502694 GQ848358 Hsieh et al. (2010)
X. schweinitzii bark / China Taiwan HAST 92092023 GU322463 GQ495957 GQ848346 Hsieh et al. (2010)
X. sicula f. major fallen leaves / China Taiwan HAST 90071613 GU300081 GQ478216 GQ844794 Hsieh et al. (2010)
Xylaria sp. 6 fallen leaves of Tibouchina semidecandra / USA JDR 258 GU300082 GQ478217 GQ844795 Hsieh et al. (2010)
X. striata branch / China HAST 304 GU300089 GQ478224 GQ844803 Hsieh et al. (2010)
X. tentaculata leaf litter or wood / Korea KA12-0530 KM077162 – – Kim et al. (2016)
X. tentaculata leaf litter or wood / Korea KA13-1324 KM077163 – – Kim et al. (2016)
X. tentaculata. leaf litter or wood / Korea KA13-1325 KM077164 – – Kim et al. (2016)
X. venosula twigs / USA HAST 94080508 EF026149 EF025617 GQ844806 Hsieh et al. (2010)
X. venustula bark / China Taiwan HAST 88113002 GU300091 GQ487699 GQ844807 Hsieh et al. (2010)
X. xylarioides wood / Iran GUM 1151 KP218909 – – Hashemi et al. (2015)
Hypoxylon fragiforme bark / France HAST 383 JN979420 AY951720 – Hsieh et al. (2005)
Camillea obularia – / Puerto Rico ATCC 28093 KY610384 KX271243 – Wendt et al. (2018)

Phylogenetic analyses

The molecular phylogeny was inferred from a combined dataset of ITS, TUB and RPB2 sequences. The sequences retrieved from open databases originated from Hsieh et al. (2005), Persoh et al. (2009), Hsieh et al. (2010), Roensch et al. (2010), Hashemi et al. (2015), Kim et al. (2016), Ma and Li (2018) and Wendt et al. (2018) (Table 1). Hypoxylon fragiforme (Pers.) J. Kickx f. and Camillea obularia (Fr.) Læssøe, J.D. Rogers & Lodge were selected as outgroup taxa. Sequences were aligned using the MAFFT online (http://mafft.cbrc.jp/alignment/server/). Alignments were optimised manually in BioEdit 7.0.5.3 (Hall 1999).

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).

Results

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 F. San Martín & J.D. Rogers and X. ficicola Hai X. Ma, Lar.N. Vassiljeva & Yu Li, X. lindericola is clustered with X. sicula Pass. & Beltr. f. major Ciccarone, but were separated from other species, as well as from each other.

Figure 1. 

Phylogenetic tree of Xylaria based on multigene alignment of ITS-TUB-RPB2 in the Bayesian analysis. Bayesian posterior probabilities (≥ 0.95, before the slash markers) and RaxML bootstrap values (≥ 50, after the slash markers) are shown. Different clades are indicated as coloured blocks.

Taxonomy

Xylaria hedyosmicola Hai X. Ma & X.Y. Pan, sp. nov.

MycoBank No: 839780
GenBank No: MZ227121, MZ221183, MZ683407 Figure 2

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.

Typification

China. Hainan Province, Lingshui County, Diaoluoshan Natural Reserve, on fallen leaves of Hedyosmum orientale (Chloranthaceae), 31 December 2020, Haixia Ma (holotype, FCATAS 856).

Etymology

“hedyosmicola” refers to the growth on leaves of Hedyosmum orientale.

Teleomorph

Stromata upright, solitary to cespitose, thread-like, unbranched or occasionally branched once at top, 2–5.5 cm total length; with a long sterile filiform apex up to 0.5–3 cm long; fertile part 3–17 mm long × 0.5–1 mm diam., usually consisting of closely packed or scattered perithecia; stipe 8–18 mm long × 0.1–0.5 mm diam., glabrous, finely longitudinally striate, the base slightly swollen; surface roughened, with half-exposed to fully exposed perithecial contours and wrinkles. Externally black, interior white. Texture soft. Perithecia subglobose, 200–470 µm diam. Ostioles papillate, 11–22 µm diam. Asci with eight ascospores arranged in uniseriate manner, cylindrical, 105–160 µm total length, the spore-bearing parts 70–100 µm long × 8–12 µm broad, the stipes 25–70 µm long, with apical apparatus bluing in Melzer’s reagent, tubular to slightly urn-shaped, 2.5–4.8 µm high × 2.5–3.5 µm broad. Ascospores brown, unicellular, ellipsoid-inequilateral, with narrowly rounded ends, smooth, (12–)13–15(–16.7) × (6–) 6.5–7.5 (–8.5) µm (M = 14 × 7 µm, n = 60), straight to slightly sigmoid germ slit spore-length or almost spore-length, with a slimy sheath on ventral side swollen at both ends to form rounded non-cellular appendages visible in Indian ink.

Figure 2. 

Xylaria hedyosmicola (FCATAS 856, holotype) a, b, e stromata on leaves (b, FCATAS 857) c stromatal surface d section through stroma, showing a perithecium f immature asci in water g, h ascal apical ring in Melzer’s reagent i, j ascospores in Melzer’s reagent k ascus in 1% SDS l, m asci and ascal apical ring in Melzer’s reagent n ascospore in Melzer’s reagent showing straight germ slit o ascospore in Melzer’s reagent showing slightly sigmoid germ slit p, q ascospore showing a slimy sheath and non-cellular appendages in India ink. Scale bars: 1 cm (a, b); 0.1 mm (c, d); 0.5 mm (e); 20 µm (f, m); 10 µm (g–l, n–q).

Additional specimen examined

China. Hainan Province, Lingshui County, Diaoluoshan Natural Reserve, on fallen leaves of Hedyosmum orientale, 31 December 2020, Haixia Ma (FCATAS 857).

Remarks

Xylaria hedyosmicola closely resembles X. vagans Petch by sharing thread-like 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 urn-shaped 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 non-cellular 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).

Xylaria lindericola Hai X. Ma & X.Y. Pan, sp. nov.

MycoBank No: 839554
GenBank No: MZ005635, MZ031978, MZ031982 Figure 3

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.

Typification

China. Hainan Province, Lingshui County, Diaoluoshan Natural Reserve, on fallen leaves of Lindera robusta (Lauraceae), 31 December 2020, Haixia Ma (holotype, FCATAS 852).

Etymology

“lindericola” refers to the growth on leaves of Lindera robusta.

Teleomorph

Stromata upright or prostrate, solitary to cespitose, unbranched or branched once or more at stipe, 3–26 cm total length; fertile part subglobose on long filiform stipes, 0.1–0.4 cm diam., the stipe 3–25 cm long × 0.1–1 mm diam., glabrous, finely longitudinally striate, the base slightly swollen; surface roughened by wrinkles and barely exposes perithecial contours. External black, interior white. Texture soft. Perithecia subglobose, 300–550 µm diam. Ostioles black, papillate. Asci with eight ascospores in uniseriate manner, cylindrical, 105–165 µm total length, the spore-bearing parts 65–115 µm long × 7.5–10.5 µm broad, the stipes 25–65 µm long, with apical apparatus bluing in Melzer’s reagent, tubular to urn-shaped, 3.9–5.5 µm high × 3–5 µm broad. Ascospores brown, unicellular, ellipsoid-inequilateral, with slightly narrowly rounded ends, aberrant ascospores with strongly pinched or beaked ends, smooth, (12.5–)13.5–15.5(–18) × (7–) 7.5–8.5 (–9.5) µm (M = 14.8 × 8 µm, n=60), with straight germ slit spore-length, without sheath or appendages visible in India ink.

Additional specimen examined

China. Hainan Province, Lingshui County, Diaoluoshan Natural Reserve, on fallen leaves of Lindera robusta, 31 December 2020, Haixia Ma (FCATAS 853).

Remarks

Xylaria lindericola is distinguished by its subglobose fertile part of stroma on a long filiform stipe and growing on fallen leaves of Lindera robusta. The species is somewhat similar to X. sicula f. major in morphology of stromatal fertile part. However, X. sicula f. major has stromata with long sterile apex, slightly smaller ascospores 9–13(–15) × (3–) 4.5–6 (–7) µm and grows on dead Olea leaves (Ciccarone 1947; Graniti 1959; Fournier 2014). In the phylogenetic tree, X. lindericola formed a fully supported clade with X. sicula f. major (Figure 1).

Xylaria hypsipoda Massee is similar to X. lindericola by sharing globose stromata and ascospores dimensions, but differs in having stromata with whitish scales, hairy stipes and urn-shaped, slightly larger apical apparatus 5–8 µm high × 2.9–5 µm broad (Rogers et al. 1987).

Xylaria ficicola resembles X. lindericola in stromatal morphology, but differs in having strongly exposed perithecial mounds of stromatal surface, larger ascospores (16–) 17.5–21(–22.7) × 6.5–8.5 µm with conspicuous hyaline noncellular appendage and grows on fallen leaves and petioles of Ficus auriculata (Ma et al. 2011). Xylaria heloidea Penz. & Sacc. from Indonesia is somewhat similar to X. lindericola in stromatal morphology, but the former has obconical, convex stromatal top, larger ascospores (14.5–) 15.5–18(–19) × (5–)5.5–6.5(–7) µm (16.7 × 6.1 µm), with a hyaline sheath swelling at both ends to form non-cellular appendages and grows on fallen fruits, twigs, petioles, and leaves of various plants (Ju et al. 2018).

Xylaria comosa (Mont.) Fr. and X. clusiae K.F. Rodrigues, J.D. Rogers & Samuels are also somewhat similar to X. lindericola in stromatal morphology. However, X. comosa has larger ascospores (21)–26–40 × 7–11 µm and larger apical ring 10.5 µm high × 7.5 µm broad (Dennis 1956) and X. clusiae has smaller stromata 1–3.5 cm, ascospores broadly ovoida1 to nearly globose (11.6–)12.8–16.7(–18) × 8–15 µm, with colorless appendage at one end (Samuels and Rogerson 1990).

Figure 3. 

Xylaria lindericola (FCATAS 852, holotype) a, b stromata on leaves c fertile part of stroma d stromatal surface e section through stroma, showing perithecia f ascal apical ring and ascospores with beaked ends in Melzer’s reagent g ascus and ascal apical ring in Melzer’s reagent h ascus in water i, j ascospores in water k, l ascospore in Melzer’s reagent m ascospore in India ink n ascospore in 1% SDS showing germ slit. Scale bars: 1.5 cm (a, b); 0.2 mm (c–e); 10 µm (f–n).

Xylaria polysporicola Hai X. Ma & X.Y. Pan, sp. nov.

MycoBank No: 839552
GenBank No: MZ005592, MZ031976, MZ031980 Figure 4

Diagnosis

Differs from X. phyllocharis by its half-exposed to fully exposed perithecial contours, the fertile part cylindrical and larger perithecia. Differs from X. phyllophila by its smaller ascospores. Differs from X. amphithele by its cylindrical stromata.

Typification

China. Hainan Province, Lingshui County, Diaoluoshan Natural Reserve, on fallen leaves of Polyspora hainanensis (Theaceae), 31 December 2020, Haixia Ma (holotype, FCATAS 848).

Etymology

“polysporicola” refers to the growth on leaves of Polyspora hainanensis.

Teleomorph

Stromata solitary, upright or prostrate, cylindrical, unbranched or occasionally branched, 1–4 cm total length, with acute sterile apex up to 2 mm long; fertile part 2–15 mm long × 0.5–1.6 mm diam., usually consists of closely packed perithecia and occasionally with scattered perithecia; the stipe 5–30 mm long × 0.3–1 mm diam., glabrous, finely longitudinally striate, the base slightly swollen; surface roughened, with half-exposed to fully exposed perithecial contours and wrinkles. Externally black, interior white. Texture soft. Perithecia subglobose, 0.4–0.6 mm diam. Ostioles papillate. Asci with eight ascospores arranged in uniseriate manner, cylindrical, 115–185 µm total length, the spore-bearing parts 75–100 µm long × 6.5–9 µm broad, the stipes 30–90 µm long, with apical apparatus bluing in Melzer’s reagent, inverted hat-shaped or urn-shaped, 2.5–4.5 µm high × 2–3.2 µm broad. Ascospores brown to dark-brown, unicellular, ellipsoidal-inequilateral, with broadly rounded ends, one end slightly pinched sometimes, smooth, (11.5–)12.5–14.5(–15) × 5.5–8 µm (M = 13.2 × 6.4 µm, n=60), with straight germ slit slightly less than spore-length, a slimy sheath or non-cellular appendages visible occasionally in Indian ink.

Additional specimens examined

China. Hainan Province, Lingshui County, Diaoluoshan Natural Reserve, on fallen leaves of Polyspora hainanensis, 31 December 2020, Haixia Ma (FCATAS 849); 5 July 2019, Haixia Ma (FCATAS 850 & 851).

Remarks

Xylaria polysporicola is morphologically similar to X. phyllocharis Mont. However, X. phyllocharis has fully immersed perithecia, the fertile part with peg-like structures and smaller perithecia 0.2–0.3 mm diam (San Martín and Rogers 1989; Fournier et al. 2020). Xylaria polysporicola is similar to Xylaria sp. (80082005) from Taiwan in stromatal morphology, but the latter has slightly smaller stroma (11–14 mm total length × 1 mm diam. vs. 10–40 mm total length × 0.5–1.6 mm diam.), hard texture, slightly larger ascospores 13.5–16.5 × 5–6 µm, with narrowly rounded ends (Ju and Rogers 1999). Xylaria phyllophila Ces. somewhat resembles X. polysporicola in stromatal morphology, but the former has larger ascospores 20 × 10 µm (Cooke 1883).

Figure 4. 

Xylaria polysporicola (FCATAS 848, holotype) a, b stromata on leaves (b, FCATAS 851) c stromatal surface d section through stroma, showing perithecia e, g asci and ascal apical ring in Melzer’s reagent f, i ascal apical ring in Melzer’s reagent h asci in black India ink j ascospore with germ slit in 1% SDS k, l ascospore in water m, n ascospore showing a slimy sheath and non-cellular appendages in India ink (FCATAS 850) o Ascospore in 1% SDS. Scale bars: 1 cm (a, b); 0.2 mm (c, d); 10 µm (e–o).

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 urn-shaped 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. 2014, 2015; Ma 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.

Key to species of Xylaria on fallen leaves in China

1 Stromata with rounded fertile apices 2
– Stromata with acute sterile apices 3
2 Stromata associated with leaves and petioles of Ficus auriculata (Moraceae), ascospores (16–)17.5–21(–22.7) × 6.5–8.5 µm X. ficicola
– Stromata associated with leaves of Lindera robusta (Lauraceae), ascospores (12.5–)13.5–15.5(–18) × (7–)7.5–8.5(–9.5) µm X. lindericola
3 Stipes tomentose X. hainanensis
– Stipes glabrous 4
4 Fertile part subglobose X. sicula f. major
– Fertile part not subglobose 5
5 Stromata cylindrical 6
– Stromata filiform 8
6 Ascospores (5.5–)6–8 × 3–3.5(–4) µm X. diminuta
– Ascospores length > 8.5 µm 7
7 Stromata with conspicuous perithecial contours, ascospores (11.5–)12.5–14.5(–15) × 5.5–8 µm X. polysporicola
– Stromata with inconspicuous perithecial contours, ascospores (8.5–)9–11 × 4–6 µm X. foliicola
8 Ascospores 16.5–20(–21.5) × 4–5(–6) µm X. jiangsuensis
– Ascospores length < 16.5 µm 9
9 Stromata associated with leaves of Betula (Betulaceae), ascospores (11.5)12–14(15) × 5–6 µm, with a straight germ slit, without appendages visible in India ink X. betulicola
– Stromata associated with leaves of Hedyosmum orientale (Chloranthaceae), ascospores (12–)13–15(–16.7) × (6–)6.5–7.5(–8.5) µm, with straight to slightly sigmoid germ slit, with appendages visible in Indian ink X. hedyosmicola

Acknowledgements

The authors thank Prof. Yu-Ming Ju (Institute of Plant and Microbial Biology, Academia Sinica, Taiwan, China) for suggestions on the manuscript. This study was supported by the National Natural Science Foundation of China (no. 31770023, 31972848, U1803232). We are also grateful to the Key Research and Development Program of Hainan (ZDYF2020062) and Hainan Basic and Applied Research Project for Cultivating High-Level Talents (2019RC305).

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