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Research Article
Addition of three new species of Xylariomycetidae fungi on bamboo from Southern China
expand article infoXin Zhou‡, Kamran Habib‡§, Wenyu Zeng‡, Yulin Ren|, Xiangchun Shen‡, Jichuan Kang, Qirui Li‡
‡ Guizhou Medical University, Gui'an, China
§ Khushal Khan Khattak University, Karak, Pakistan
| Guizhou Medical University, Gui’an, China
¶ Guizhou University, Guiyang, China

Corresponding author: Jichuan Kang ( jckang@gzu.edu.cn )

Corresponding author: Qirui Li ( lqrnd2008@163.com )

Academic editor: Nalin Wijayawardene
© 2024 Xin Zhou, Kamran Habib, Wenyu Zeng, Yulin Ren, Xiangchun Shen, Jichuan Kang, Qirui Li.
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: Zhou X, Habib K, Zeng W, Ren Y, Shen X, Kang J, Li Q (2024) Addition of three new species of Xylariomycetidae fungi on bamboo from Southern China. MycoKeys 109: 109-129. https://doi.org/10.3897/mycokeys.109.128020
Open Access

Abstract

In our ongoing research on bambusicolous Xylariomycetidae fungi, three new microfungi taxa were collected and identified as members of the genera Amphibambusa, Arecophila, and Nigropunctata. Amphibambusa aureae sp. nov., Arecophila gaofengensis sp. nov., and Nigropunctata xiaohensis sp. nov. are introduced based on morphological comparisons and phylogenetic analyses using combined ITS, LSU, tub2, and tef1α loci. Comprehensive morphological descriptions, illustrations, and a phylogenetic tree showcasing the placement of these new taxa are provided. Additionally, keys to Amphibambusa and Nigropunctata are provided.

Key words

Cainiaceae, Guizhou, systematics, Xylariales

Introduction

Bamboo, as the largest member of the grass family Poaceae, plays an important role in local economies worldwide, being distributed across diverse climates, from cold mountainous regions to hot tropical areas. Bamboos exhibit high diversity and are particularly abundant in Asia, notably in China. China boasts plentiful bamboo resources, with its bamboo species constituting of more than 50% of the world’s total (Liu et al. 2018). Due to their low natural toxicity, bamboos are susceptible to fungi and insect infestations resulting in abundant microfungi inhabiting their culms and leaves (Dai et al. 2018; Wang et al. 2018; Jiang et al. 2022; Hyde et al. 2023a, b).

Dai et al. (2018) reported an association of more than 1300 fungi with bamboo, consisting of 150 basidiomycetes and 800 ascomycetes species. Among these, 240 and 110 taxa have been reported as hyphomycetous and coelomycetous, respectively. The taxonomic placements of bamboo-associated ascomycetous fungi are highly diverse, comprising more than 1150 species, in 120 families and 400 genera (Dai et al. 2018). Among these families, Xylariaceae and Hypocreaceae are the most abundant, with 74 (belonging to 18 genera) and 63 (belonging to 14 genera) species, respectively (Dai et al. 2018; Wijayawardene et al. 2022). The genus Phyllachora holds the highest number of species occurring on bamboo, followed by Nectria and Hypoxylon (Dai et al. 2018). Most bambusicolous ascomycetous taxa in China are known from Taiwan, with 144 species, followed by Hong Kong with 139 species, Yunnan with 133 species, Guangdong with 53 species, Zhejiang with 37 species, Jiangsu with 36 species, and Sichuan with 35 species (Jiang et al. 2022). Jiang et al. (2022) reported 512 bambusicolous ascomycetous taxa from China, associated with 16 bamboo genera. These species are distributed across 50 orders, 116 families, and 279 genera (including 45 genera without any higher rank) and represent more than one-third of the known bambusicolous ascomycetes in the world. Most reported bambusicolous fungi lack detailed morphology or sequence data thus, still require further study (Dai et al. 2018).

During the investigation of bambusicolous Xylariomycetidae fungi, we observed specimens that could not be readily assigned to any known species. To better understand their taxonomic position, we conducted a phylogenetic analysis using a multi-marker approach (internal transcribed spacer ITS, large subunit LSU, β-tubulin tub2, and translation elongation factor tef1α). Their distinct morphological characteristics distinguish them from the known species. As a result, we propose these specimens as new species.

Materials and methods

Sample collection and morphological study

The specimens were collected during surveys conducted in Guizhou province, and Guangxi Zhuang Autonomous Region in China during 2023. All related habitat information was 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 morphological characterization and isolation. To preserve the freshness of the specimens, they were dried using a portable fan drier. The dried specimens were carefully labeled and stored. After this preparation, the specimens were ready for both morphological and molecular studies. 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

Macroscopic features (ostiole, clypeus, etc.) of the specimens were examined using an Olympus SZ61 stereomicroscope and photographed using a Canon 700D digital camera. Microscopic morphological features (ascomata, peridium, paraphyses, asci, ascospore, etc.) were observed using an optical microscope (Nikon Ni) and photographed using a Canon 700D digital camera attached. Melzer’s iodine reagent was used to test the apical apparatus structures for amyloid reaction. Asci and ascospores of the samples were measured using Tarosoft Image Framework (v. 0.9.0.7). Images were polished using Adobe Photoshop CS6 (Adobe Systems, USA). Pure cultures were obtained by single-ascospore isolation (Long et al. 2019) and maintained at 25 °C for 1–5 weeks on PDA (potato dextrose agar) and oatmeal-agar (OA) medium.

DNA extraction, PCR amplification and sequencing

Mycelium was scraped from pure culture plates using a sterilized scalpel and was used for DNA extraction with the methods following the manufacturer’s instructions 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), large subunit LSU, β-tubulin (tub2), and translation elongation factor (tef1α) were amplified by PCR with primers ITS1/ITS4 (White et al. 1990; Gardes and Bruns 1993), LR0R/LR5 (Vilgalys and Hester 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-983F/EF1-2218R (Rehner and Buckley 2005), respectively. The components of a 25 μL volume PCR mixture was: 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 Golden View, 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 (Tables 1, 2). These sequences were compared with each other and all the known sequences in GenBank using the BLASTN algorithm for precise identification. The molecular phylogeny was inferred from a combined dataset of ITS, LSU, tub2 and tef1α sequences. The reference sequences retrieved from open databases originated from recent published literature, and the Blastn results of close matches. 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) where necessary. 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). The phylogenetic analyses were also performed for Bayesian inference in MrBayes v. 3.2.1 (Ronquist et al. 2012) online. The Markov Chain Monte Carlo (MCMC) sampling in MrBayes v.3.2.2 (Ronquist et al. 2012) was used to determine the posterior probabilities (PP). Six simultaneous Markov chains were run for 1,000,000 generations, and trees were sampled every 1,000th generation. The phylogenetic tree was visualized in FIGTREE v.1.4.4 (Rambaut 2018). All analyses were run on the CIPRES Science Gateway v 3.3 webportal (Miller et al. 2010).

Table 1.
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Taxa and corresponding GenBank accession numbers of sequences used in the phylogenetic analysis of Fig. 1.

Species Strain number GenBank Accession Numbers References
ITS LSU
Amphibambusa aureae GMB4550T PQ066508 PQ066514 The study
Amphibambusa aureae GMB4561 PQ066509 PQ066515 The study
Amphibambusa bambusicola MFLLUCC 11-0617T KP744433 KP744474 Liu et al. (2015)
Amphibambusa hongheensis KUN-HKAS 112723T MW892971 MW892969 Jiang et al. (2021a)
Amphibambusa hongheensis KUNMCC 20-0334T MW892972 MW892970 Jiang et al. (2021a)
Arecophila australis GZUCC0124 MT742125 MT742132 Li et al. (2022)
Arecophila australis GZUCC0112T MT742126 MT742133 Li et al. (2022)
Arecophila bambusae HKUCC 4794 NA AF452038 Kang et al. (1999)
Arecophila clypeata GZUCC0127 MT742128 MT742135 Li et al. (2022)
Arecophila clypeata GZUCC0110T MT742129 MT742136 Li et al. (2022)
Arecophila gaofengensis GMB4541T PQ066512 PQ066516 The study
Arecophila gaofengensis GMB4559 PQ066513 PQ066517 The study
Arecophila miscanthii MFLU 19-2333T NR171235 NG088086 Hyde et al. (2020a)
Arecophila miscanthii FU31025 MK503821 MK503827 Hyde et al. (2020a)
Arecophila muroiana GZUCC0122 MT742127 MT742134 Li et al. (2022)
Arecophila zhaotongensis ZHKU 23-0260 OR995738 OR995745 Han et al. (2024)
Arecophila zhaotongensis ZHKU 23-0259 OR995735 OR995742 Han et al. (2024)
Arecophila zhaotongensis GMBCC1145T OR995740 OR995747 Han et al. (2024)
Arecophila sp. HKUCC 6487 NA AF452039 Jeewon et al. (2003)
Arecophila xishuangbannaensis ZHKU 23-0280 OR995737 OR995744 Han et al. (2024)
Arecophila xishuangbannaensis GMB-W1283T OR995736 OR995743 Han et al. (2024)
Atrotorquata lineata Mt25 AF009807 NA Kang et al. (1998)
Barrmaelia macrospora CBS 142768T NR167684 NA Jaklitsch et al. (2014)
Barrmaelia rhamnicola CBS 142772T NR153497 NA Voglmayr et al. (2018)
Cainia anthoxanthis MFLUCC 15-0539T NR138407 NG070382 Senanayake et al. (2015)
Cainia desmazieri CAI KT949896 NA Jaklitsch et al. (2016)
Cainia globosa MFLUCC 13-0663T NR171724 KX822123 Hyde et al. (2016)
Cainia graminis CBS 136.62 MH858123 MH869701 Vu et al. (2019)
Endocalyx cinctus JCM 7946 LC228648 LC228704 Delgado et al. (2022)
Endocalyx cinctus NBRC 31306 MZ313191 MZ313152 Delgado et al. (2022)
Endocalyx grossus JCM 5164T MZ313160 MZ313138 Delgado et al. (2022)
Endocalyx grossus JCM 5165 MZ313159 MZ313158 Delgado et al. (2022)
Endocalyx grossus JCM 5166 MZ313179 MZ313171 Delgado et al. (2022)
Endocalyx indumentum JCM 5171T MZ313153 MZ313161 Delgado et al. (2022)
Endocalyx indumentum JCM 8042 MZ313162 MZ313157 Delgado et al. (2022)
Endocalyx melanoxanthus CBS 147393 MW718204 NA Delgado et al. (2022)
Endocalyx melanoxanthus CBS 147394 MW718203 NA Delgado et al. (2022)
Endocalyx metroxyli MFLUCC 15-0723B MT929163 MT929314 Konta et al. (2021)
Endocalyx metroxyli MFLUCC 15-0723AT NR176745 MT929313 Konta et al. (2021)
Endocalyx metroxyli MFLUCC 15-0723C NA MT929315 Konta et al. (2021)
Endocalyx ptychospermatis ZHKUCC 21 0008T MZ493352 OK569894 Phukhamsakda et al. (2022)
Endocalyx ptychospermatis ZHKUCC 21 0009T MZ493353 OK569895 Phukhamsakda et al. (2022)
Endocalyx ptychospermatis ZHKUCC 21 0010T MZ493354 OK569896 Phukhamsakda et al. (2022)
Longiappendispora chromolaenae MFLUCC 17-1485T MT214370 MT214464 Mapook et al. (2020)
Requienella fraxini RS7 KT949911 NA Jaklitsch et al. (2016)
Requienella fraxini CBS 140475 NR138415 NA Jaklitsch et al. (2016)
Requienella seminuda CBS 140502T NR154630 MH878683 Jaklitsch et al. (2016)
Seynesia erumpens SMH 1291 NA AF279410 Bhattacharya et al. (2000)

Notes: Type specimens are marked with T; “NA”: indicates no sequence available in GenBank; newly generated sequences are indicated in bold.
Table 2.
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Taxa and corresponding GenBank accession numbers of sequences used in the phylogenetic analysis of Fig. 2.

Species Strain number GenBank Accession Numbers References
ITS LSU tub2 tef1α
Alloanthostomella rubicola MFLUCC 16-0479 KX533455 KX533456 NA NA Daranagama et al. (2016)
Anthostomella obesa MFLUCC 14-0171 KP297405 KP340546 KP406616 NA Daranagama et al. (2015)
Melanographium phoenicis MFLUCC 18-1481T MN482677 MN482678 NA MN481518 Hyde et al. (2020b)
Melanographium smilacis MFLU 21-0075 MZ538514 MZ538548 NA NA Boonmee et al. (2021)
Nigropunctata bambusicola MFLU 19-2134T MW240662 MW240592 NA MW759547 Samarakoon et al. (2022)
Nigropunctata bambusicola MFLU 19-2145T MW240664 MW240594 NA MW759548 Samarakoon et al. (2022)
Nigropunctata complanata HHUF 30674T LC760560 LC760580 NA LC760613 Sugita et al. (2024)
Nigropunctata complanata HHUF 30675T LC760561 LC760581 NA LC760614 Sugita et al. (2024)
Nigropunctata complanata HHUF 30676T LC760562 LC760582 NA LC760615 Sugita et al. (2024)
Nigropunctata complanata HHUF 30677T LC760563 LC760583 NA LC760616 Sugita et al. (2024)
Nigropunctata hydei CMUB 40018T OR507150 OR507163 NA NA Samarakoon et al. (2023)
Nigropunctata hydei MFLU 23-0410T OR507151 OR507164 NA NA Samarakoon et al. (2023)
Nigropunctata khalidii GMB1156T PP153389 NA PP209114 NA Li et al. (2024)
Nigropunctata nigrocircularis MFLU 19-2130T MW240661 MW240591 MW775612 MW759546 Samarakoon et al. (2022)
Nigropunctata saccata MFLU 19-2144T MW240663 MW240593 MW775613 NA Samarakoon et al. (2023)
Nigropunctata saccata MFLU 18-0804 MW240658 MW240588 MW775611 NA Samarakoon et al. (2023)
Nigropunctata thailandica MFLU 19-2118T MW240659 MW240589 NA MW759544 Samarakoon et al. (2022)
Nigropunctata thailandica HKAS 106975 MW240660 MW240590 NA MW759545 Samarakoon et al. (2022)
Nigropunctata xiaohensis GMB4503T PQ066510 PQ066518 PQ083530 PQ083532 The study
Nigropunctata xiaohensis GMB4552 PQ066511 PQ066519 PQ083531 PQ083533 The study
Pseudoanthostomella conorum CBS 119333 EU552099 EU552099 NA NA Daranagama et al. (2016)
Pseudoanthostomella delitescens MFLUCC 16-0477 KX533451 KX533452 KX789490 NA Daranagama et al. (2016)
Pseudoanthostomella pini-nigrae MFLUCC 16-0478T KX533453 KX533454 NA NA Daranagama et al. (2016)
Pseudoanthostomella pini-nigrae MFLU 18-0877 MW240654 MW240584 MW820918 MW759541 Daranagama et al. (2016)
Pseudoanthostomella pini-nigrae MFLU 15-3608 MW240655 MW240585 MW820919 MW759542 Daranagama et al. (2016)
Pseudoanthostomella pini-nigrae HKAS 102309 MW240656 MW240586 MW820920 NA Daranagama et al. (2016)
Pseudoanthostomella senecionicola MFLUCC 15-0013 MW240674 MW240604 MW820913 MW759554 Daranagama et al. (2016)
Virgaria nigra CBS 128006 MH864744 MH876180 NA NA Vu et al. (2019)

Notes: Type specimens are marked with T; “NA”: indicates no sequence available in GenBank; newly generated sequences are indicated in bold.

Results

Phylogeny

Analyses 1: Placements of Amphibambusa and Arecophila

The aligned data set of phylogram (Fig. 1) comprised 1250 (ITS/LSU) characters, after the exclusion of ambiguously aligned regions and long gaps. Barrmaelia macrospora (Nitschke) Rappaz and B. rhamnicola Rappaz were chosen as the outgroup taxa. The sequences of our collection Amphibambusa aureae formed a clade, exhibiting a firmly established sister relationship with Amphibambusa bambusicola D.Q. Dai & K.D. Hyde (94/1.00 ML/BI, Fig. 1). Newly generated sequences from Arecophila gaofengensis strains formed a sister branch to those of A. xishuangbannaensis L.S. Han & D.Q. Dai with a low support value (69/0.86 ML/BI, Fig. 1). Amphibambusa aureae and Arecophila gaofengensis are described as two new species.

Figure 1. 

Molecular phylogenetic analysis of Amphibambusa aureae, Arecophila gaofengensis and related taxa based on a combined ITS and LSU sequences. Bootstrap support values for maximum likelihood (ML) greater than 75% and Bayesian posterior probabilities (BPP) greater than 0.95 are displayed above or below the respective branches (ML/BI). The newly described species are marked red. Holotype and ex-type materials are in bold.

Analyses 2: Placement of Nigropunctata

The aligned dataset of Nigropunctata (Fig. 2) comprised 2730 (ITS/LSU/tub2/tef1α) characters, after exclusion of ambiguously aligned regions and long gaps. Virgaria nigra (Link) Nees was chosen as the outgroup taxon. In the phylogram (Fig. 2), the sequences of our collection Nigropunctata xiaohensis formed a well-supported (100/1.00, ML/BI) distinct clade on the basal of Nigropunctata. Nigropunctata xiaohensis is described as a new species.

Figure 2. 

Molecular phylogenetic analysis of Nigropunctata and related taxa based on a combined ITS, LSU, tub2 and tef1α sequences. Bootstrap support values for maximum likelihood (ML) greater than 75% and Bayesian posterior probabilities (BPP) greater than 0.95 are displayed above or below the respective branches (ML/BI). The newly described species are marked red. Holotype and ex-type materials are in bold.

Taxonomy

Amphibambusa D.Q. Dai & K.D. Hyde Fungal Diversity 72: 9, 2015.

MycoBank No: 550940

Notes

The genus Amphibambusa was introduced by Liu et al. (2015) which is characterized by immersed, solitary, scattered, globose to subglobose ascomata, ostiole at the centre, surrounded by white margin, unitunicate, cylindrical, short-pedicellate asci with a J+, subapical ring, and fusiform, subhyaline, longitudinally striated, 1-septate ascospores surrounded by a gelatinous sheath. Currently, the genus comprises two species: A. hongheensis H.B. Jiang & Phookamsak and A. bambusicola D.Q. Dai & K.D. Hyde (Liu et al. 2015, Jiang et al. 2021a). In this study, we introduce a new species of Amphibambusa from Guangxi Zhuang Autonomous Region, China.

Amphibambusa aureae X. Zhou, K. Habib & Q. R. Li, sp. nov.

MycoBank No: 853721
Fig. 3

Etymology

Named after the host-specific epithet “Phyllostachys aureae Rivière & C. Rivière” from which the fungus was isolated.

Type

China • Guangxi Zhuang Autonomous Region, Liangfengjiang Forest Park (22°43'24.91"N, 108°26'56.39"E), altitude: 99 m, on Phyllostachys aureae, 15 August 2023, Xin Zhou, Wenyu Zeng, 2023LFJ9 (GMB4550, holotype; GMBC4550, ex-type); ibid KUN-HKAS 134919, isotype.

Description

Saprobic on dead culms of bamboo, forming black circular spots on the host surface. Sexual morph: Ascomata 660–860 μm wide, 520–630 μm high, immersed under host epidermis, solitary, scattered, globose to subglobose, visible as a black dot, ostiole at the center, with a neck, with an underdeveloped clypeus. Ostioles are centrally located, black, surrounded by white margin. Peridium 13–30 μm thick, outer brown to hyaline inner, cells textura angularis. Paraphyses 2–4.8 μm (x̄ = 3.7 μm, n = 20) wide, longer than the asci, numerous, filamentous, colorless, branched, septate. Asci 90–190 × 9–18 μm (x̄ = 148.5 × 13.1 μm, n = 20), 8-spored, unitunicate, cylindrical, short-pedicellate, apically rounded, with a J+ subapical ring, 1.4–1.9 × 2.5–3.6 μm (x̄ = 1.7 × 3.1 μm, n = 6). Ascospores 15–22.5 × 5–7.9 μm (x̄ = 19 × 6.6 μm, n = 40), L/W 3.4, 1–2 seriate, fusiform, subhyaline, 1-septate in the middle, slight constricted at the septum, with round ends, with longitudinal striations along the entire length of the ascospore, and enveloped by a gelatinous sheath 2.5–7 μm (x̄ = 5.2 μm, n = 20), lacking appendage. Asexual morph: Undetermined.

Culture characteristics

Cultured on PDA medium at 27 °C for 4–5 weeks, the colony diameter measures 4–4.5 cm, round, slightly raised in the center, with a neat margin. The mycelium at the colony edge is degraded, appearing white and glossy. A portion of the colony center is brown.

Paratype

CHINA • Guangxi Zhuang Autonomous Region, Liangfengjiang Forest Park (22°43'20.90"N, 108°26'33.52"E), altitude: 99 m, on Phyllostachys aureae, 15 August 2023, Xin Zhou, Wenyu Zeng, 2023LFJ190 (GMB4561; paratype; GMBC4561, ex-paratype).

Notes

In the phylogram, Amphibambusa aureae (ex-type: GMBC4550) clustered in a distinct clade close to A. bambusicola D.Q. Dai & K.D. Hyde (ex-type: MFLLUCC 11–0617). The genus Amphibambusa is represented by two species, A. hongheensis H.B. Jiang & Phookamsak and A. bambusicola. Amphibambusa aureae shares similarities with both species, such as ascomata immersed in a black clypeus, ostiolar openings surrounded by a white margin, cylindrical asci with a J+ subapical ring, and fusiform, longitudinally striated ascospores enveloped by a distinct mucilaginous sheath (Liu et al. 2015, Jiang et al. 2021a). However, A. aureae can be distinguished from A. bambusicola by its smaller ascospores (15–22.5 × 5–7.9 μm compared to 25–27 × 5.5–6 μm in A. bambusicola) (Liu et al. 2015). Additionally, ascospores of A. aureae have rounded ends and are slightly constricted at the septum, whereas those of A. bambusicola have pointed end cells and are deeply constricted at the septum. Amphibambusa hongheensis differs from A. aureae by having smaller asci (118–160 × 14–18 μm vs. 90–190 × 9–18 μm) and larger ascospores (25.5–33 × 5.5–7.2 μm vs. 15–22.5 × 5–7.9 μm) (Jiang et al. 2021a).

Figure 3. 

Amphibambusa aureae (GMB4550, holotype) A type material B ascoma immersed under the surface of host C cross-section of ascoma D, E longitudinal sections of ascomata F peridium G paraphyses H, I asci J a J+ subapical ring bluing in Melzer’s reagent K–M ascospores N culture on PDA. Scale bars: 0.5 mm (C, D); 100 μm (E); 10 μm (F–M).

Key to the Amphibambusa species

1 Ascospore > 22 µm long 3
2 14.7–21.47 μm long ascospore A. aureae
3 Ascospore 25–27 μm long, with pointed end cells, deeply constricted at the septum A. bambusicola
– Ascospore 25.5–33 μm long, with round end cells, and slightly constricted at the septum A. hongheensis

Arecophila K.D. Hyde, Nova Hedwigia 63(1–2): 82 (1996)

MycoBank No: 27653

Notes

The genus Arecophila was introduced by Hyde (1996). The genus is characterized by immersed ascomata with blackened clypeus, ostiole at the centre, unitunicate, long-cylindrical asci with a J+, apical ring, and 1-septate ascospores with striations, and covered with a thick mucilaginous sheath (Hyde 1996; Li et al. 2022; Han et.2024). In this study, we introduce a new species of Arecophila from Guizhou Province, China.

Arecophila gaofengensis X. Zhou, K. Habib & Q. R. Li, sp. nov.

MycoBank No: 853722
Fig. 4

Etymology

The specific epithet “gaofengensis” refers to the geographical location, Gaofeng Village, where the holotype specimen was collected.

Type

China • Guizhou Province, Anshun City, Pingba District, Gaofeng Town, 26°33'96.54"N, 106°54'20.37"E, altitude: 1250 m, on dead culms of bamboo, 30 October 2023, Yulin Ren, 2023GFZ15 (GMB4541, holotype; GMBC4541, ex-type); ibid KUN-HKAS 134920, isotype.

Description

Saprobic on the surface of dead bamboo culms, forming black round spots. Sexual morph: Ascomata 400–600 µm high, 600–900 µm diam, globose to subglobose, solitary, scattered, sometimes gregarious, immersed beneath blackened clypeus; clypeus well developed, black, coriaceous, ostiole at the center, weakly papillate. Peridium 13–20 μm wide, composed of thick walled, hyaline to brown cells, texture angularis. Paraphyses 2–3 µm (x̄ = 2.6 µm, n = 20) wide, hyaline, numerous, filamentous, branched, septate. Asci 126–210 × 10–13.5 µm (x̄ = 165 × 12.5 µm, n = 20), 8-spored, unitunicate, long-cylindrical, short-pedicellate, with a J+, trapezoidal shape apical ring, bluing in Melzer’s reagent, 2.2–3.4 μm high, 3.6–4.2 μm diam. Ascospores 19–24.5 × 7–9.5 µm (x̄ = 21.6 × 7.8 µm, n = 30), uniseriate, fusiform, brown, 1-septate, septate at the center, slightly constricted septum, tapering at the ends, with longitudinal and sulcate striations, covered with a thick mucilaginous sheath measuring 3–8 µm (x̄ = 6.3 µm, n = 10). Asexual morph: Undetermined.

Culture characteristics

Ascospores germinating on PDA within 36 hours and germ tubes produced from upper cells. Colonies growing fast on PDA, reaching 2 cm in 1 week at 28 °C, effuse, velvety to hairy, nearly circular, irregular at the margin, white from above, pale yellowish white from below. Mycelium immersed in the media, composed of branched, septate, smooth-walled, hyaline, hyphae.

Figure 4. 

Arecophila gaofengensis (GMB4541, holotype) A, B ascomata immersed in bamboo host C Cross-section of ascoma D, E longitudinal sections of ascomata F peridium G paraphyses H a J+ subapical ring staining by Melzer’s reagent I–K asci with ascospores L–N ascospores surrounded by a gelatinous sheath. Scale bars: 0.5 mm (B–D); 100 μm (E); 10 μm (F–N).

Paratype

China • Guizhou Province, Anshun City, Pingba District, Gaofeng Town, 26°33'95.44"N, 106°54'30.27"E, altitude: 1250 m, on dead culms of bamboo, 30 October 2023, Yulin Ren, 2023GFZ530 (GMB 4559; paratype; GMBC4559, ex-paratype).

Notes

In the phylogram (Fig. 1), Arecophila gaofengensis formed a sister branch with A. xishuangbannaensis L.S. Han & D.Q. Dai with a low bootstrap values (69/0.86 ML/BI, Fig. 1). Arecophila gaofengensis differs from A. xishuangbannaensis by its smaller ascospores (19–24.5 × 7–9.5 µm vs. 23–27 × 8.5–9.5 μm) and smaller asci (126–210 × 10–13.5 µm vs. 180–270 × 12–14 μm) (Han et al. 2024). The analysis of ITS sequences for these two species reveals a sequence length of 471 base pairs, with a 92.8% similarity, and a 2.1% gap presence, indicating 437 matching positions. Morphologically, the new taxon is close to A. bambusae, but can be distinguished from A. bambusae by having larger asci (126–210 × 10.3–13.7 µm vs. 132.5–140 × 7.5–8 µm) and wider ascospores (19–24.5 × 7.1–9.5 µm vs. 19–22.5 × 5.5–7 µm) (Umali et al. 1999). Morphologically, the new species also resembles A. muroiana (I. Hino & Katum.) You Z. Wang et al. However, clypeus is absent in A. muroiana (Li et al. 2022), while blackened clypeus was observed in A. gaofengensis. So, here we introduced it as a new species of Arecophila.

Nigropunctata Samarak. & K.D. Hyde, Fungal Diversity 112: 68, 2022.

MycoBank No: 558737

Notes

The genus Nigropunctata, typified by N. bambusicola Samarak. & K.D. Hyde, has recently been classified into Xylariales. The genus is characterized by immersed, solitary or scattered ascomata appearing as small black dots, unitunicate, cylindrical asci with a J+, discoid apical ring (Samarakoon et al. 2022). The genus is represented by seven species (https://www.indexfungorum.org/Names/Names.asp; Accessed June 21, 2024). In this study, we introduce a new species of Nigropunctata from China.

Nigropunctata xiaohensis X. Zhou, K. Habib & Q. R. Li, sp. nov.

MycoBank No: 853723
Fig. 5

Etymology

The specific epithet “xiaohensis” refers to the geographical location, Xiaohe Village, where the holotype specimen was collected.

Type

China • Guizhou Province, Guiyang City, Huaxi District, Xiaohe Village, (25°33'10.46"N, 105°38'22.57"E), altitude: 120 m, on bamboo, 1 June 2023, Xin Zhou, Wenyu Zeng, 2023XHC1 (GMB4503, holotype, no culture was obtained); ibid KUN-HKAS 134921, isotype.

Description

Saprobic on decaying bamboo culms. Sexual morph: Ascomata 320–380 × 340–400 μm (x̄ = 352.7 × 360 μm, n = 10), immersed, solitary or scattered, appearing as small black dots, solitary, in cross-section globose to subglobose with a flattened base. Ostioles centrally, slightly, papillate, black, flush with the surface of the host. Peridium 15–25 µm thick, comprised of several layers, composed of thick-walled, dense, brown to hyaline, cells of textura angularis. Paraphyses 2.8–4.3 μm (x̄ = 3.6 μm, n = 20) wide, longer than the asci, numerous, filamentous, curving, contain white intracellular material. Asci 85.5–140 × 11–18.5 μm (x̄ = 120.2 × 15.5 μm, n = 20) 8-spored, unitunicate, cylindrical, short-pedicellate, apically rounded, with a J+, discoid apical ring, measures 1.3–2.4 μm high, 3.5–5.0 μm wide (x̄ = 1.8 × 4.4 μm, n = 10). Ascospores 11–21 × 6.5–10.5 μm (x̄ = 17.8 × 8.1 μm, n = 30), L/W 2.2, uniseriate, unicellular, ellipsoid to broadly ellipsoid, dark brown to black, with rounded ends, covered with a thick mucilaginous sheath measuring 5–8 µm (x̄ = 6.2 µm, n = 10), with a germ slit extending across the entire spore. Asexual morph: Undetermined.

Figure 5. 

Nigropunctata xiaohensis (GMB4503, holotype) A material B ascoma on the surface of host C cross-section of ascoma D, E longitudinal sections of ascomata F peridium G paraphyses H–J asci K a wedge-shaped, J+ apical ring bluing in Melzer’s reagent L, M ascospores N ascospores with germ slits. Scale bars: 0.5 mm (C, D); 100 μm (E); 10 μm (F–N).

Paratype

China • Guizhou Province, Guiyang City Huaxi District, Xiaohe Village (25°33'20.34"N, 105°38'32.23"E), altitude: 120 m, on bamboo, 4 June 2023, Xin Zhou, Wenyu Zeng, 2023XHC340 (GMB4552, paratype).

Notes

In the phylogram (Fig. 2), Nigropunctata xiaohensis formed a separate clade in Nigropunctata s. str. Morphologically, N. xiaohensis resembles N. complanate R. Sugita & Kaz. Tanaka (Sugita et al. 2024) as both share similar size ascospore. However, N. complanate is distinguished by thick clypeus (75–90 μm high, 270–410 μm diam.), larger asci (130–175 × 13–20 μm), and an inverted hat-shaped apical ring. The ITS sequences analysis of N. complanate and N. xiaohensis reveals a sequence length of 496 base pairs, with an 84.3% identity, and 9.1% gap presence. Nigropunctata nigrocircularis Samarak. & K.D. Hyde differs in having larger ascomata (450–535 × 455–560 μm), longer asci (125–170 μm) and smaller ascospore averaging 15.5 × 6.4 μm with a 3–4.5 μm mucilaginous sheath (Samarakoon et al. 2022). The type species of the genus, N. bambusicola Samarak. & K.D. Hyde differs in having smaller ascomata measuring 285–315 × 260–340 μm, smaller discoid-inverted hat-shaped ascal apical rings (1.7–2 × 4–4.8 μm), and ascospores measuring 13.5–17 × 5.5–9.5 μm, with a 2–6 μm mucilaginous sheath (Samarakoon et al. 2022). A recently reported new species from China, N. khalidii Y. P. Wu & Q. R. Li, differs by possessing larger ascomata (608–782 × 762–830 μm vs. 320–380 × 340–400 μm in N. xiaohensis), larger asci (146–173 × 8.6–13.6 µm vs. 85.5–140 × 11–18.5 μm in N. xiaohensis), and slightly smaller ascospores (14.8–18 × 6.3–9 µm) lacking a germ slit (Li et al. 2024).

Key to the Nigropunctata species

1 Ascospores lacking a germ slit 4
2 Ascospores with germ slit 5
3 Lacking mucilaginous sheath around ascospores N. saccata
4a peridium 11–16 µm wide, ascomata 606–782 × 762–830 µm N. khalidii
4b Peridium 16.5–31 µm wide, ascomata 400–520 × 485–575 µm N. hydei
5a Ascomata > 450 µm diam 6
5b Ascomata 260–340 μm diam, asci 95–140 µm long, ascal apical apparatus 1.7–2 × 4–4.8 µm N. bambusicola
5c Ascomata 390–450 µm diam, asci 130–175 µm long, ascal apical apparatus 2.5–3 × 4.5–5 µm N. complanata
5d Ascomata 340–400 µm diam, asci 85.5–140 µm long, ascal apical apparatus 1.3–2.4 × 3.5–5 µm N. xiaohensis
6a Ascomata 450–535 × 455–560 μm, ascal apical apparatus 3.2–3.6 µm wide N. nigrocircularis
6b Ascomata 615–830 × 770–965 μm, ascal apical apparatus 4.5–6 μm wide N. thailandica

Discussion

In this paper, three new species of Amphibambusa, Arecophila, and Nigropunctata associated with bamboo were introduced, which were collected from karst areas of China. Recent studies have expanded our understanding of bambusicolous fungi from southern China. Han et al. (2024) introduced three new species from the family Cainiaceae, including a novel genus Paramphibambusa and two new Arecophila species. Jiang et al. (2021b) described two new species, Occultibambusa hongheensis and Seriascoma bambusae, and reported Occultibambusa kunmingensis from new habitats. Yu et al. (2023) identified three new species in the Savoryellaceae family and two new records from Sichuan Province. Our discoveries have enriched the research on bambusicolous fungal diversity in southern China.

Amphibambusa has a widespread distribution, reported in both Thailand and China. All known species of Amphibambusa have been found exclusively on decorating bamboo, indicating a potential host specificity (Liu et al. 2015; Jiang et al. 2021a). Phylogenetic analysis conducted in this study reveals a close relationship between Amphibambusa and Arecophila. However, Amphibambusa possesses hyaline ascospores pointed at both ends, which distinguishes it from Arecophila (Liu et al. 2015). The longitudinal stripes on the surface of Amphibambusa ascopores are not easily visible under an optical microscope and can be easily overlooked. Special attention should be paid when observing and describing morphology. Here one new species of Amphibambusa aureae was introduced as the third species of the genus.

Currently, there are 20 Arecophila epithets in Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, July 2024), but only six species and one strain of Arecophila sp. have molecular data on Genbank. Arecophila clustered into two clades through phylogenetic analysis (Li et al. 2022, Han et al. 2024). Our study also identifies Arecophila as comprising two clades. Morphologically, we cannot find a clear difference between these two clades. At the same time, the morphological characteristics of the species in both branches conform to definitions of Arecophila (Hyde 1996). This may indicate that Arecophila is a polyphyletic group that has undergone convergent evolution. It may also indicate that the genes currently used to construct phylogenetic trees cannot serve as good DNA barcoding for distinguishing Arecophila from its approximate genera. In summary, the use of more samples, gene sequences, and morphological features is essential for the future accurate identification of Arecophila.

Ascospores are the main identifying feature of ascomycetous fungi (Webster and Weber 2007). Currently, there are eight Nigropunctata species published including our new introduction. However, the shapes, dimensions, and colors of the ascospores of all species in the genus Nigropunctata are similar with very little variation (Samarakoon et al. 2022, 2023; Li et al. 2024; Sugita et al. 2024). The presence or absence of germ slits and mucilaginous sheaths of ascospores is used as the main basis for distinguishing Nigropuntata khalidii, N. hydei, N. saccata from similar species (Samarakoon et al. 2023; Li et al. 2024). In terms of ascospores’ size, the mean value of ascospores of all eight species was 15–18 μm. For example, the ascospores of N. thailandica measure 15–18.5 × 7–11.5 μm (mean = 17 × 9 μm, n = 25), while those of N. complanata measure 14.5–19.5 × 7.5–10 μm (Samarakoon et al. 2022; Sugita et al. 2024). The averages of the ascospore sizes of these two species differed by only 0.1 µm. The ascospore colors of all eight species are brown to dark brown, and the ascospore shapes of all eight species are ellipsoidal (Samarakoon et al. 2022; Samarakoon et al. 2023; Li et al. 2024; Sugita et al. 2024). Except for the ascospores, there are also relatively small morphological differences among the Nigropunctata species (Samarakoon et al. 2022; Samarakoon et al. 2023; Li et al. 2024; Sugita et al. 2024). However, there are significant differences in their DNA sequences. Hence, we believe that DNA sequence should be a primary feature for the species identification of Nigropunctata.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (32170019 and 31960005); the Guizhou Medical University High-Level Talent Launch Fund Project (2023-058); the Guizhou Provincial Scientific and Technologic Innovation Base (No. [2023]003); the High-level Innovation Talents of Guizhou (No. GCC [2023]048); National Natural Science Foundation of China (12132006); the Guizhou Provincial Natural Science Foundation for High-Level Innovative Talents and Teams (2016-5676, 2015-4021).

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author contributions

Conceptualization: Jichuan Kang, Qirui Li, Xiangchun Shen. Collection and morphological examinations: Xin Zhou, Wenyu Zen, Yulin Ren. Molecular sequencing, and phylogenetic analyses: Xin Zhou, Kamran Habib. Specimen identification: Xin Zhou, Qirui Li. Original draft preparation: Xin Zhou, Qirui Li. Review and editing, supervision: Xiangchun Shen, Jichuan Kang, Kamran Habib. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Kamran Habib https://orcid.org/0000-0003-2572-0306

Yulin Ren https://orcid.org/0009-0003-9063-425X

Jichuan Kang https://orcid.org/0000-0002-6294-5793

Qirui Li https://orcid.org/0000-0001-8735-2890

Data availability

The datasets generated during and/or analyzed during the current study are available in the MycoBank repository (included in the manuscript), and GenBank (included in Tables 1, 2). And also, the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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