Chiang Mai Journal of Science

Roussoella yangjiangensis sp. nov. (Roussoellaceae, Pleosporales), A Holomorphic Fungus in Guangdong Province, China

1. INTRODUCTION

     Roussoellaceae was established by Liu et al. [1] to accommodate three roussoella-like genera, namely Neoroussoella, Roussoella (type genus), and Roussoellopsis. However, Jaklitsch and Voglmayr [2] synonymized Roussoellaceae under Thyridariaceae because their phylogenetic analyses showed that Roussoellaceae and Thyridariaceae formed a highly supported clade, which represent the same family. Therefore, Thyridariaceae was suggested based on the principle of priority according to the year of establishment. Subsequently, Tibpromma et al. [3] proposed that Roussoellaceae should be treated as a distinct family based on an updated phylogenetic analysis, and the broader taxon sampling also supported this conclusion [4–8]. Roussoellaceae is now regarded as a well-resolved family in Pleosporales [9,10]. According to “A comprehensive overview of genera in Dothideomycetes” and “The 2024 Outline of Fungi and fungus-like taxa”, the family comprises 11 genera, viz., Cytoplea, Elongatopedicellata, Neoroussoella, Nothoroussoella, Pararoussoella, Pseudoneoconiothyrium, Pseudoroussoella, Roussoella, Roussoellopsis, Setoarthopyrenia, and Xenoroussoella [9,11].
     Roussoella was established by Saccardo and Paoletti [12] to accommodate the type species R. nitidula, which was collected from dead stems of Bambusa sp. in Malaysia. Roussoella is characterized by immersed, clypeate ascostromata, trabeculate pseudoparaphyses, cylindrical asci with a distinct ocular chamber, and fusiform to ellipsoidal, ornamented, 1-septate, brown to dark brown ascospores, and an asexual morph producing pycnothyrial conidiomata, holoblastic conidiogenous cells, and oblong to ellipsoidal, brown conidia [6,10]. Most Roussoella species are lignicolous saprobes in terrestrial habitats [13,14], with the exception of R. mangrovei in the mangrove habitats, R. aquatica, R. intermedia, and R. minutella in the freshwater habitats [6], and R. terrestris in the soil [4,15], while R. neopustulans can cause human infections, such as fungal keratitis [16]. They were often reported from decaying culms of bamboo (Poaceae) across various regions worldwide, such as Chile, China, France, Japan, Jawa, Malaysia, Thailand, and Vietnam [1,12,17–21]. To date, 56 species of Roussoella have been reported worldwide, of which 22 species have been documented from China.
     In this study, samples of decaying culms of Phyllostachys edulis and Bambusa sinospinosa were collected from Guangdong Province, China. The phylogenetic position of our collections was determined within Roussoella through a multi-locus phylogenetic analysis based on LSU, ITS, tef1-α, and rpb2 sequences. Based on morphological and phylogenetic evidences, a new species, Roussoella yangjiangensis, is established.

2. MATERIALS AND METHODS

2.1 Collecting, Isolation and Morphological Examination
      Dead bamboo samples were collected from Yangjiang City, Guangdong Province, China, during the spring (9 April 2023) and winter (9 December 2023) seasons. These specimens were carefully placed in zip-lock bags and brought to the laboratory for morphological examination. A stereomicroscope (Chongqing Optec Instrument Co., Chongqing, China) was used to examine the fruiting bodies on natural substrate. The micro-fungal structures were examined with a compound microscope (Nikon Eclipse Ni-U, Japan) equipped with a digital camera (Canon 750D, Japan). Measurements were carried out using the TaroSoft (R) Image Frame Work program v. 0.9.7. The photographs were processed by Adobe Photoshop CS6 Extended v. 13.0 (Adobe Systems, U.S.A.).
     Single spore isolations were performed according to the methodology described by Senanayake et al. [22]. The herbarium specimens are deposited at the Mycological Herbarium of Zhongkai University of Agriculture and Engineering, Guangzhou, China (MHZU), while the living cultures are deposited in the Zhongkai University of Agriculture and Engineering Culture Collection, Guangzhou, China (ZHKUCC). The newly described species was registered in the database Index Fungorum 2025 [23] and assigned to Facesoffungi number following the method described by Jayasiri et al. [24].

2.2 DNA Extraction, PCR Amplification and Sequencing
     The germinated single spore was transferred onto potato dextrose agar (PDA) medium at room temperature (25–31 °C and 19–26 °C) for two weeks. After culturing, genomic DNA was extracted from fresh mycelia using the Maglso plant DNA isolation kit (Magen, China) according to the manufacturer's instructions. DNA amplification was performed using the polymerase chain reaction (PCR) with a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA).
     This study focused on two partial gene regions and two protein-coding genes: the large subunit of the nuclear ribosomal RNA gene (LSU), the internal transcribed spacer (ITS), the translation elongation factor-1 alpha (tef1-α), and the second largest subunit of RNA polymerase II (rpb2). The primer pairs employed in the amplifications were as follows: LR0R/LR5 [25], ITS5/ITS4 [26], EF1-983F/EF1-2218R [27], and fRPB2-5F/fRPB2-7cR [28].
     The PCR reactions were conducted in a 25 μL volume consisting of 9.5 μL of ddH₂O, 12.5 μL of 2 × FastTaq PCR Master Mix (Vazyme Co., China), 1 μL of each primer (10 μM), and 1 μL of DNA template. The PCR thermal cycling program for amplifying LSU, ITS, and tef1-α consisted of an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, and extension at 72 °C for 60 s, with a final extension at 72 °C for 10 min. For rpb2 amplification, the annealing temperature was adjusted to 56 °C.
     The PCR products were checked using 1% agarose gel electrophoresis stained with ethidium bromide (EB). Products exhibiting bright bands were sent to Tianyi Hui-yuan Biotechnology Co. (Guangzhou, China) for sequencing.

2.3 Sequence Alignment and Phylogenetic Analyses
     SeqMan v. 7.0.0 [29] was employed to assemble sequences and generate consensus sequences. These consensus sequences were submitted to analysis using the basic local alignment search tool (BLAST) to obtain a preliminary assessment of their classification (https://www.ncbi.nlm.nih.gov/). The reference sequences were downloaded from GenBank (Table 1), the phylogenetic tree was constructed referring to the recent publication [10]. Sequence alignments were performed with MAFFT v. 7.0362 [30] using default settings and subsequently manually adjusted with BioEdit v. 7.0 [31]. The aligned datasets (LSU+ITS+tef1-α+rpb2) were concatenated by Mesquite v. 3.81 [32]. The data were converted from the FASTA format to the PHYLIP format for RAxML analysis and to the NEXUS format for Bayesian analysis using the Alignment Transformation Environment online program (https://sing.ei.uvigo.es/ALTER/) [33]. Phylogenetic analysis was conducted using maximum likelihood (ML) [34] and Bayesian inference (BI) approaches [35].
     The ML tree was carried out using RAxML-HPC2 on XSEDE v. 8.2.8, with the default settings but adapted: the GAMMA nucleotide substitution model and 1,000 rapid bootstrap replicates [34] on the CIPRES Science Gateway platform [36]. Bayesian analysis was performed using MrBayes v. 3.1.2 [35]. For the BI approach, the evolutionary model of nucleotide substitution for each locus was independently selected using MrModeltest v. 2.3 [37]. Markov chain Monte Carlo sampling (MCMC) was run for 1,000,000 generations, and trees were sampled every 100^th generation. The first 25% of the trees, which represented the burn-in phase, were discarded. The remaining 75% of the trees were utilized to calculate the posterior probabilities (PP) for the majority rule consensus tree. The resulting trees were visualized in FigTree v. 1.4.0 (Institute of Evolutionary Biology, University of Edinburgh, UK) [38] and further edited using Microsoft Office PowerPoint 2007 (Microsoft Corporation, USA).

3. RESULTS AND DISCUSSION

3.1 Phylogenetic Analyses
     The combined sequence alignments comprised 95 taxa (Table 1) with Torula herbarum (CBS 111855) and T. hollandica (CBS 220.69) as the outgroup taxa. The final dataset comprised 1,511 characters for ITS, 920 characters for LSU, 943 characters for tef1-α, and 1,060 characters for rpb2. The combined alignment (ITS+LSU+tef1-α+rpb2) consisted of 4,848 characters in total, including alignment gaps. The RAxML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of -39064.023328. The matrix had 2,194 distinct alignment patterns, with 42.31% undetermined characters or gaps. The estimated base frequencies were as follows: A = 0.241453, C = 0.265726, G = 0.270618, T = 0.222203; substitution rates were AC = 1.302460, AG = 3.839038, AT = 1.571410, CG = 1.061699, CT = 7.946120, GT = 1.000000; gamma distribution shape parameter was α = 0.682161. Bayesian posterior probabilities from Bayesian inference analysis were assessed with a final average standard deviation of split frequencies = 0.080520. In the phylogenetic tree (Figure 1), phylogenetic analyses showed that the new collections ZHKUCC 25-0204 and ZHKUCC 25-0278 belong to the genus Roussoella. The new species was identified and established following the guidelines specified in [39,40].

Taxonomy
Roussoella yangjiangensis H.J. Zhao, W. Dong & K.D. Hyde, sp. nov. (Figures 2, 3).
     Index Fungorum number: IF 904304; Facesoffungi number: FoF 18203
     Etymology: “yangjiangensis” refers to Yangjiang City, where the holotype was collected.
     Holotype: MHZU 24-0033
     Saprobic on decaying culms of bamboo. Sexual morph: Ascostromata immersed under the host epidermis, gregarious, raised, visible as a black dome-shaped on the host surface, coriaceous, glabrous, brown to dark brown, dull, bi-loculate. Locules \(280\text{--}445~\mu\text{m}\) high, \(300\text{--}760~\mu\text{m}\) diam., (\(\bar{x} = 355.8 \times 498.8~\mu\text{m},\ n = 5\)), immersed in the ascomata, subglobose to ampulliform, brown to dark brown, ostiolate. Peridium \(10\text{--}30~\mu\text{m}\) thick, slightly thinner at the base, two-layered, outer layer composed of dark brown to black, thick-walled cells of textura angularis, inner layer composed of hyaline to pale brown, thin-walled, flattened cells of textura prismatica, merging with the inner wall tissue. Pseudoparaphyses \(1.3\text{--}2.2~\mu\text{m}\) (\(\bar{x} = 1.6~\mu\text{m},\ n = 20\)) wide, dense, aseptate, branched, trabeculate, embedded in a gelatinous matrix. Asci \(70\text{--}97 \times 5.5\text{--}7.8~\mu\text{m}\) (\(\bar{x} = 77.8 \times 6.6~\mu\text{m},\ n = 20\)), 8-spored, bitunicate, cylindrical, short pedicellate, with furcate or knob-like pedicel, apically rounded with indistinct ocular chamber. Ascospores \(10.5\text{--}14.5 \times 3\text{--}4.5~\mu\text{m}\) (\(\bar{x} = 12 \times 3.6~\mu\text{m},\ n = 30\)), uni-seriate, usually overlapping bi-seriate near the apex, fusiform with rounded ends, initially subhyaline to pale brown, becoming yellowish brown to dark brown at maturity, 1-septate, constricted at the septum, rough-walled, with longitudinal striae, 1–3 guttulate in each cell, surrounded by a large, entire mucilaginous sheath. Asexual morph: Coelomycetous. Pseudostromata \(50\text{--}90~\mu\text{m}\) high, \(135\text{--}310~\mu\text{m}\) diam., (\(\bar{x} = 71.5 \times 269~\mu\text{m},\ n = 10\)), scattered to aggregated, pycnidial, immersed under a clypeus, appearing as dark dot-shaped on the host surface, surrounded by a conspicuous concentric ring, comprising brown to dark brown fungal material growing in the cortex of host cells, ampulliform in section, uni-loculate, with a central, black ostiolate. Peridium \(7\text{--}18~\mu\text{m}\) wide, composed of thin-walled, brown cells of textura angularis, fusing with the host cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells \(3.7\text{--}6.5 \times 2.3\text{--}3.5~\mu\text{m}\) (\(\bar{x} = 4.6 \times 3~\mu\text{m},\ n = 15\)), enteroblastic, monophialidic, determinate, terminal, ampulliform, hyaline, smooth-walled. Conidia \(4.5\text{--}6 \times 2\text{--}3~\mu\text{m}\) (\(\bar{x} = 5.3 \times 2.6~\mu\text{m},\ n = 40\)), straight, cylindrical to oblong, rounded at both ends, hyaline when young, brown at maturity, aseptate, smooth-walled, 1–3-guttulate.
     Culture characteristics: Ascospores germinated on PDA within 24 h. Colonies on PDA reach 50 mm diam. after 18 days at \(25\text{--}31^\circ\text{C}\), circular, raised, dark brown in the middle, with dense white aerial hyphae on the surface; in reverse view, greenish-brown, with white wavy margin, not producing pigments. Conidia germinated on PDA within 24 h. Colonies on PDA reach 10 mm diam. after 13 days at \(19\text{--}26^\circ\text{C}\), circular, brown in the middle, white, cottony from above, with white, even margin; in reverse view, greenish-brown in the middle, with white margin, not producing pigments.
     Material examined: CHINA, Guangdong Province, Yangjiang City, on decaying culms of Phyllostachys edulis (Carrière) J. Houz., 9 April 2023, H.J. Zhao, YG053 = MHZU 24-0033, holotype; ex-type culture, ZHKUCC 25-0204; ibid., Maoming City, on decaying culms of Bambusa sinospinosa McClure, 9 December 2023, H.J. Zhao, CZ006-1 = MHZU 25-0212, paratype; living culture, ZHKUCC 25-0278.
     Notes: The single gene comparison reveals that our sexual strain (from Phyllostachys edulis) and asexual strain (from Bambusa sinospinosa) share the identical nucleotides. We can confirm that the two strains represent the same species, Roussoella yanjiangensis, in different states based on the sexual and asexual morphological characteristics of Roussoella [6,10]. Our multi-locus phylogenetic analysis further shows that R. yanjiangensis is closely related to R. aseptata and R. yunnanensis with 100% MLBS and 1.00 BYPP (Figure 1). Roussoella yanjiangensis resembles R. aseptata in having cylindrical to oblong, aseptate conidia, but it can be distinguished by its immersed conidiomata beneath a clypeus, which appear as dark, dot-like structures surrounded by a conspicuous concentric ring, and brown conidia, in contrast with the linear arrangement of conidiomata, the absence of a clypeus, and the hyaline conidia in the latter species [8]. Roussoella yanjiangensis is similar to R. yunnanensis in having immersed ascostromata, short pedicellate asci, and fusiform ascospores with longitudinal striae [5]. However, R. yanjiangensis can be distinguished from R. yunnanensis in having yellowish brown to dark brown ascospores surrounded by a large, entire mucilaginous sheath, whereas R. yunnanensis possesses yellowish ascospores without a sheath [5]. In addition, R. yanjiangensis has larger ascospores (\(10.5\text{--}14.5 \times 3\text{--}4.5~\mu\text{m}\)) compared to R. yunnanensis (\(9.2\text{--}10.4 \times 2.7\text{--}3.7~\mu\text{m}\)), and slightly larger asci (\(70\text{--}97 \times 5.5\text{--}7.8~\mu\text{m}\) vs. \(63\text{--}81 \times 4.1\text{--}5.3~\mu\text{m}\)).
     Roussoella yanjiangensis is also similar to R. neopusulans and R. striata in having cylindrical asci and longitudinally striated, 1-septate ascospores surrounded by a sheath, as well as similar dimensions of asci and ascospores [1,8,41]. However, R. neopusulans and R. striata differ from R. yanjiangensis in having very dark, shiny, unilocular ascomata (brown to dark brown, dull, bi-loculate in R. yanjiangensis). In addition, the ascospores of R. striata possess a gelatinous sheath that appears clear in water, which differs from the sheath of R. yanjiangensis that is only clearly seen in Indian Ink and exhibits an indistinct margin. Our phylogenetic analysis also shows that these three species are distantly related. Therefore, R. yanjiangensis is introduced as a new species based on morphological characteristics and phylogenetic analysis.

4. DISCUSSION

     Bamboo hosts a remarkable diversity of Roussoella species in tropical and subtropical regions, particularly in Asia. In China, a total of 24 species have been documented within the genus, among which 22 species are isolated from bamboo substrates in either terrestrial [5,7,8,10,14,19,21,42–46] or aquatic habits [47]. It is worth noting that only two bamboo-associated species are reported from South China, including Hong Kong [42] and Guangdong provinces, whereas most species are discovered from Southwest China, mainly in Guizhou, Sichuan, and Yunnan provinces [5,7,8,10,14,21,43–47]. It indicates that the research on Roussoella species associated with bamboo in South China remains largely scarce. In this study, we introduce an additional Roussoella species in its sexual and asexual morphs from decaying bamboo culms of Phyllostachys edulis and Bambusa sinospinosa in Guangdong Province, which enriches the known diversity of Roussoella species in South China. In addition, we provide the valuable data for the understanding of taxonomy and phylogeny of Roussoella. Two dichotomous keys to the sexual and asexual morphs (Figures 2,3) of Roussoella species from China are also provided separately to facilitate the identification of this group.
     Our phylogenetic analysis reveals that Roussoella is polyphyletic (Figure 1), which is consistent with the previous studies [20,48–52]. So far, many species have been excluded from the genus based on morphological and phylogenetic analyses. For instance, R. acaciae has been transferred to Thyridaria (as T. acacia) because its conidiogenous cells exhibit prominent periclinal thickening and percurrent proliferations at the apex, which fit Thyridaria rather than Roussoella [2]. Roussoella mangrovei and R. mukdahanensis lack a clypeus and dome-shaped surface of ascomata, therefore, they have been transferred to Pararoussoella (as P. mangrovei and P. mukdahanensis) [53,54]. Roussoella solani has been transferred to Neoroussoella (as N. solani) due to the annellidic conidiogenous cells [55]. However, the placements of several Roussoella species, such as R. arundinacea, R. chinensis and R. mexicana, remain doubtful as they morphologically belong to Roussoella, but are phylogenetically distant from the core members of Roussoella (Figure 1).
     Phylogenetically, Roussoella arundinacea (CBS 146088) is closed to Neoroussoella and Nothoroussoella (Figure 1). Roussoella arundinacea resembles Nothoroussoella irregulanis in having phialidic, ampulliform conidiogenous cells and aseptate, brown conidia [58,59], which differs from Neoroussoella by annellidic conidiogenous cells and hyaline conidia [1]. R. arundinacea can be distinguished from Nothoroussoella irregulanis by subcylindrical conidia, whereas latter has ellipsoidal to oblong conidia [58,59]. Although pervious study suggested that R. arundinacea should be transferred to Neoroussoella [49], it appears to be more consistent with Nothoroussoella. However, as Nothoroussoella is currently represented by a single species (Notho. irregulanis), whose phylogenetic placement remains uncertain [10], additional specimens are required to further clarify the taxonomic relationship between Nothoroussoella and Roussoella arundinacea. Hence, Roussoella arundinacea is provisionally maintained in Roussoella, pending additional collections and phylogenetic analyses.
     Roussoella chinensis was collected from a decaying pod of Wisteria sp. in China, and characterized by uni-seriate, ellipsoidal to fusiform, light yellowish to brown, 1-septate, smooth-walled ascospores without a sheath [50]. In their phylogenetic analysis, although R. chinensis was related to Neoroussoella, it was identified in Roussoella based on morphological characteristics. Our updated phylogenetic analysis reveals that R. chinensis forms a basal lineage to the clade containing Elongatopedicellata, Roussoella, Roussoellopsis and Setoarthopyrenia (Figure 1). The natural classification of R. chinensis requires reevaluation in the future. Roussoella mexicana was collected from the leaf spot of Coffea arabica in Mexico, and it was classified in Roussoella due to the phialidic, globose to ampulliform conidiogenous cells, and ellipsoidal, brown, aseptate conidia [59]. However, it is phylogenetically related to Pseudoneoconiothyrium, Pseudoroussoella, and Xenoroussoella (Figure 1). In addition, Elongatopedicellata and Roussoellopsis cluster within the clade of Roussoella in our phylogenetic analysis and previous studies [1–5,10,13,60–62]. However, their distinct morphological characteristics do not allow us to synonymize the two genera under Roussoella. Therefore, a more accurate generic concept of Roussoella is necessary to establish a natural classification of Roussoella and roussoella-like taxa.

ACKNOWLEDGEMENTS

     Wei Dong thanks the National Natural Science Foundation of China (grant no. 32200015), the Foreign Expert Program (Flexible Talent Introduction) of the Department of Science and Technology of Guangdong Province (Grant No. 252025061951000003) and the foundation of Guangzhou Municipal Science and Technology Bureau (grant no. 2023A04J1425). Mingkwan Doilom acknowledges the Foundation of Guangzhou Municipal Science and Technology Bureau (Grant No. 2023A04J1426). Kevin D. Hyde would like to thank the National Research Council of Thailand (NRCT) Grant “Total fungal diversity in a given forest area with implications towards species numbers, chemical diversity and biotechnology” (Grant no. N42A650547) and the Mushroom Research Foundation, Thailand, for funding this work. Hai-Jun Zhao would like to thank Mae Fah Luang University for awarding a scholarship. We would also like to acknowledge Dr. Shaun Pennycook, Manaaki Whenua–Landcare Research, for helping with the new species name.

CONFLICT OF INTEREST STATEMENT

     The authors declare that they hold no competing interests.

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