Savoryella claviformis (Savoryellaceae), a new freshwater hyphomycetous species from the Tibetan Plateau, China

In recent years, there has been a significant increase in research focusing on the taxonomy and phylogeny of saprobic fungi worldwide. These include an extensive examination of the diversity of lignicolous freshwater fungi in China^[1−8]. The Tibetan Plateau, as the largest and most unique geographical region on Earth encompasses a remarkable range of endemic diversity^[9−11]. Recent advancements in the study of lignicolous freshwater fungi on the Tibetan Plateau have led to the discovery of an increasing number of species, underscoring its critical importance for global biodiversity conservation and scientific research^[12−14].

Savoryella was introduced by Jones & Eaton^[15] with S. lignicola as the type species. The sexual morphs of Savoryella are characterized by immersed, partly immersed, or superficial, globose, subglobose, or ellipsoidal ascostromata, typically 8-spored, occasionally 2-spored, cylindrical or clavate, unitunicate asci, and ellipsoidal, three-septate ascospores^[16−18]. By contrast, the asexual morphs are characterized by glistening, punctiform colonies; micronematous, mononematous conidiophores; holoblastic, determinate, integrated, terminal, and intercalary conidiogenous cells, and solitary or aggregated, pyriform to obovoid, septate conidia^[19]. Zhang et al.^[19] synonymized Trichocladium nypae with Savoryella nypae and introduced an asexual species, S. sarushimana, into the genus Savoryella, based on morphological and phylogenetic analyses. Subsequently, Tian et al.^[20] reported two additional asexual species, S. cocois, and S. chiangraiensis, collected from decaying leaves of the Arecaceae, based on phylogenetic analysis and morphological characters. Savoryella, recognized as a holomorphic genus predominantly inhabits submerged, decaying woody debris within both aquatic and terrestrial ecosystems. It has been systematically described and illustrated by mycologists worldwide^{[15,18,21−25]}.

During the investigation of the diversity of lignicolous freshwater fungi on the Tibetan Plateau, two collections were made from freshwater habitats of taxa in their hyphomycetous forms. Multigene phylogenetic analysis showed that these two isolates belong to Savoryella. In this study, one new species, Savoryella claviformis, is introduced with morphological description and phylogenetic placement. These discoveries further add to the diversity of freshwater fungi on the Tibetan Plateau.

Submerged decaying wood samples were collected from freshwater habitats in the Tibetan Plateau, China. Samples were obtained from freshwater lakes and rivers, encompassing various substrates such as parts of tree trunks, branches, twigs, and litter. The specimens were studied following the methods of Senanayake et al.^[26]. Microscopic structures were examined by using a stereomicroscope (SteREO Discovery.V12, Carl Zeiss Microscopy GmBH, Germany), photographed by using a Nikon ECLIPSE 80i compound microscope fitted with a NikonDS-Ri2 digital camera, macro morphological characters were examined by using a dissection microscope (Nikon SMZ745T, Nikon Instruments Inc., Japan), photographed by using a Canon 6D Mark II camera, measured by using the Tarosoft (R) Image Frame Work program. The illustrated figures were processed by using Adobe Photoshop CS6 v. 10.0 software (Adobe Systems, San Jose, CA, USA).

Single spores were isolated on potato dextrose agar (PDA) plates using the techniques outlined in Senanayake et al.^[26]. Both the holotype and pure cultures were deposited at the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS), and the Kunming Institute of Botany Culture Collection (KUNCC), Kunming, China. Taxonomic novelties were submitted to the Faces of Fungi database^[27] and Index Fungorum 2024^[28].

DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing

Fresh mycelia were scraped from colonies grown on PDA plates and transferred to a 1.5 mL microcentrifuge tube using a sterilized lancet for genomic DNA extraction. Fungal genomic DNA was extracted using the TOLOBIO Plant Genomic DNA Extraction Kit (Shanghai Co. Ltd, China), following the protocols in the manufacturer's instructions.

PCR amplifications were undertaken using the following primer pairs: ITS5/ITS4 for the internal transcribed spacer ribosomal DNA (rDNA) region, encompassing the 5.8S rDNA coding region (ITS); LR0R/LR5 for the 28S rDNA of the nuclear ribosomal large subunit (LSU); NS1/NS4 for the 18S rDNA of the nuclear ribosomal small subunit (SSU)^[29,30]. DNA preparation was conducted in a 25 μL mixture, which included 21 μL of 1× Power Taq PCR Master Mix, 1 μL of each primer from a 10 μL stock, and 2 μL of genomic DNA, and amplification was performed in the BioTeke GT9612 thermocycler (Beijing, China). The PCR conditions for ITS, LSU, and SSU involved an initial denaturation at 98 °C for 3 min, followed by 35 cycles of 98 °C for 20 s for denaturation, 53 °C for 10 s for annealing, and 72 °C for 20 s for extension, and then the final extension at 72 °C for 5 min.

The PCR products were examined using 1% agarose gel electrophoresis with ethidium bromide staining. The presence of distinct bands was confirmed using the Compact Desktop UV Transilluminator Analyzer GL-3120 gel documentation system. The PCR products were sequenced by Tsingke Company (Beijing, China).

Phylogenetic analyses

Newly sequences were blasted to search for closely related taxa in GenBank database (www.ncbi.nlm.nih.gov/blast). Sequences generated from the ITS, LSU, and SSU gene regions were verified before further analyses, using BioEdit v. 7.0.9^[31]. Sequences with high similarity percentages were determined to find the closest matches with taxa and from recently published data in Table 1^[19−21]. Multiple sequence alignments were aligned with MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html)^[32] and automatically trimmed by using TrimA1 (http://phylemon2.bioinfo.cipf.es/index.html)^[33]. A combined sequence dataset was performed with the SquenceMatrix v. 1.7.8^[34].

A maximum likelihood (ML) analysis was conducted using RAxML-HPC2 v. 8.2.12^[35] on the CIPRES Science Gateway web server^[36] (www.phylo.org/portal2), employing 1,000 rapid bootstrap replicates and the GTRGAMMA + I model.

The model of evolution for the Bayesian inference (BI) analysis was performed by using MrModeltest v. 2.3^[37,38]. GTR + I + G was selected as the best-fitting model for the ITS, LSU, and SSU dataset. For the nucleotide substitution model BI analysis was conducted by Markov chain Monte Carlo sampling (BMCMC) to assess posterior probabilities (PP) by using MrBayes v. 3.2.7^[38]. Six simultaneous Markov chains were run for random trees for 1,000,000 generations and trees were sampled every 200^th generation. Bootstrap support values for ML equal to or greater than 75% and Bayesian posterior probabilities (PP) equal to or greater than 0.95 were given above the nodes in the phylogenetic tree (Fig. 1). Phylograms were created using FigTree v. 1.4.0^[39] and subsequently modified in Adobe Photoshop CS6 (Adobe Systems, USA). The completed alignments and phylogenetic trees were then submitted to TreeBASE, with the submission ID 31293 (www.treebase.org).

Figure 1. 

RAxML tree based on analysis of a combined LSU, SSU, and ITS sequences for Savoryellaceae. Bootstrap support values for maximum likelihood (ML) equal to or greater than 75% were given above the nodes (left). Bayesian posterior probability (BIPP) equal to or greater than 0.95 were given above the nodes (right) and hyphen (−) were marked as values below 0.95. The tree was rooted to Pleurotheciella aquatica (MFLUCC 17-0464) and P. erumpens (CBS 142447)^[18]. Two new isolates were shown in red, and ex-type strains are bold.

Best-scoring RA x ML tree for Savoryellaceae based on analysis of the combined LSU, SSU, and ITS datasets. The combined dataset comprised 51 strains with 3,268 characters including gaps (LSU: 1–1,845 bp; SSU: 1,846–2,759 bp; ITS: 2,760–3,268 bp). The tree is rooted with Pleurotheciella aquatica (MFLUCC 17-0464) and P. erumpens (CBS 142447)^[18] and has a final ML likelihood value of –22,514.850213. The matrix had 1,326 distinct alignment patterns, with 36.62% undetermined characters or gaps. The estimated base frequencies were A = 0.226977, C = 0.268195, G = 0.306489, T = 0.198339; the substitution rates were AC = 1.307677, AG = 2.608024, AT = 1.966100, CG = 0.982783, CT = 6.087648, and GT = 1.000000; and the gamma distribution shape parameter α = 0.0010000000. The tree topologies of combined sequence data obtained from ML. BI analyses were not significantly different (Fig. 1).

Phylogenetic analysis showed that Savoryella claviformis (KUNCC 10408 and KUNCC 10495) formed a distinct lineage within the genus, and formed a distinct and sister group (100% ML, 1.00 BIPP) with S. bambusicola (UESTCC 22-0057 and CGMCC 3.23775) (Fig. 1).

Taxonomy

Savoryella claviformis R.J. Xu, S. Boonmee, K.D. Hyde & Q. Zhao, sp. nov. (Fig. 2)

MycoBank: MB853230; Facesoffungi number: FoF 15685

Etymology: The specific epithet 'claviformis' refers to the claviform conidia.

Holotype: HKAS 133191

Figure 2. 

Savoryella claviformis (HKAS 133191, holotype). (a) Colony on nature substrates. (b), (c) Conidiophores and apical conidia. (d) Conidiophore. (e)–(g) Conidia. (h) Culture colony on PDA medium, from surface. (i) Culture colony on PDA medium, from reverse. Scale bars: (b)–(g) = 50 μm.

Saprobic on decaying stems of wood submerged in a freshwater stream habitat. Asexual morph: Colonies effuse, scattered, brown to dark brown. Mycelium immersed, subhyaline to pale brown, composed of branched, septate hyphae. Conidiophores 66–151 × 3–6 μm ($ \overline{x} $ = 99 × 4 μm, n = 20), semi-micronematous, mononematous, straight or slightly flexuous, solitary, cylindrical, smooth-walled, septate, unbranched, pale brown to brown, guttulate, truncate at the apex. Conidiogenous cells holoblastic, monoblastic, terminal, determinate, cylindrical, dark brown to brown, smooth. Conidia 55–160 × 6–12 μm ($ \overline{x} $ = 102 × 9 μm, n = 25), acrogenous, solitary, fusiform, claviform, rostrate, straight or slightly curved, tapering at apex, truncated at the base, 4–6-septate, smooth, guttulate, brown when young, dark brown to black when mature. Sexual morph: Undetermined.

Culture characteristics: Conidium germinated on PDA within 48 h. Mycelia superficial, velvet, irregular, circular, raised near the center, surface villiform, dense, grey mycelium in the center, brown to grey from above, dark brown from below, becoming sparse and paler at the entire margin.

Material examined: CHINA, Xinjiang Province, Bayingoleng Mongolian Autonomous Prefecture, Kaidu River, 41°52'4.8" N, 86°43'39.8" E, 1049 msl, saprobic on submerged decaying wood in freshwater habitats, 22 July 2021, R.J. Xu, MD-325 (HKAS 133191, holotype), ex-type culture KUNCC 10408. Bayingoleng Mongolian Autonomous Prefecture, Bosten Lake, 42°3'4.69" N, 87°8'47.71" E, 1051 msl, saprobic on submerged decaying wood in freshwater habitats, 22 July 2021, R.J. Xu, MD-376 (HKAS 133192), living culture KUNCC 10495.

Notes: Savoryella claviformis can be distinguished from all asexual species in Savoryella by its semi-micronematous conidiophores, terminal, determinate conidiogenous cells, and acrogenous, solitary, fusiform, claviform, rostrate conidia^[19,20]. Additionally, phylogenetic analysis showed that S. claviformis clustered into a distinct subclade and is sister to S. bambusicola, with (100% ML/1.00 BIPP) bootstrap support (Fig. 1). Since only the sexual morph of S. bambusicola has been discovered, it is not possible to compare their morphological differences^[18]. Further comparisons of ITS sequences demonstrate a 7.9% (33/417 bp, excluding gaps) difference between S. claviformis and S. bambusicola. Therefore, following the guidelines of Chethana et al.^[40,41], S. claviformis has been identified as a new species, supported by both morphological and phylogenetic evidence.

Savoryellaceae currently comprises seven genera: Aquabispora, Ascotaiwania, Bactrodesmium, Canalisporium, Dematiosporium, Neoascotaiwania, and Savoryella^{[7,17,18,21,42−44]}. The asexual morphs of these genera are typically characterized by micronematous, often reduced to undifferentiated hyphal conidiophores and monodictys-like conidia. Specifically, Ascotaiwania, which has terminal, blastic, globose, dictyosporous conidia; Bactrodesmium known for its sporodochium-like conidiomata; Canalisporium is characterized by dark brown and muriform conidia; Dematiosporium is known for monodictys-like conidia; and Neoascotaiwania, recognized for its fusiform, dark brown, transversely septate conidia^{[17,21,42−45]}.

The specimens described in this study were collected from freshwater habitats on the Tibetan Plateau in China, increasing our understanding of fungal diversity in this region and enabling comparisons across a north-south gradient in Asia^[1]. In addition, among the 17 accepted species in Savoryella, only four species, including S. cocois, S. chiangraiense, S. nypae, and S. sarushimana, are known as asexual morphs^[19,20]. Savoryella limnetica was observed producing an asexual morph in culture, as reported by Réblová et al.^[46]. However, based on molecular data and culture characteristics, it was synonymized with Neoascotaiwania limnetica by Hernández-Restrepo et al.^[47].

This study exposes a new ascomycetous taxon from the freshwater ecosystems of the Kaidu River and Bosten Lake in Xinjiang, China, marking a new discovery in the region's fungal diversity. Originating from elevations above 4,000 m on the Tibetan Plateau, the unique geographical and climatic conditions of the Kaidu River and Bosten Lake create distinctive niches that support a rich biodiversity, including previously undocumented fungal species. This discovery enriches our understanding of ascomycetes in freshwater ecosystems.

The authors confirm contribution to the paper as follows: study conception and design: Xu RJ, Hyde KD, Zhao Q; data collection: Xu RJ, Guo YY, Yang QY; analysis and interpretation of results: Xu RJ, Dong W, Boonmee S; draft manuscript preparation: Xu RJ. All authors reviewed the results and approved the final version of the manuscript.

The authors of the manuscript confirm that data supporting our study findings are available in the article. Data regarding species/specimen DNA sequences are publically available on the accession provided in Table 1, in the GenBank data base of NCBI.