Morphological and Multi-Gene Phylogenetic Analyses Reveal Pseudotubeufia gen. nov. and Two New Species in Tubeufiaceae from China
by
Jian Ma
1,2,3, 
Xing-Juan Xiao
1,
Ning-Guo Liu
1,
Saranyaphat Boonmee
2,3,
Yuan-Pin Xiao
1 and 
Yong-Zhong Lu
1,* 1
School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550003, China
2
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
3
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
*
Author to whom correspondence should be addressed.
J. Fungi 2023, 9(7), 742; https://doi.org/10.3390/jof9070742
Submission received: 6 June 2023
/
Revised: 9 July 2023
/
Accepted: 10 July 2023
/
Published: 12 July 2023
(This article belongs to the Special Issue Freshwater Fungal Diversity)
Abstract
:Three helicosporous hyphomycete collections representing two species were obtained from rotting wood found in freshwater and terrestrial habitats in the Guizhou and Guangxi Provinces, China. A new genus Pseudotubeufia (Tubeufiaceae, Tubeufiales), comprising Ps. hyalospora sp. nov. and Ps. laxispora sp. nov., was introduced with morphological characteristic and molecular data. In addition, the molecular evidence showed that Helicomyces sp. (G.M. 2020-09-19.1), H. roseus (CBS: 102.76), and the new genus Pseudotubeufia clustered together with high support based on a multi-gene (LSU, ITS, tef1α, and rpb2) phylogenetic analysis. Detailed descriptions, illustrations, and notes of the three new collections are provided.
1. Introduction
Tubeufia was first introduced by Penzig and Saccardo [1], which included the type species T. javanica and two other species (T. anceps and T. coronata). Based on Tubeufia, the family Tubeufiaceae and order Tubeufiales were subsequently established [2,3]. The latest comprehensive study on Tubeufiaceae was carried out by Lu et al. [4]. Currently, there are 46 accepted genera in the family Tubeufiaceae, including Acanthohelicospora, Acanthophiobolus, Acanthostigma, Acanthostigmina, Acanthotubeufia, Aquaphila, Artocarpomyces, Berkleasmium, Bifrontia, Boerlagiomyces, Chaetosphaerulina, Chlamydotubeufia, Dematiohelicoma, Dematiohelicomyces, Dematiohelicosporum, Dematiotubeufia, Dictyospora, Helicangiospora, Helicoarctatus, Helicodochium, Helicohyalinum, Helicoma, Helicomyces, Helicosporium, Helicotruncatum, Helicotubeufia, Kamalomyces, Kevinhydea, Manoharachariella, Muripulchra, Neoacanthostigma, Neochlamydotubeufia, Neohelicoma, Neohelicomyces, Neohelicosporium, Neomanoharachariella, Neotubeufia, Parahelicomyces, Pleurohelicosporium, Podonectria, Pseudohelicoon, Tamhinispora, Thaxteriella, Thaxteriellopsis, Tubeufia, and Zaanenomyces [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Among them, five genera, viz. Acanthophiobolus, Bifrontia, Boerlagiomyces, Podonectria, and Thaxteriella, only have morphological data available, and their systematic evolutionary relationships have not been confirmed by molecular data.
Helicosporous hyphomycetes are asexual fungi that produce various forms of coiled two- or three-dimensional hollow conidia, which is the most common asexual morph in the family Tubeufiaceae [4,17,18,19,20,21]. The classification of helicosporous hyphomycetes has been studied for more than 200 years [22,23,24]. These fungi are widely distributed in tropical and subtropical regions, mostly acting as saprobes on plant litter, rotten wood, and decaying twigs in freshwater and terrestrial habitats [4,20,21]. However, there have been rare reports of endophytic fungi with coiled conidia [25,26].
In this study, three new collections from the family Tubeufiaceae were obtained during a survey of helicosporous hyphomycetes from the Guizhou and Guangxi Provinces, China. Based on detailed morphological comparisons and multi-gene phylogenetic analyses, we introduced a new genus named Pseudotubeufia, which comprises two new species, Ps. hyalospora and Ps. laxispora.
2. Materials and Methods
2.1. Sample Collection, Specimen Examination, and Isolation
Fresh specimens of submerged rotting wood were collected from May to August 2021 in the Guizhou and Guangxi provinces in southern China. The newly collected samples were processed following the method described by Boonmee et al. [3]. The colonies on the host surfaces were examined and observed with stereomicroscopes (SMZ 745 and SMZ 800N, Nikon, Tokyo, Japan). Their micro-morphological characters were studied using an ECLIPSE Ni compound microscope (Nikon, Tokyo, Japan) and a Canon 90D digital camera. Measurements were made with the Tarosoft (R) Image Frame Work program. Photo-plates were made using Adobe Illustrator CC 2019 (Adobe Systems, San Jose, CA, USA).
Single spores were isolated on potato dextrose agar (PDA) medium and the germinated conidia were aseptically transferred to fresh PDA plates, as described in Senanayake et al. [27]. Fungal colonies growing on the PDA were incubated at 25 °C for 28 or 42 days, and their morphological characteristics, including color and size, were recorded. Dried fungal specimens were deposited in the herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (Herb. HKAS), Kunming, China, and in the herbarium of the Guizhou Academy of Agriculture Sciences (Herb. GZAAS), Guiyang, China. Ex-type living cultures were deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, and the Guizhou Culture Collection, China (GZCC). Facesoffungi numbers (FoF) and Index Fungorum numbers were determined according to the guidelines of Jayasiri et al. [28] and the Index Fungorum (2023) [29], respectively.
2.2. DNA Extraction, PCR Amplification, and Sequencing
Fresh fungal mycelia were scraped using the methods described by Lu et al. [30]. Genomic DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, Shanghai, China), according to the manufacturer’s protocol. The large subunit of the ribosomal DNA (LSU), the internal transcribed spacer (ITS), the translation elongation factor 1 alpha (tef1α), and the RNA polymerase II second largest subunit (rpb2) gene regions were amplified using LR0R/LR5, ITS5/ITS4, EF1-983F/EF1-2218R, and fRPB2-5F/fRPB2-7cR primer pairs, respectively [31,32,33,34]. PCR amplification was performed in a reaction volume of 50 μL, including 44 μL 1.1 × T3 Supper PCR Mix (Qingke Biotech, Chongqing, China), 2 μL of each forward and reverse primer, and a 2 μL DNA template. The LSU, ITS, tef1α, and rpb2 amplification reactions were carried out according to the following reference method (Table 1) [4,13,30,35,36,37].
The quality of the PCR products was checked on 1% agarose gel electrophoresis stained with ethidium bromide. The purification and sequencing of the PCR products were carried out at Tsingke Bio-logical Engineering Technology and Services Co., Ltd. (Chongqing, China).
2.3. Phylogenetic Analyses
The original sequences of our newly obtained strains were checked and assembled using BioEdit v 7.0.5.3 and SeqMan v. 7.0.0 (DNASTAR, Madison, WI) [38,39]. The closest taxa to our strains were determined by blast searches in GenBank (http://www.ncbi.nlm.nih.gov/, accessed on 10 May 2023). The other sequences used in the phylogenetic analysis (Table 2) were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/, accessed on 10 May 2023). The sequence alignments for each locus were performed using the online multiple alignment program MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/, accessed on 10 May 2023) [40], and auto-adjusted by trimAl v1.2 [41]. The multigenic sequences (LSU-ITS-tef1α-rpb2) were merged using the SequenceMatrix-Windows-1.7.8 software, and the sequences were exported to CIPRES for RAxML analyses [42]. The aligned Fasta and Phylip format file was converted to a Nexus format file for Bayesian inference (BI) and Maximum Parsimony (MP) analyses by using AliView v. 1.27 [43]. A phylogenetic tree, which infers phylogenetic relationships, was reconstructed based on a concatenated LSU, ITS, tef1α, and rpb2 dataset using the online CIPRES Science Gateway (https://www.phylo.org/portal2/home.action, accessed on 10 May 2023) to construct the Maximum Likelihood (ML), Maximum Parsimony (MP), and Bayesian inference (BI), respectively.
The maximum likelihood (ML) analysis was carried out with the RAxML-HPC2 tool on XSEDE (8.2.12) using a GTRGAMMA approximation with a rapid bootstrap analysis, followed by 1000 bootstrap replicates [44].
The maximum parsimony (MP) analysis was performed by using PAUP on the XSEDE (4.a168) tool. A heuristic search with 1000 random taxa was added to infer MP trees. The value of the MaxTrees, which collapsed branches of zero length and saved all the multiple parsimonious trees, was set to 5000. The parsimony score values of the tree length (TL), consistency index (CI), retention index (RI), and homoplasy index (HI) were calculated for the trees generated under different optimum criteria. The clade stability was estimated using a bootstrap analysis with 1000 replicates, and the taxa were added for a random stepwise of each with 10 replicates [45].
The Bayesian inference (BI) analysis was conducted in MrBayes on XSEDE (3.2.7a) [46]. The best-fit substitution model GRT + I + G was determined for the LSU, ITS, tef1α, and rpb2 matrix using MrModeltest 2.3 [47] under the Akaike Information Criterion (AIC). Four simultaneous Markov chains were run for 10,000,000 generations, and trees were sampled every 1000th generation. The burn-in phase was set at 25% and the remaining trees were used to calculate the posterior probabilities (PP).
The phylogenetic tree and photo-plates were created using FigTree v. 1.4.4., Adobe Illustrator CC 2019 v. 23.1.0 (Adobe Systems, San Jose, CA, USA), and Adobe PhotoShop CC 2019 (Adobe Systems, San Jose, CA, USA).
3. Results
3.1. Phylogenetic Analysis
The partial LSU-ITS-tef1α-rpb2 nucleotide sequences were used to determine the phylogenetic positions of the newly obtained isolates. These sequences were concatenated to generate a sequence matrix consisting of LSU (1–843 bp), ITS (844–1548 bp), tef1α (1549–2460 bp), and rpb2 (2461–3505 bp) regions. The resulting matrix comprised a total of 3505 characters for 105 taxa and two outgroups, Botryosphaeria agaves (MFLUCC 10–0051) and B. dothidea (CBS 115476). The total characters analyzed in the concatenated dataset were 3505, out of which, 2002 characters were constant, 273 variable characters were parsimony-uninformative, and 1230 characters were parsimony-informative. The ML, MP, and BI analyses of the concatenated LSU-ITS-tef1α-rpb2 dataset yielded similar tree topologies, and the ML tree is shown in Figure 1.
In the phylogenetic analyses (Figure 1), the newly isolated strains GZCC 22–2011 and GZCC 22–2012 clustered together (95% ML/100% MP/1 PP) without a significant branch length, indicating that they are phylogenetically the same species, as Pseudotubeufia laxispora sp. nov. Pseudotubeufia hyalospora sp. nov. formed a sister clade with Ps. laxispora with 91% ML/100% MP/0.97 PP supports. In addition, the three strains of Pseudotubeufia clustered with Helicomyces sp. (G.M. 2020-09-19.1), Helicomyces roseus (CBS 102.76), and Dematiohelicoma pulchrum (MUCL 39827) with weak support.
3.2. Taxonomy
Pseudotubeufia J. Ma & Y.Z. Lu, gen. nov.
Index Fungorum number: IF900553; Facesoffungi number: FoF 03700.
Etymology: “Pseudotubeufia”, referring to the genus morphologically similar to the helicosporous asexual morph of Tubeufia.
Saprobic on the decaying wood in a freshwater stream. The sexual morph was undetermined. The asexual morph was helicosporous hyphomycetes. The colonies on the substratum were superficial, effuse, gregarious, and white. The mycelium were partly immersed, composed of hyaline to pale brown, septate, branched, and smooth hyphae. The conidiophores were macronematous, mononematous, erect or procumbent, flexuous, cylindrical, branched or unbranched, septate, hyaline to brown, and smooth-walled. The conidiogenous cells were holoblastic, mono- to polyblastic, integrated, sympodial, repeatedly geniculate, intercalary or terminal, irregularly cylindrical, denticulate, hyaline to pale brown, and smooth-walled. The conidia were solitary, acropleurogenous, helicoid, rounded at the tip, coiled 2–3 times, became loose in water, indistinctly septate, guttulate, hyaline, and smooth-walled.
Type species: Pseudotubeufia hyalospora J. Ma & Y.Z. Lu.
Notes: Morphologically, Pseudotubeufia is the most similar to Tubeufia as it has flexuous, cylindrical conidiophores, cylindrical, denticulate, hyaline to pale brown conidiogenous cells, and hyaline helicoid conidia [4]. However, the phylogenetic analysis result showed that Pseudotubeufia has a close affinity with the species of Dematiohelicoma and Helicomyces, and is distant from the group of Tubeufia (Figure 1). However, Dematiohelicoma can be distinguished from Pseudotubeufia by its erect conidiophores and multi-septate, brown to dark brown conidia. Pseudotubeufia is also easily distinguished from Helicomyces by its repeatedly geniculate conidiogenous cells [4]. Therefore, the new genus Pseudotubeufia is introduced to accommodate two species, Ps. hyalospora and Ps. laxispora.
Pseudotubeufia hyalospora J. Ma & Y.Z. Lu., sp. nov., Figure 2.
Index Fungorum number: IF900554; Facesoffungi number: FoF 14268.
Etymology: The epithet “hyalospora”, referring to hyaline helicoid conidia.
Holotype: HKAS 125885.
Saprobic on the decaying wood in a freshwater stream. The sexual morph was undetermined. The asexual morph was helicosporous hyphomycetes. The colonies on the substratum were superficial, effuse, gregarious, and white. The mycelium were partly immersed, composed of hyaline to pale brown, septate, branched, and smooth hyphae. The conidiophores were 31–46 μm long, 3–5.5 μm wide, macronematous, mononematous, procumbent, flexuous, cylindrical, branched, septate, hyaline to pale brown, and smooth-walled. The conidiogenous cells were 5.5–27.5 μm long, 3–5 μm wide, holoblastic, mono- to polyblastic, integrated, sympodial, repeatedly geniculate, intercalary or terminal, irregularly cylindrical, denticulate, hyaline to pale brown, and smooth-walled. The conidia were solitary, acropleurogenous, helicoid, rounded at the tip, 35–58 μm in diam. and had conidial filaments 4–5.5 μm wide ( = 48 × 4.5 μm, n = 20), 201–316 μm long, coiled 2–3 times, became loose in water, were indistinctly septate, guttulate, and hyaline.
Culture characteristics: The conidia germinated on the PDA within 10 h. The colonies on the PDA were irregular, with a flat surface, edge undulate, were pale brown to brown from above and below, and reached a 28 mm diam. after 42 days of incubation at 25 °C.
Material examined: China, Guizhou Province, Qiandongnan Miao and Dong Autonomous Prefecture, Zhenyuan City, 27°18′ N, 108°21′ E, on rotting wood in a freshwater stream, 1 May 2021, Xing-Juan Xiao, XXJ11.2 (HKAS 125885, holotype; GZAAS 22–2010, isotype), ex-type living cultures, CGMCC, GZCC 22–2010.
Notes: Morphologically, Ps. hyalospora is similar to Ps. laxispora (HKAS 125868), as it has flexuous, branched conidiophores, repeatedly geniculate conidiogenous cells, and acropleurogenous, guttulate, hyaline helicoid conidia. However, Pseudotubeufia hyalospora differs from Ps. laxispora (HKAS 125868) in having shorter conidiophores (31–46 μm vs. up to 155 μm), shorter conidiogenous cells (5.5–27.5 μm vs. up to 39 μm), and a different colony morphology in PDA (irregular, undulate edge vs. circular, entire edge). In addition, the phylogenetic analysis result showed that they are a distinct species. In accordance with the recommendations of Jeewon and Hyde [48] for species delimitation, we analyzed the pairwise dissimilarities of the DNA sequences between Ps. hyalospora (GZCC 22–2010) and Ps. laxispora (GZCC 22–2011) and found 60/905 bp (6.6%) differences in the tef1α gene. Therefore, we propose Pseudotubeufia hyalospora as a new species.
Pseudotubeufia laxispora J. Ma & Y.Z. Lu, sp. nov., Figure 3.
Index Fungorum number: IF900555; Facesoffungi number: FoF 14269.
Etymology: The epithet “laxispora”, referring to loosely coiled conidia.
Holotype: HKAS 125868.
Holotype: Saprobic on dead bamboo culms in a freshwater stream. The sexual morph was undetermined. The asexual morph was helicosporous hyphomycetes. The colonies on the substratum were superficial, effuse, gregarious, and white. The mycelium were partly immersed, composed of hyaline to pale brown, septate, and abundantly branched hyphae. The conidiophores were 30–155 μm long, 3.5–6.5 μm wide, macronematous, mononematous, procumbent, flexuous, irregular cylindrical, branched, septate, hyaline to pale brown, and smooth-walled. The conidiogenous cells were 10–39 μm long, 3.5–6 μm wide, holoblastic, mono- to polyblastic, integrated, sympodial, intercalary or terminal, cylindrical, repeatedly geniculate, hyaline to pale brown, and smooth-walled. The conidia were solitary, acropleurogenous, helicoid, rounded at the tip, 35–56 μm in diam. and had conidial filaments that were 3–6.5 μm wide ( = 45 × 4.5 μm, n = 20), 242–327 μm long, loosely coiled 21/4–23/4 times, became loosely coiled in water, were indistinctly multi-septate, guttulate, hyaline, and smooth-walled; Paratype (Figure 4): Saprobic on the decaying wood in a terrestrial habitat. The sexual morph was undetermined. The asexual morph was helicosporous hyphomycetes. The colonies on the substratum were superficial, effuse, gregarious, and white. The mycelium were partly immersed, composed of hyaline to pale brown, septate, and abundantly branched hyphae. The conidiophores were 21–184 μm long, 3.5–9 μm wide, macronematous, mononematous, erect, flexuous, cylindrical, branched, septate, with the lower part dark brown and the upper part hyaline to pale brown, and smooth-walled. The conidiogenous cells were 4.5–33.5 μm long, 3–5.5 μm wide, holoblastic, mono- to polyblastic, integrated, sympodial, with arising tiny bladder-like protrusions, intercalary or terminal, cylindrical, truncate at apex after conidial secession, hyaline to pale brown, and smooth-walled. The conidia were solitary, acropleurogenous, helicoid, rounded at the tip, 36–50.5 μm in diam. and had conidial filaments that were 3.5–6 μm wide ( = 42 × 4.5 μm, n = 20), 189–231 μm long, coiled 11/2–21/2 times, became loosely coiled in water, were indistinctly multi-septate, guttulate, hyaline, and smooth-walled.
Culture characteristics: Holotype: The conidia germinated on the PDA within 10 h. The colonies on the PDA were circular, with a flat surface, edge entire, pale brown to brown from above and below, and reached 33 mm in diam. after 42 days of incubation at 25 °C; Paratype: The conidia germinated on the PDA within 10 h. The colonies on the PDA were circular, with a flat surface, edge entire, dark brown from above and below, and reached 22 mm in diam. after 28 days of incubation at 25 °C.
Material examined: China, Guangxi Province, Liuzhou City, Luzhai County, 24°46′ N, 109°53′ E, on dead bamboo culms in a freshwater stream, 4 May 2021, Jian Ma, LZ6.2 (HKAS 125868, holotype; GZAAS 22–2011, isotype), ex-type living cultures, CGMCC, GZCC 22–2011; China, Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Sandu City, 25°56′ N, 107°57′ E, on decaying wood in a terrestrial habitat, 12 August 2021, Jingyi Zhang, SD12 (GZAAS 22–2012; paratype), living culture GZCC 22–2012.
Notes: Two collections, HKAS 125868 and GZAAS 22–2012, were obtained from freshwater and terrestrial habitats in southern China. Morphologically, HKAS 125868 has procumbent and hyaline conidiophores, while GZAAS 22–2012 has erect and brown conidiophores. Additionally, GZAAS 22–2012 has smaller conidia compared to HKAS 125868 (189–231 μm vs. 242–327 μm). However, based on pairwise nucleotide comparisons of ITS, LSU, tef1α, and rpb2, GZCC 22–2011 only differs from GZCC 22–2012 in a few genetic markers (2/469 bp for ITS, 1/824 bp for LSU, 1/916 bp for tef1α, and 13/1113 bp for rpb2). Furthermore, the phylogenetic analysis did not show any significant differences between these two strains (Figure 1). Therefore, despite their distinct morphology, we introduce these two isolates as one species named Pseudotubeufia laxispora.
4. Conclusions
In this study, we introduced a new genus, Pseudotubeufia, based on multi-gene phylogenetic analyses and morphological characteristics. Morphologically, the asexual morphs of Ps. hyalospora and Ps. laxispora (HKAS 125868) are most similar to the species of Tubeufia, while Ps. laxispora (GZAAS 22–2012) resembles the species of Parahelicomyces. However, the multi-gene phylogenetic analyses showed that they did not cluster with Tubeufia or Parahelicomyces. Instead, they formed a distinct sister clade with the strains Helicomyces sp. (G.M. 2020-09-19.1, GenBank: MW276143) and H. roseus (CBS: 102.76), with 100% ML/100% MP/1 PP supports (Figure 1).
The ITS sequences of Ps. hyalospora and Ps. laxispora were searched using BLASTn in NCBI GenBank, and they exhibited the highest similarities to Helicomyces sp. (G.M. 2020-09-19.1: 58% query cover, 97.49% similarity and 100% query cover, 97.53% similarity), Helicomyces roseus (CBS 102.76: 58% query cover, 97.11% similarity and 100% query cover, 97.35% similarity), and Tubeufia sp. (MFLUCC 17–1520 and KUMCC 21–0472: 97% query cover, 84.98% similarity and 99% query cover, 86% similarity), respectively. In order to confirm the phylogenetic positions of the newly isolated strains, we performed single-gene and multi-gene phylogenetic analyses, including all species of the genera Tubeufia, Parahelicomyces, Helicomyces, and other related taxa, and obtained the same conclusion as shown in Figure 1. It is worth noting that Helicomyces sp. (G.M. 2020-09-19.1) and H. roseus (CBS 102.76) currently lack morphological descriptions and only have molecular data [49]. Their taxonomic positions require further molecular data and morphological descriptions for clarification.
Morphological differences can vary widely, even within the same species of helicosporous hyphomycetes. For instance, two collections (MFLU 16–2544 from decaying wood in China and MFLU 17–1091 from decaying wood in Thailand) have been identified as the same species, namely Tubeufia aquatica [4,50]. However, MFLU 16–2544 has larger conidiophores (109.5–189.5 μm) than those of MFLU 17–1091 (18–40 μm). Additionally, the conidiophores of MFLU 16–2544 are multi-septate, branched, and brown to dark brown, while those of MFLU 17–1091 are 0–1-septate, unbranched, and pale brown [4,50]. Similarly, our two collections of Ps. laxispora (HKAS 125868 and GZAAS 22–2012) showed significant differences in their conidiophores (Figure 3 and Figure 4). We speculate that such differences may be attributable to variations in their habitats and geographical regions.
Author Contributions
Morphological data, photo-plates, and phylogenetic analyzes were completed by J.M. and X.-J.X. The original draft was written by J.M. and N.-G.L., S.B., Y.-P.X. and Y.-Z.L. revised the paper. All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by Guizhou Provincial Key Technology R&D Program (grant No. Qian Ke He Zhi Cheng [2021] Generally 200), Guizhou Province High-level Talent Innovation and Entrepreneurship Merit Funding Project (No. 202104), and Youth Science and Technology Talent Development Project from Guizhou Provincial Department of Education (QJHKYZ [2021]263).
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
All sequences generated in this study were submitted to GenBank database.
Acknowledgments
We would like to thank Shaun Pennycook (Manaaki Whenua Landcare Research, New Zealand) for advising on fungal nomenclature.
Conflicts of Interest
All authors declare no conflict of interest.
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Figure 1.
Phylogram generated from the maximum likelihood analysis based on a combined of LSU, ITS, tef1α, and rpb2 sequence data. Bootstrap support values of maximum likelihood (ML) and maximum parsimony (MP) equal to or greater than 75%, and Bayesian posterior probabilities (PP) equal to or greater than 0.95 are given near the nodes as ML/MP/PP. Botryosphaeria agaves (MFLUCC 10–0051) and B. dothidea (CBS 115476) were used as outgroup taxa. The newly generated sequences are shown in red bold.
Figure 1.
Phylogram generated from the maximum likelihood analysis based on a combined of LSU, ITS, tef1α, and rpb2 sequence data. Bootstrap support values of maximum likelihood (ML) and maximum parsimony (MP) equal to or greater than 75%, and Bayesian posterior probabilities (PP) equal to or greater than 0.95 are given near the nodes as ML/MP/PP. Botryosphaeria agaves (MFLUCC 10–0051) and B. dothidea (CBS 115476) were used as outgroup taxa. The newly generated sequences are shown in red bold.
Figure 2.
Pseudotubeufia hyalospora (HKAS 125885, holotype). (a,b) Colonies on the host surface. (c–g,i,m,n) Conidiophores with attached conidia. (h,j–l,q,r) Conidiophores and conidiogenous cells. (o,p,s–u) Conidia. (v) Germinating conidium. (w,x) Colonies on PDA at 42 days old (from above and below). Scale bars: (c–i,m,o,p,s–v) 20 µm, (j–l,n,q,r) 10 µm.
Figure 2.
Pseudotubeufia hyalospora (HKAS 125885, holotype). (a,b) Colonies on the host surface. (c–g,i,m,n) Conidiophores with attached conidia. (h,j–l,q,r) Conidiophores and conidiogenous cells. (o,p,s–u) Conidia. (v) Germinating conidium. (w,x) Colonies on PDA at 42 days old (from above and below). Scale bars: (c–i,m,o,p,s–v) 20 µm, (j–l,n,q,r) 10 µm.
Figure 3.
Pseudotubeufia laxispora (HKAS 125868, holotype). (a,b) Colonies on the host surface. (c–i) Conidiophores with attached conidia. (j,k,m) Conidiogenous cells. (l) Germinating conidium. (n–p) Conidia. (q,r) Colonies on PDA at 42 days old (from above and below). Scale bars: (c–i,l,m) 20 µm, (j,k,n–p) 10 µm.
Figure 3.
Pseudotubeufia laxispora (HKAS 125868, holotype). (a,b) Colonies on the host surface. (c–i) Conidiophores with attached conidia. (j,k,m) Conidiogenous cells. (l) Germinating conidium. (n–p) Conidia. (q,r) Colonies on PDA at 42 days old (from above and below). Scale bars: (c–i,l,m) 20 µm, (j,k,n–p) 10 µm.
Figure 4.
Pseudotubeufia laxispora (GZAAS 22–2012, paratype). (a,b) Colonies on host surface. (c–g,j) Conidiophores with attached conidia. (h,i) Conidiogenous cells. (m–p) Conidia. (q) Germinating conidium. (k,l) Colonies on PDA at 28 days old (from above and below). Scale bars: (c–f) 20 µm, (g,h,j–q) 10 µm, (i) 5 µm.
Figure 4.
Pseudotubeufia laxispora (GZAAS 22–2012, paratype). (a,b) Colonies on host surface. (c–g,j) Conidiophores with attached conidia. (h,i) Conidiogenous cells. (m–p) Conidia. (q) Germinating conidium. (k,l) Colonies on PDA at 28 days old (from above and below). Scale bars: (c–f) 20 µm, (g,h,j–q) 10 µm, (i) 5 µm.
Table 1.
PCR protocols.
| Locus | Primer | Initial Denaturation | Denaturation | Annealing | Elongation | Final Extension | Hold |
|---|---|---|---|---|---|---|---|
| LSU | LR0R/LR5 | 95 °C/3 min | 94 °C/30 s | 51 °C/50 s | 72 °C/1 min | 72 °C/7 min | 4 °C |
| 30 cycles | |||||||
| ITS | ITS5/ITS4 | 95 °C/3 min | 95 °C/30 s | 51 °C/1 min | 72 °C/45 s | 72 °C/10 min | |
| 34 cycles | |||||||
| tef1α | EF1-983F/EF1-2218R | 95 °C/3 min | 94 °C/30 s | 55 °C/50 s | 72 °C/1 min | 72 °C/7 min | |
| 40 cycles | |||||||
| rpb2 | fRPB2-5F/fRPB2-7cR | 95 °C/3 min | 95 °C/30 s | 54 °C/40 s | 72 °C/1 min | 72 °C/7 min | |
| 34 cycles | |||||||
Table 2.
Taxa used in this study and their GenBank accession numbers.
| Taxon | Strain | GenBank Accessions | |||
|---|---|---|---|---|---|
| LSU | ITS | tef1α | rpb2 | ||
| Acanthohelicospora aurea | NBRC 7098 | AY856894 | AY916478 | – | – |
| Acanthohelicospora guianensis | UAMH 1699 | AY856891 | AY916479 | – | – |
| Acanthohelicospora pinicola | MFLUCC 10-0116 T | KF301534 | KF301526 | KF301555 | – |
| Acanthohelicospora scopula | ANM 386 | GQ850489 | GQ856141 | – | – |
| Acanthostigma chiangmaiensis | MFLUCC 10-0125 T | JN865197 | JN865209 | KF301560 | – |
| Acanthostigma perpusillum | UAMH 7237 | AY856892 | AY916492 | – | – |
| Acanthostigmina minutum | ANM 238 | GQ850487 | – | – | – |
| Acanthostigmina minutum | ANM 880 | GQ850486 | – | – | – |
| Acanthotubeufia filiforme | ANM 101 T | GQ850495 | – | – | – |
| Acanthotubeufia filiforme | ANM 514 | GQ850494 | GQ856146 | – | – |
| Aquaphila albicans | MFLUCC 16-0010 | KX454166 | KX454165 | KY117034 | MF535255 |
| Aquaphila albicans | MFLUCC 16-0020 | KX454168 | KX454167 | – | MF535256 |
| Berkleasmium aquaticum | MFLUCC 17-0049 T | KY790432 | KY790444 | KY792608 | MF535268 |
| Berkleasmium fusiforme | MFLUCC 17-1987 T | MH558822 | MH558695 | MH550886 | MH551009 |
| Berkleasmium guangxiense | MFLUCC 17-0042 T | KY790436 | KY790448 | KY792612 | MF535270 |
| Berkleasmium latisporum | MFLUCC 16-0019 T | KY790437 | KY790449 | KY792613 | MF535271 |
| Berkleasmium longisporum | MFLUCC 17-1999 T | MH558825 | MH558698 | MH550889 | MH551012 |
| Berkleasmium thailandicum | MFLUCC 15-1248 T | MH558829 | KX454176 | KY792614 | MH551017 |
| Boerlagiomyces macrospora | MFLUCC 12-0388 | KU764712 | KU144927 | KU872750 | – |
| Botryosphaeria agaves | MFLUCC 10-0051 | JX646807 | JX646790 | – | – |
| Botryosphaeria dothidea | CBS 115476 | DQ678051 | KF766151 | DQ767637 | DQ677944 |
| Chlamydotubeufia cylindrica | MFLUCC 16-1130 T | MH558830 | MH558702 | MH550893 | MH551018 |
| Chlamydotubeufia krabiensis | MFLUCC 16-1134 | KY678759 | KY678767 | KY792598 | MF535261 |
| Dematiohelicoma pulchrum | MUCL 39827 | AY856872 | AY916457 | – | – |
| Dematiohelicomyces helicosporus | MFLUCC 16-0213 T | KX454170 | KX454169 | KY117035 | MF535258 |
| Dematiohelicomyces helicosporus | MFLUCC 16-0003 | MH558831 | MH558703 | MH550894 | MH551019 |
| Dematiohelicosporum guttulatum | MFLUCC 17-2011 T | MH558833 | MH558705 | MH550896 | MH551021 |
| Dematiotubeufia chiangraiensis | MFLUCC 10-0115 T | JN865188 | JN865200 | KF301551 | – |
| Dictyospora thailandica | MFLUCC 16-0001 T | KY873622 | KY873627 | KY873286 | MH551023 |
| Dictyospora thailandica | MFLUCC 18-0641 | MH558834 | MH558706 | MH550897 | MH551022 |
| Dictyospora thailandica | MFLUCC 16-0215 | KY873623 | KY873628 | KY873287 | – |
| Helicangiospora lignicola | MFLUCC 11-0378 T | KF301531 | KF301523 | KF301552 | – |
| Helicoarctatus aquaticus | MFLUCC 17-1996 T | MH558835 | MH558707 | MH550898 | MH551024 |
| Helicodochium aquaticum | MFLUCC 17-2016 T | MH558837 | MH558709 | MH550900 | MH551026 |
| Helicodochium aquaticum | MFLUCC 18-0490 | MH558838 | MH558710 | MH550901 | MH551027 |
| Helicohyalinum aquaticum | MFLUCC 16-1131 T | KY873620 | KY873625 | KY873284 | MF535257 |
| Helicohyalinum aquaticum | MFLUCC 16-1133 T | MH558840 | MH558712 | MH550903 | MH551029 |
| Helicoma guttulatum | MFLUCC 16-0022 T | KX454172 | KX454171 | MF535254 | MH551032 |
| Helicoma inthanonense | MFLUCC 11-0003 T | JN865199 | JN865211 | – | – |
| Helicoma siamense | MFLUCC 10-0120 T | JN865192 | JN865204 | KF301558 | – |
| Helicoma tectonae | MFLUCC 12-0563 T | KU764713 | KU144928 | KU872751 | – |
| Helicomyces sp. | G.M. 2020-09-19.1 | – | MW276143 | – | – |
| Helicomyces chiayiensis | BCRC FU30842 T | – | LC316604 | – | – |
| Helicomyces colligatus | MFLUCC 16-1132 | MH558853 | MH558727 | MH550918 | MH551043 |
| Helicomyces hyalosporus | MFLUCC 17-0051 T | MH558857 | MH558731 | MH550922 | MH551047 |
| Helicomyces hyalosporus | GZCC 16-0070 | MH558854 | MH558728 | MH550919 | MH551044 |
| Helicomyces hyalosporus | GZCC 16-0073 | MH558855 | MH558729 | MH550920 | MH551045 |
| Helicomyces hyalosporus | GZCC 16-0075 | MH558856 | MH558730 | MH550921 | MH551046 |
| Helicomyces roseus | CBS: 102.76 | MH872733 | MH860964 | – | – |
| Helicomyces torquatus | MFLUCC 16-0217 | MH558858 | MH558732 | MH550923 | MH551048 |
| Helicosporium aquaticum | MFLUCC 17-2008 T | MH558859 | MH558733 | MH550924 | MH551049 |
| Helicosporium luteosporum | MFLUCC 16-0226 T | KY321327 | KY321324 | KY792601 | MH551056 |
| Helicosporium setiferum | MFLUCC 17-1994 T | MH558861 | MH558735 | MH550926 | MH551051 |
| Helicosporium viridiflavum | MFLUCC 17-2336 T | – | MH558738 | MH550929 | MH551054 |
| Helicotruncatum palmigenum | NBRC 32663 | AY856898 | AY916480 | – | – |
| Helicotubeufia hydei | MFLUCC 17-1980 T | MH290026 | MH290021 | MH290031 | MH290036 |
| Helicotubeufia jonesii | MFLUCC 17-0043 T | MH290025 | MH290020 | MH290030 | MH290035 |
| Kamalomyces bambusicola | MFLU 11-0228 T | MF506880 | – | – | – |
| Kamalomyces thailandicus | MFLUCC 11-0158 | MF506881 | MF506883 | MF506885 | – |
| Kamalomyces thailandicus | MFLUCC 13-0233 T | MF506882 | MF506884 | MF506886 | – |
| Kevinhydea brevistipitata | MFLUCC 18-1269 T | MH747115 | MH747102 | – | – |
| Manoharachariella tectonae | MFLUCC12-0170 T | KU764705 | KU144935 | KU872762 | – |
| Muripulchra aquatica | KUMCC 15-0276 | KY320551 | KY320534 | KY320564 | MH551058 |
| Muripulchra aquatica | MFLUCC 15-0249 T | KY320549 | KY320532 | – | – |
| Neoacanthostigma fusiforme | MFLUCC 11-0510 T | KF301537 | KF301529 | – | – |
| Neochlamydotubeufia fusiformis | MFLUCC 16-0016 T | MH558865 | MH558740 | MH550931 | MH551059 |
| Neochlamydotubeufia khunkornensis | MFLUCC 10-0118 T | JN865190 | JN865202 | KF301564 | – |
| Neohelicoma fagacearum | MFLUCC 11-0379 T | KF301532 | KF301524 | KF301553 | – |
| Neohelicomyces aquaticus | MFLUCC 16-0993 T | KY320545 | KY320528 | KY320561 | MH551066 |
| Neohelicomyces grandisporus | KUMCC 15-0470 T | KX454174 | KX454173 | – | MH551067 |
| Neohelicomyces hyalosporus | GZCC 16-0086 T | MH558870 | MH558745 | MH550936 | MH551064 |
| Neohelicomyces submersus | MFLUCC 16-1106 T | KY320547 | KY320530 | – | MH551068 |
| Neohelicosporium astrictum | MFLUCC 17-2004 T | MH558872 | MH558747 | MH550938 | MH551070 |
| Neohelicosporium fusisporum | MFUCC 16-0642 T | MG017613 | MG017612 | MG017614 | – |
| Neohelicosporium hyalosporum | GZCC 16-0076 T | MF467936 | MF467923 | MF535249 | MF535279 |
| Neohelicosporium krabiense | MFLUCC 16-0224 T | MH558879 | MH558754 | MH550945 | MH551077 |
| Neohelicosporium ovoideum | GZCC 16-0064 T | MH558881 | MH558756 | MH550947 | MH551079 |
| Neohelicosporium parvisporum | MFLUCC 17-1523 T | MF467939 | MF467926 | MF535252 | MF535282 |
| Neotubeufia krabiensis | MFLUCC 16-1125 T | MG012024 | MG012031 | MG012010 | MG012017 |
| Parahelicomyces aquaticus | MFLUCC 16-0234 T | MH558891 | MH558766 | MH550958 | MH551092 |
| Parahelicomyces hyalosporus | MFLUCC 15-0343 T | KY320540 | KY320523 | – | – |
| Parahelicomyces indicus | CBS 374.93 | AY856885 | AY916477 | – | – |
| Parahelicomyces paludosus | CBS 120503 | DQ341103 | DQ341095 | – | – |
| Parahelicomyces roseus | KUMCC 15-0411 | KY320544 | KY320527 | KY320560 | – |
| Parahelicomyces talbotii | MFLUCC 17-2021 | MH558890 | MH558765 | MH550957 | MH551091 |
| Pleurohelicosporium parvisporum | MFLUCC 17-1982 T | MH558889 | MH558764 | MH550956 | MH551088 |
| Pseudohelicoon gigantisporum | BCC 3550 | AY856904 | AY916467 | – | – |
| Pseudohelicoon subglobosum | BCRC FU30843 T | LC316610 | LC316607 | – | – |
| Psedotubeufia laxispora | GZCC 22-2011 T | OR030831 | OR030838 | OR046675 | OR046682 |
| Psedotubeufia laxispora | GZCC 22-2012 | OR030832 | OR030839 | OR046676 | OR046683 |
| Psedotubeufia hyalospora | GZCC 22-2010 T | OR030833 | OR030840 | OR046677 | – |
| Tamhinispora indica | NFCCI 2924 T | KC469283 | KC469282 | – | – |
| Tamhinispora srinivasanii | NFCCI 4231 T | MG763745 | MG763746 | – | – |
| Thaxteriellopsis lignicola | MFLUCC 10-0121 | JN865193 | JN865205 | – | – |
| Thaxteriellopsis lignicola | MFLUCC 10-0124 | JN865196 | JN865208 | KF301561 | – |
| Tubeufia abundata | MFLUCC 17-2024 T | MH558894 | MH558769 | MH550961 | MH551095 |
| Tubeufia aquatica | MFLUCC 16-1249 T | KY320539 | KY320522 | KY320556 | MH551142 |
| Tubeufia bambusicola | MFLUCC 17-1803 T | MH558896 | MH558771 | MH550963 | MH551097 |
| Tubeufia brevis | MFLUCC 17-1799 T | MH558897 | MH558772 | MH550964 | MH551098 |
| Tubeufia brunnea | MFLUCC 17-2022 T | MH558898 | MH558773 | MH550965 | MH551099 |
| Tubeufia inaequalis | MFLUCC 17-1998 T | MH558916 | MH558791 | MH550984 | MH551117 |
| Tubeufia javanica | MFLUCC 12-0545 T | KJ880036 | KJ880034 | KJ880037 | – |
| Tubeufia latispora | MFLUCC 16-0027 T | KY092412 | KY092417 | KY117033 | MH551119 |
| Tubeufia roseohelicospora | MFLUCC 15-1247 T | KX454178 | KX454177 | – | MH551144 |
| Tubeufia rubra | GZCC 16-0081 T | MH558926 | MH558801 | MH550994 | MH551128 |
| Zaanenomyces moderatricis-academiae | CPC 41273 T | OK663762 | OK664723 | – | OK651167 |
| Zaanenomyces versatilis | CPC 41224 T | OK663769 | OK664730 | – | – |
Note: Newly generated sequences in this study are indicated in blue bold. “T” denotes ex-type strain. “–” as meaning no data available in GenBank.
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Ma, J.; Xiao, X.-J.; Liu, N.-G.; Boonmee, S.; Xiao, Y.-P.; Lu, Y.-Z. Morphological and Multi-Gene Phylogenetic Analyses Reveal Pseudotubeufia gen. nov. and Two New Species in Tubeufiaceae from China. J. Fungi 2023, 9, 742. https://doi.org/10.3390/jof9070742
AMA Style
Ma J, Xiao X-J, Liu N-G, Boonmee S, Xiao Y-P, Lu Y-Z. Morphological and Multi-Gene Phylogenetic Analyses Reveal Pseudotubeufia gen. nov. and Two New Species in Tubeufiaceae from China. Journal of Fungi. 2023; 9(7):742. https://doi.org/10.3390/jof9070742
Chicago/Turabian StyleMa, Jian, Xing-Juan Xiao, Ning-Guo Liu, Saranyaphat Boonmee, Yuan-Pin Xiao, and Yong-Zhong Lu. 2023. "Morphological and Multi-Gene Phylogenetic Analyses Reveal Pseudotubeufia gen. nov. and Two New Species in Tubeufiaceae from China" Journal of Fungi 9, no. 7: 742. https://doi.org/10.3390/jof9070742
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