Morphological and Multi-Gene Phylogenetic Analyses Reveal Pseudotubeufia gen. nov. and Two New Species in Tubeufiaceae from China

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.

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.

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.
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

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 parsimonyuninformative, 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.
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. 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 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.
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. 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.