Morpho-Molecular Characterization Reveals a New Genus, Three Novel Species and Two New Host Records in Xylariomycetidae

Xylariomycetidae comprises extremely diverse taxa that are widespread on decaying wood worldwide. An investigation of the diversity of microfungi on oil tree plantations in Sichuan Province was conducted during 2020–2021. Twelve saprobic taxa representing five species were identified as members of Amphisphaeriales and Xylariales through morphological comparisons. Phylogenetic analyses of combined ITS, LSU, rpb2, tub2 and tef1 sequence data indicated a distinct clade formed by three strains within Xylariomycetidae, unrelated to any currently recognized families. Thus, a novel anthostomella-like genus, Bicellulospora, is proposed and treated as Xylariales genera incertae sedis. Bicellulospora is characterized by dark brown to black, immersed, subglobose ascomata with a clypeus, cylindrical asci, and hyaline to yellowish brown, inequilaterally ellipsoidal ascospores with a large upper cell and a dwarf lower cell. Two new species of Amphisphaeria, namely A. oleae and A. verniciae, are introduced based on multi-gene phylogenetic analyses (ITS, LSU, rpb2 and tub2) coupled with morphological characteristics. Amphisphaeria micheliae and Endocalyx ptychospermatis are reported as new host records.

Cainiaceae was introduced by Krug [11] and was recently subsumed to Xylariales [1,12].Based on the molecular data, Endocalyx was transferred from Apiosporaceae to Cainiaceae [13,14].Endocalyx is a well-resolved genus in Cainiaceae, in which five species are accepted [14,15].Endocalyx has a strong host specificity to palm but also occurs on dead vines, lilies or twigs of woody trees [16][17][18].
During an investigation of fungal diversity on oil tree plantations in Sichuan Province from 2020 to 2021, a new anthostomella-like genus, Bicellulospora, was established and classified in Xylariales as genera incertae sedis, with B. elaeidis as the type species.Two new species of Amphisphaeria, namely A. oleae and A. verniciae, are introduced and justified by their morphological characteristics coupled with phylogenetic analyses.Two new host records, Amphisphaeria micheliae and Endocalyx ptychospermatis, are presented with detailed descriptions and illustrations.

Sample Collection, Isolation and Morphology
Specimens were collected from decayed stems or twigs and were placed in paper bags or envelopes when taken to the laboratory.Macro-micro morphological observations were carried out, as mentioned in Samarakoon et al. [27].Single-spore isolation was performed as described in Senanayake et al. [28].Germinated spores were individually transferred to potato dextrose agar (PDA) plates and grown at 25 • C in the dark.Measurements were made with the Tarosoft (R) Image Framework program v. 0.9.7, following the procedures outlined by Liu et al. [8].Photo plates representing fungal structures were processed in Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA).
Herbarium specimens were deposited in the herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China, and the herbarium of the University of Electronic Science and Technology (HUEST), Chengdu, China.The isolates obtained in this study were deposited in China General Microbiological Culture Collection Center (CGMCC), Beijing, China, and the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China.MycoBank numbers were registered as outlined in MycoBank (http://www.MycoBank.org,accessed on 21 April 2023).
ML and BI were performed on the CIPRES Science Gateway platform [38].ML analyses were made with RAxML-HPC2 on XSEDE v 8.2.8 with default parameters and bootstrapping with 1000 replicates [39].MrBayes analyses were conducted in CIPRES with MrBayes on XSEDE 3.2.7a.Four simultaneous Markov chains were run for 20,000,000 generations, and trees were sampled every 1000th generation.The first 20% of the trees of the generations were discarded as burn-in, and the posterior probabilities (PP) were calculated from the remaining 80% of the trees [40].BI posterior probabilities and maximum likelihood bootstrap values equal to or greater than 0.95/75% are indicated near each node of the phylogenetic tree.
Phylograms were visualized on FigTree v.1.4.0 [41], and the layouts of the trees were drawn in the Adobe Illustrator CS6 software (Adobe Systems, USA).All newly generated sequences in the study were deposited in GenBank, and relevant sequences for phylogenetic analyses are included in the Table S1.

Phylogenetic Analyses
Two phylogenetic analyses were conducted to resolve the relationships among taxa in Amphisphaeria (Amphisphaeriaceae) and Xylariomycetidae.
Notes: The phylogenetic analyses revealed that three new collections (CGMCC 3.24962, UESTCC 23.0127 and UESTCC 23.0128) formed a distinct lineage in Xylariales.They clustered with Barrmaeliaceae (Barrmaelia and Entosordaria) and Castellaniomyces rosae (MFLUCC 15-0536), with poor statistical support.Species of Barrmaelia can be separated from Bicellulospora by their one-celled ascospores without a dwarf cell.Entosordaria possesses ellipsoid to allantoid, two-celled ascospores consisting of a dark brown larger cell and a small, hyaline dwarf cell [10].Castellaniomyces rosae differs from Bicellulospora in having elongated fusiform, brown to dark brown, and equally two-celled ascospores surrounded by a thick mucilaginous sheath [44].
Culture characteristics: Ascospores germinating on PDA within 48 h at 25 • C. Colonies growing on PDA reaching 1.9−2.2cm diam.after two weeks at 25 • C in the dark, circular, velvety, pinkish brown in the middle, white at the outer rings, with dense mycelia in the inner rings, sparse at the entire margin; in reverse, brown in the center, pale yellow at the middle ring and white at the margin.Culture characteristics: Ascospores germinating on PDA within 48 h at 25 °C.Colonies growing on PDA reaching 1.9−2.2cm diam.after two weeks at 25 °C in the dark, circular, velvety, pinkish brown in the middle, white at the outer rings, with dense mycelia in the inner rings, sparse at the entire margin; in reverse, brown in the center, pale yellow at the middle ring and white at the margin.(488/567 bp, 25 gaps) and 85.69% (491/573 bp, 26 gaps) similarity with Ba. oxyacanthae (BO) and E. quercina (CBS 142774), respectively.The multi-gene analyses indicated that Bi. elaeidis (CGMCC 3.24962, UESTCC 23.0127 and UESTCC 23.0128) formed a distinct clade that is a sister to the clade containing Barrmaelia, Castellaniomyces and Entosordaria (Figure 2).However, they have significant differences in morphology.

Discussion
Xylariomycetidae is a taxonomically complex fungal group, and most of its taxa were classified based on their morphological characteristics before the wide application of molecular data [50][51][52].However, phylogenetic studies have indicated that fungi with similar spore-bearing structures have not evolved from the same ancestral lineages [53].
Stromatic characteristics, such as the length of the ascus stipe [51], amyloid reactions [54], and the shape and size of the ascus apical ring [55], are used to delimit xylarialean taxa.The number of ascospores per ascus [56], color [57], septation [23], appendage [54,58] and character of the germ slit [1,12] also play a vital role in the identification of xylarialean taxa.Though morphological characteristics are used for traditional generic circumscriptions (such as stromal morphology), they do not commonly reflect phylogenetic relationships.Several anthostomella-like genera (e.g., Anthostomella, Apiospora, Entosordaria, Occultitheca, Pyriformiascoma and Vamsapriya), with dwarf cells of ascospores, were shown to be morphologically comparable but are phylogenetically distinct throughout Xylariomycetidae [1].Therefore, the morphology of dwarf cells does not reflect phylogenetic relationships at the familial level but could be informative at the generic level.The new genus Bicellulospora has inequilateral ascospores with an olivaceous brown dwarf cell, distinguishing it from other similar genera such as Entosordaria and Castellaniomyces rosae (Xylariales genera incertae sedis), as they differ in morphology.Based on the molecular data and morphological comparison, a new monotypic genus, Bicellulospora, with type species B. elaeidis, is hereby established and placed in Xylariales as genera incertae sedis.
Samarakoon et al. [23] considered that the number of septa cannot be used as a basis for classification at the genus level.Consequently, Lepteutypa was synonymized to Amphisphaeria based on its holomorphic morphology and multi-gene phylogeny.In our study, two new species (A.oleae and A. verniciae) and A. micheliae were isolated from woody oil plants in Sichuan Province, China.Amphisphaeria oleae and A. micheliae share one-septate ascospores, while A. camelliae and A. verniciae clustered close to A. curvaticonidia, as they all have three-septate ascospores.Though the number of septa has less taxonomic significance for generic delimitation, it can properly reflect interspecific relationships.
Endocalyx was introduced by Berkeley and Broome [59] and is characterized by sporodochial or synnematous, cylindrical to cup-shaped conidiomata enclosed by yellow or brown sterile peridial hyphae; hyaline to subhyaline, basauxic conidiophores bearing unicellular, elliptical and brown conidia, with a smooth or echinulate surface; and a hyaline germ slit.Delgado et al. [14] conducted a comprehensive assessment of Endocalyx based on the molecular data from specimens and strains collected in Japan, Hawaii and the continental U.S.A. Four species were eventually accepted in Endocalyx (E.cinctus, E. indumentum, E. grossus and E. melanoxanthus).Recently, a fifth species, E. ptychospermatis, was reported after its identification in a dead petiole of Ptychosperma macarthurii (palm) in China [15].Here, we provide another collection of E. ptychospermatis from Trachycarpus fortunei (Palmae) as a new host record.

Figure 1 .
Figure 1.Phylogram generated from RAxML analysis based on the combined ITS, LSU, rpb2 and tub2 sequence data of Amphisphaeria isolates.Bootstrap values for maximum likelihood of ≥75% and Bayesian posterior probabilities of ≥0.95 are given near the nodes as ML/PP.Isolates from this study are marked in red, and ex-type strains are in bold.

Figure 1 .
Figure 1.Phylogram generated from RAxML analysis based on the combined ITS, LSU, rpb2 and tub2 sequence data of Amphisphaeria isolates.Bootstrap values for maximum likelihood of ≥75% and Bayesian posterior probabilities of ≥0.95 are given near the nodes as ML/PP.Isolates from this study are marked in red, and ex-type strains are in bold.

Figure 2 .
Figure 2. Phylogram generated from RAxML analysis based on the combined ITS, LSU, rpb2, tub2 and tef1 sequence matrix of Xylariomycetidae.Bootstrap values for maximum likelihood of ≥75% and Bayesian posterior probabilities of ≥0.95 are given near the nodes as ML/PP.Isolates obtained from this study are in red, and ex-type strains are in bold.

Figure 2 .
Figure 2. Phylogram generated from RAxML analysis based on the combined ITS, LSU, rpb2, tub2 and tef1 sequence matrix of Xylariomycetidae.Bootstrap values for maximum likelihood of ≥75% and Bayesian posterior probabilities of ≥0.95 are given near the nodes as ML/PP.Isolates obtained from this study are in red, and ex-type strains are in bold.