﻿ Stromatolinea, a new diatrypaceous fungal genus (Ascomycota, Sordariomycetes, Xylariales, Diatrypaceae) from China

﻿Abstract Molecular phylogeny and morphological characteristics of collections of diatrypaceous fungi from Guizhou Province, China, lead to the establishment of a new genus, Stromatolinea, and the identification of four new species and two new combinations. The taxa were found growing on the dead culms of Phyllostachys bamboo. The new genus is distinguished by its well-developed, discrete linear stromata with yellow interior tissue and allantoid subhyaline ascospores. The newly described species are Stromatolineagrisea, S.guizhouensis, S.hydei, and S.xishuiensis. Additionally, two new combinations, Stromatolinealinearis and S.phaselina, are proposed based on comparative analysis and morphology. Phylogenetic analyses were conducted using ITS and TUB2 sequences. The study includes comprehensive morphological descriptions, illustrations, and a phylogenetic tree depicting the placement of the new taxa.

Diatrypaceae species are distributed worldwide and are commonly found on deadwood and the bark of various plant species.The family is characterized by black or dark brown, immersed to erumpent, pseudostromatic or eustromatic stromata, polysporous or 8-spored asci, hyaline to light brown allantoid ascospores, and a libertella-like asexual morph (Senanayake et al. 2015;Wijayawardene et al. 2017).
Bamboo, as the largest member of the grass family Poaceae, plays an important role in local economies worldwide, being distributed across diverse climates, from cold mountainous regions to hot tropical areas.China boasts plentiful bamboo resources, with its bamboo species constituting over 50% of the world's total (Liu et al. 2018).There are more than 1300 fungal species associated with bamboo, including 150 basidiomycetes, 800 ascomycetes.The taxonomic placements of bamboo-associated ascomycetous fungi are highly diverse, comprising over 1,150 species, in 120 families and 400 genera (Dai et al. 2018;Hyde et al. 2023).Jiang et al. (2022) reported 512 bambusicolous ascomycetous taxa from China, associated with 16 bamboo genera, representing more than one-third of the known bambusicolous ascomycetes in the world.
In an investigation into the diversity of bambusicolous fungi in Guizhou, China, four previously undescribed species of diatrypaceous fungi were discovered.Morphological analyses revealed their close affinity to the genus Alloeutypa.However, phylogenetic analyses did not support their placement within this genus.Following detailed morphological examinations and comparative analyses, we propose a new genus, Stromatolinea, within the family Diatrypaceae, which includes four new species and one new combination.The findings contribute significantly to the understanding of diatrypaceous fungal diversity and taxonomy.

Sample collection and morphological study
The specimens of bamboo were collected during surveys conducted in the Guizhou province, China.All related habitat information, including details about elevation, climatic conditions, and geographical features, was recorded.The photos of the collected materials were taken using a Canon G15 camera (Canon Corporation, Tokyo, Japan).Materials were placed in paper bags and were taken to the lab for examination.To preserve the freshness of the specimens, they were dried at room temperature.The dried specimens were carefully labeled and stored in an ultra-low freezer at -80 °C for one week to eliminate any insects and their eggs.After this preparation, the specimens were ready for both morphological and molecular studies.Macroscopic characteristics were observed under an Olympus SZ61 stereomicroscope and photographed with a Canon 700D digital camera fitted to a light microscope (Nikon Ni).Morphological characteristics of specimens were examined, and photomicrographs were taken as described in Senanayake et al. (2020).Materials were mounted in water for anatomical examination and added Melzer's reagent where necessary.More than 30 ascospores and 30 asci were measured using the Tarosoft image framework (v.0.9.0.7).Images were arranged using Adobe Photoshop CS6 (Adobe Systems, USA).
Isolates were derived by single spore isolation following the method of Chomnunti et al. (2014).Germinating spores were observed with a Stereo Zoom microscope and transferred to potato dextrose agar (PDA; 39 g/l distilled water, Difco potato dextrose).The cultures were incubated at 25-30 °C for 1-4 weeks, with frequent observations.Cultural characteristics, such as mycelium colour, shape, texture and growth rate, were recorded after incubating at 25 °C under normal light for a week.
Herbarium materials were deposited at the herbarium of Guizhou Medical University (GMB) and the Herbarium of Cryptogams, Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS), and living cultures were deposited at the Guizhou Medical University Culture Collection (GMBC).

DNA extraction, PCR amplification, and sequencing
Fungal DNA was directly extracted from the contents of stromata and perithecia using the BIOMIGA fungus genomic DNA extraction kit, following the manufacturer's instructions.The DNA samples were stored at -20 °C.Internal transcribed spacers (ITS), and β-tubulin (TUB2), were amplified by PCR with primers ITS1/ITS4 (White et al. 1990;Gardes and Bruns 1993), and T1/T22 (Glass and Donaldson 1995;O'Donnell and Cigelnik 1997), respectively.The components of a 25 μL volume PCR mixture was: 9.5 μL of double distilled water, 12.5 μL of PCR Master Mix, 1 μL of each primer and 1 μL of template DNA.Qualified PCR products were checked through 1.5% agarose gel electrophoresis stained with GoldenView, and sent to Sangon Co., China, for sequencing (Xie et al. 2020).

Phylogenetic analyses
All newly generated sequences from this study were deposited in GenBank (https:// www.ncbi.nlm.nih.gov/;accessed on March 28, 2024; Table 1).These sequences were compared with each other and all the known sequences in the GenBank by using the BLAST algorithm for identification.The sequences retrieved from open databases originated from Li et al. (2023), Ma et al. (2023), and the BLASTn results of close matches and other Diatrypaceae representatives.The molecular phylogeny was inferred from a combined dataset of ITS and TUB2 sequences.Sequences were aligned using the MAFFT v.7.110 online programme (Katoh et al. 2019) with the default settings, respectively.Alignment was adjusted manually using BioEdit v.7.0.5.3 (Hall 1999) where necessary.The start and end of alignment were trimmed to nearly equal number of sites for all sequences.The combined sequence data was used to perform maximum likelihood (ML) and Bayesian inference analysis (BI).The ML analysis was implemented in RAxML v.8.2.12 using the GTRGAMMA substitution model with 1,000 bootstrap replicates (Stamatakis 2014).

Phylogenetic analyses
After the exclusion of ambiguously aligned regions and long gaps, the final combined data matrix contained 1,450 characters.Kretzschmaria deusta (CBS 826.72) and Xylaria hypoxylon (CBS 122620) were added as the outgroup.The tree topology derived from Maximum Likelihood (ML) analysis closely resembled that of Bayesian Inference (BI) analysis.The best-scoring RAxML tree is shown in Fig. 1.The topology of the phylogenetic tree is similar to those in previous studies (Li et al. 2023;Ma et al. 2023) Description.Saprobic on dead bamboo culms, forming black parallel elongate ascostromata on the host, surrounded by grey or black patches like pseudostromata.Pseudostromata grey or black, spreading between stromata and across the host surface.Sexual morph: Stromata parallel elongate, linear, consistent to inconsistent in thickness, fusiform, high, solitary to confluent, slit to non-slit, black or grey on its sides, exposing black ostioles.Upper cells of stromata near the perithecial ostiole black, thick-walled.Stromatic tissue completely yellow or yellow above and white between/below perithecia, compact.Ascomata perithecial, few to frequent, immersed in stromata, globose to subglobose, ostiolate centrally, with a neck, opening to outer surface, slight erumpent over stromata, appearing as black shinny spots.Peridium composed of elongate cell, texture angularis, outer thick layer, dark brown, inner hyaline, surrounded by yellow or white and yellow stromatic tissue.Hamathecium paraphyses, filiform, hyaline, long.Asci 8-spored, clavate, with a long and thin pedicel, apically rounded to truncate, J-apical ring.Ascospores allantoid, aseptate, straight to slightly curved, rounded at both ends, subhyaline, with oil droplets in both ends.Asexual morph: undetermined.
Culture characteristics.Ascospores germinating on PDA within 24 hours, colonies on PDA, white when young, became pale, thinning toward the edge, white from above, reverse pale, no pigmentation, and no sporulation produced on the PDA medium.
Notes.It is morphologically similar to Stromatolinea hydei and S. xishuiensis but can be easily distinguished by the appearance of stromata and pseudostromata color.The pseudostromata of the latter two species are black, whereas grey in S. grisea.The stromata of S. hydei and S. xishuiensis are consistent in thickness and possess frequent ascomata, whereas the stromata of S. grisea are inconsistent in thickness, thin in between, and possess usually 2 or 3 ascomata.Moreover, stromatic tissue is yellow above and white between or below perithecia in S. grisea, while it is completely yellow in S. hydei and S. xishuiensis.The comparison of ITS sequences revealed 94% and 98% similarity to S. hydei and S. xishuiensis, respectively, while TUB2 sequences displayed 93% and 94% similarity to S. hydei and S. xishuiensis, respectively.Differentiation from other known species of the genus is discussed in the note section of the below described species.Etymology.The epithet "guizhouensis" refers to the locality of the collection, Guizhou province.
Culture characteristics.Ascospores germinating on PDA within 24 hours, colonies on PDA, white when young, became pale, dense at centre, thinning toward the edge, reverse white at the margin, pale at the centre, no pigmentation, and no sporulation produced on the PDA medium.

Stromatolinea xishuiensis
Etymology.The epithet "xishuiensis" refers to the locality of the collection, Xishui County.

Stromatolinea linearis
Notes.Stromatolinea phaselina was first described and illustrated by Montagne in 1855 based on a collection from Guyana.Rappaz (1987) conducted a de-tailed analysis of various species that had been reported under different names (given above as synonyms).He reviewed their descriptions and type materials, though some of type material were lost.Rappaz (1987) synonymized E. kusanoi and E. bambusina with Eutypa hypoxantha and grouped these along with Sphaeria phaselina and Eutypella hypoxantha under the broader name Diatrype phaselina (Mont.)Rappaz.The morphological character of this taxon also aligns with those of Stromatolinea, providing compelling support for its placement within the Stromatolinea taxonomic framework.
Morphologically, Stromatolinea phaselina resembles S. grisea in having yellow entostromatic tissue above and white tissue between or below the perithecia.However, there are no reports of pseudostromata presence, detailed stromata morphology, or the number of perithecia per stromata in the published description of Diatrype phaselina (Rappaz 1987).Furthermore, descriptions and synonymized accounts of this species report very short asci sizes, measuring 25-35 × 5-7 μm.This is problematic because the family is known to typically possess long asci.This gap in detailed morphological data limits our ability to fully understand and differentiate Diatrype phaselina from other species.Without a detailed description and access to type material or DNA data, we cannot definitively classify it within the key of the genus.
The synonyms of this species are not updated in Index Fungorum and Mycobank, where they are still listed as separate species.Given Rappaz (1987) thorough analysis of historical descriptions and most of the original materials, we consider his classification/synonyms of the species to be well-founded and reliable.

Discussion
The generic concepts of Diatrypaceae have been unstable; several new genera within the family have been reported through a combination of morphological characteristics and multi-locus phylogeny.Early classification systems of Diatrypaceae were mainly based on stromatal features including the degree of stromatal development, structure of perithecial necks, and type of host tissue (Glawe and Jacobs 1987;Rappaz 1987).However, the morphological variability of stromata has caused significant confusion within Diatrypaceae.Many genera, including Neoeutypella, Allodiatrype, Diatrype, Diatrypella, Allocryptovalsa, Cryptovalsa, Eutypella, and Paraeutypella, exhibit similar stromatal characteristics, limiting their utility for species identification (Li et al. 2023).This confusion has led to polyphyletic genera, where species have often been transferred between genera (Shang et al. 2017(Shang et al. , 2018;;Phookamsak et al. 2019;Konta et al. 2020;Ma et al. 2023).In this study, we introduce a new genus that phylogenetically forms a well-supported distinct clade and morphologically distinguished by its linear stromata and yellow interior tissue.This new genus includes four new species namely S. grisea, S. guizhouensis, S. hydei, and S. xishuiensis.Additionally, Stromatolinea linearis and S. phaselina are proposed for Eutypa linearis and D. phaselina, respectively, based on morphological characteristics and comparative analysis.
Species within Stromatolinea can be differentiated by key morphological features, including the presence or absence and color of pseudostromata; stromata size, color, slit presence, and interior tissue color; ascomata number, arrangement, and measurements; and ascospores dimensions.Furthermore, significant phylogenetic distances in the ITS and TUB2 regions also serve as valuable tools for species discrimination.Notably, all Stromatolinea species have been reported as saprobes on dead bamboo, implying a potential host specificity confined to bamboo.

Table 1 .
GenBank Accession Numbers used in this study.The newly generated sequences are marked bold.T indicates type strain.

Stromatolinea xishuiensis GMB4535 Stromatolinea xishuiensis GMB4522 Stromatolinea xishuiensis GMB4514 Stromatolinea guizhouensis GMB4523 Stromatolinea guizhouensis GMB4515 Diatrypaceae sp. Stromatolinea hydei GMB4538 Stromatolinea hydei GMB4521
. The new genus Stromatolinea, including four species, formed a distinct clade that represents its monophyletic status.The strains ZHKUCC 21-0114 and S21 clustered in the clade of the new genus, are deposited in NCBI under the name Diatrypella sp., but remain unpublished.The details provided in NCBI for these two strains (ZHKUCC 21-0114 and S21) were searched, but no records were found.Figure1.A-C.Phylogenetic tree generated from maximum likelihood analysis (RAxML) based on combined ITS and TUB2 sequences data.Bootstrap support values for maximum likelihood (ML) greater than 70% and Bayesian posterior probabilities (BPP) greater than 0.90 are displayed above or below the respective branches (ML/BPP).The species obtained in this study are in red and ex-type taxa are in bold.