Taxonomic and phylogenetic contributions to Diatrypaceae from southeastern Tibet in China

In this study, we investigated the diversity of diatrypaceous fungi from southeastern Tibet in China. The phylogenetic analyses were carried out based on ITS and β-tubulin sequences of 75 taxa of Diatrypaceae from around the world. Based on a combination of morphological features and molecular evidence, a new genus—Alloeutypa, with three new species—A. milinensis, Diatrype linzhiensis, and Eutypella motuoensis, and a new combination—A. flavovirens, were revealed by the materials in China. Alloeutypa is characterized by stromatal interior olivaceous buff, stromata producing well-developed discrete, and ascospores allantoid, subhyaline. These characteristics separate the new genus from the similar genus Eutypa. Comprehensive morphological descriptions, illustrations, and a phylogenetic tree to show the placement of new taxa are provided. All novelties described herein are morphologically illustrated and phylogeny investigated to better integrate taxa into the higher taxonomic framework and infer their phylogenetic relationships as well as establish new genera and species. Our results indicate that the diatrypaceous fungi harbor higher species diversity in China.

The genus Diatrype Fr. was established by Fries (1849) and typified with D. disciformis (Hoffm.) Fr. The genus was characterized by stromata widely effuse or verrucose, flat or slightly convex, with discoid or sulcate ostioles at the surface, eight-spored and long-stalked asci and hyaline or brownish, allantoid ascospores (Rappaz, 1987;Vasilyeva and Stephenson, 2004;Vasilyeva and Stephenson, 2009;Senanayake et al., 2015). Recently, Zhu et al. (2021) included a new species, and Yang et al. (2022) introduced two new taxa with polysporous asci as members in Diatrype based on the phylogenies inferred from the dataset of ITS and β-tubulin.
Diatrype, Eutypa, and Eutypella are all unresolved lineages, and phylogenetic studies indicated that the three genera do not form monophyletic groups, even though they clustered within Diatrypaceae Wijayawardene et al., 2020;Long et al., 2021;Yang et al., 2022). In an investigation of the diversity of wood-decaying fungi in southeastern Tibet of China, three undescribed species of diatrypaceous fungi were collected. In order to further the knowledge of species diversity and taxonomy of Diatrypaceae, we carried out complete morphological and molecular phylogenetic studies on these specimens with an emphasis on diatrypaceous fungi. In this study, we introduce a new genus, three new species, and a new combination of Diatrypaceae occurring on decaying wood.

Specimen collection
The specimens studied in this article were collected from Motuo County and Milin County in Linzhi City of southeastern Tibet, China. In situ photographs of the specimens were taken with a Canon G16 camera (Tokyo, Japan). Fresh specimens were dried and deposited following Yang et al. (2022).

Morphological examination
The studied specimens were macromorphologically observed with the aid of a VHX-600E microscope of Keyence Corporation (Osaka, Japan) up to ×200. The microscopic procedure followed Song et al. (2022). Specimen sections were mounted in water, 10% potassium hydroxide (KOH), and Melzer's reagent (1.5 g potassium iodide, 0.5 g crystalline iodine, and 22 g chloral hydrate dissolved in 20 ml distilled water), and then microscopic examinations were carried out with an Olympus IX73 inverted fluorescence microscope (Tokyo, Japan) at magnifications up to × 1,000.

DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from dried specimens using CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) and RaPure Plant DNA Mini Kit (Magen Biotechnology) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region and β-tubulin (TUB2) were amplified with primer pairs ITS5/ITS4 (White et al., 1990) and T1/ T22 (O'Donnell and Cigelnik, 1997), respectively. Polymerase chain reaction (PCR) was performed following Song et al. (2022). DNA sequencing was performed at BGI tech, Guangzhou, China. All newly generated sequences in this study including eight ITS sequences and six β-tubulin sequences were deposited in GenBank (Table 1). 1

Phylogenetic analyses
Sequencher 4.6 (GeneCodes, Ann Arbor, MI, United States) was used to edit the DNA sequence. Sequences were manually cut and orientation adjusted using BioEdit software (Hall, 1999). Sequences were aligned using the "G-INS-i" strategy at the MAFFT 7 (http:// mafft.cbrc.jp/alignment/server/) website and manually corrected using BioEdit. The sequences of Kretzschmaria deusta (Hoffm.)     Frontiers in Microbiology 07 frontiersin.org P.M.D. and Xylaria hypoxylon (L.) Grev. were obtained from GenBank as out-groups. Maximum likelihood analyses were performed in raxmlGUI 2.0 selecting ML + rapid bootstrap analysis and GTRGAMMA+G as the surrogate model Song et al., 2022). Branch support (BS) for ML analysis was determined by 1,000 bootstrap replicates. MrModeltest 2.3 (Nylander, 2004) was used to determine the best-fit evolution model for each dataset for Bayesian inference (BI). Bayesian inference was calculated with MrBayes 3.1.2 with a general time reversible (GTR + I + G) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck, 2003). Four simultaneous Markov chains were run for 2000000 generations, and every 100 generations were sampled as a tree. The first one-fourth generations were discarded as burn-in. The majority rule consensus tree of all remaining trees is computed. Branches were considered as significantly supported if they received maximum likelihood bootstrap (BS) ≥ 70% and Bayesian posterior probabilities (BPP) ≥ 0.95.

Molecular phylogeny
The contribution of the molecular phylogenetic tree based on 197 sequences of two DNA loci (116 ITS and 81 β-tubulin sequences) was composed of 116 samples representing 75 strains of Diatrypaceae ( Table 1). The concatenated dataset had an aligned length of 1936 characteristics, including gaps (609 for ITS and 1,327 for TUB2). Bayesian obtained a topology similar to ML, with an average standard deviation of split frequencies = 0.007766 (BI). Only the ML tree is provided in Figure 1 with the likelihood bootstrap values (≥ 70%, before the slash) and Bayesian posterior probabilities (≥ 0.95, behind the slash) labeled along the branches.
The topology of the phylogenetic tree is similar to those in previous studies (Konta et al., 2020;Zhu et al., 2021 (Figure 1), Eutypa species are distributed in two distinct clades Eutypa sensu stricto and Eutypa 1, indicating that the genus is polyphyletic. The type species, E. lata clusters in Eutypa clade1 which can be regarded as Eutypa sensu stricto. However, it is hard to justify Eutypa 1 as a new genus without examining old types of species and identified fresh collections with molecular data.
The sexual morphology of Eutypa sensu stricto (as Eutypa taxonomic species 2) comprises wide-spreading stromata that embedded in decorticated wood or bark, usually poorly developed with ill-defined margins, surface black, interior white or blackened, eight-spore asci spindle-shaped, long-stipitate, ascospores allantoid, subolivaceous (Glawe and Rogers, 1984). The Chinese collection in this study is clearly different from members of Eutypa sensu stricto based on the green interior of the stromata, discrete, Diatrype-like.
Based on the morpho-molecular differences, the new genus Alloeutypa is introduced to accommodate Alloeutypa milinensis. Alloeutypa is typified by A. milinensis, which was found on dead branches of angiosperm plant from southeastern Tibet in China. Eutypa flavovirens resembles A. milinensis in having well-developed discrete, Diatrype-like stromata with yellow-green to olive-green interior tissue, asci spindle-shaped, long-stipitate, ascospores allantoid, and subhyaline to subolivaceous. The phylogenetic analyses based on ITS and β-tubulin sequence data also supported Alloeutypa as a monophyletic genus in the Diatrypaceae, and A. milinensis and A. flavovivens as separate lineages within Alloeutypa. Thus, based on morphological evidence and phylogenetic analyses, we accommodate Alloeutypa as a new genus with A. milinensis as the type, and E. flavovirens was transferred to Alloeutypa as A. flavovirens comb. nov.

Frontiers in
The sequence comparison showed that there are 97.22 and 95%, respectively, similarities in ITS and TUB2 between A. milinensis from China (FCATAS 4309) and A. flavovirens (E. flavovirens) from Italy (MFLU19-0911), while 97.13 and 94.12 between A. milinensis from China (FCATAS 4309) and A. flavovirens (E. flavovirens) from France (E48C, CBS 272.87). Unfortunately, TUB2 sequences of the Indian and Thailand collections are not available in GenBank. However, the ITS sequence comparison showed that there are both 92% similarities between A. milinensis from China (FCATAS 4309) and A. flavovirens (E. flavovirens) from India (PUFNI 310) and Thailand (MFLUCC 13-0625). Therefore, we described the Chinese material as a new species. Notes: Alloeutypa flavovirens is one of the most common fungi and found throughout the world and appears to have a wide host range Rogers, 1982, 1984;Rappaz, 1987). It is characterized by having yellow-greenish stromatic tissues, spindle-shaped asci with refractive apical invaginations, allantoid ascospores subhyaline to subolivaceous (Glawe and Rogers, 1984). It is most similar to A. milinensis in having the green interior of the stromata. There are no sequence data for the type of A. flavovirens, but there are two putatively named collections, CBS 272.87 and MFLU 19-0911, from France and Italy, respectively (Rolshausen et al. 2006;Boonmee et al., 2021). Based on the morphological and molecular analyses that the two collections were the records of A. flavovirens (E. flavovirens) by Senanayake et al. (2015) and Boonmee et al. (2021), in our phylogenetic tree, the two strains of A. flavovirens (E. flavovirens) clustered together with A. milinensis with strong support (95% ML, 1.00 BYPP; Figure 1) and maybe the same genus because of its location. However, morphological differences on size of stromata, perithecium, and ascus can distinguish the two species from each other (Senanayake et al., 2015;Boonmee et al., 2021). Etymology: referring to the locality (Linzhi City) of the type specimens.

Discussion
The species diversity, taxonomy, and phylogeny of diatrypaceous fungi were intensively studied recently by many authors, and a large number of new taxa were described (Mehrabi et al., 2019;Konta et al., 2020;Dayarathne et al., 2020a,b;Dissanayake et al., 2021;Long et al., 2021;Peng et al., 2021;Zhu et al., 2021;Yang et al., 2022). This study furthers the knowledge of these fungi with the addition of a new genus, three new species, and a new combination in the Diatrypaceae. Morpho-molecular analyses confirmed the introduction of the newly described genus, Alloeutypa, for accommodating the new species A. milinensis and the new combination A. flavovirens. Our phylogenetic analyses on the species of Diatrype and Eutypella also confirmed that they are all polyphyletic genera, agreeing with the previous studies (Acero et al., 2004;Trouillas et al., 2011;Mehrabi et al., 2019;Konta et al., 2020;Dayarathne et al., 2020a,b;Long et al., 2021;Zhu et al., 2021).
In our phylogenetic trees, most taxa of Diatrype (Diatrype sensu stricto) formed a main clade with high support values (Figure 1), including D. disciformis, the type species of the genus. The new species, D. linzhiensis, from China also was included in this group. In the molecular analyses of ITS and β-tubulin sequences performed by Zhu et al. (2021), Eutypa flavovirens (Pers.) Tul. & C. Tul. grouped in a clade with two Cryptosphaeria taxa by no supported values. In our analyses (Figure 1), E. flavovirens appeared in a strongly supported clade along with the new species A. milinensis, suggesting the new species is closely related to E. flavovirens. The novel diatrypacous genus, Alloeutypa, is therefore introduced in the present study and will help to stabilize the classification of Diatrypaceae. However, the other species of Eutypa formed two distinct clades in the family and the generic position remains unresolved, which may need to be studied in the future.
The Eutypella species analyzed were distributed in two main separate clades (El sensu stricto and El 1), one mixed with taxa of Paraeutypella and Allocryptovalsa (El 1) and another related to a species of Anthostoma (Eutypella sensu stricto). Eutypella motuoensis formed a sister subclade with El. persica with no support values.
The molecular evidence has brought significant changes and increased our understanding of the taxonomy and phylogeny of Diatrypaceae. However, the phylogenetic trees show that the classification of these diatrypaceous fungi in many genera is confusing. To determine more important and useful morphological characteristics for distinguishing those species and to resolve infragenera and infra-specific phylogeny, more specimens of these species from their original regions and more taxa from other regions should be included in future phylogenetic studies.

Funding
The study was supported by the National Natural Science Foundation of China (No. 31770023 and 31972848) and the Central Public-Interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630032022001, 1630052022003).

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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