Paraeutypella guizhouensis gen. et sp. nov. and Diatrypella longiasca sp. nov. (Diatrypaceae) from China

Abstract Background In this study, we introduce a novel genus, Paraeutypella, of the family Diatrypaceae comprising three species viz. Paraeutypella guizhouensis sp. nov. and P. citricola (basionym: Eutypella citricola) and P. vitis (basionym: Sphaeria vitis). Diatrypella longiasca sp. nov. is also introduced, which forms a distinct clade in Diatrypella sensu stricto. The discovery of this new genus will contribute to expanding the knowledge and taxonomic framework of Diatrypaceae (Xylariales). New information Generic delimitations in Diatrypaceae are unsettled because the phylogeny has yet to be resolved using extensive taxon sampling and sequencing of ex-type cultures. During an investigation of xylarialean fungi, we collected eutypella-like fungi which is distinct from Eutypella sensu stricto in our phylogenetic analyses (ITS and β-tubulin), thus, introduced as Paraeutypella guizhouensis gen. et sp. nov.. Paraeutypella is characterised by having 4–25 perithecia in a stroma each with 3–6 sulcate, long ostiolar necks. Paraeutypella citricola comb. nov. (basionym: Eutypella citricola) is introduced on Acer sp. from China. Diatrypella longiasca sp. nov. is introduced as a new species in Diatrypella sensu stricto. which has 2–5 ascomata per stroma and long ascospores, unusual when compared to other Diatrypella species and distinct phylogenetically.

Members of Diatrypaceae are saprobes, pathogens or endophytes, associated with a wide range of hosts in terrestrial and aquatic environments (Mehrabi et al. 2019, Dayarathne et al. 2020a, Dayarathne et al. 2020b  have been reported as causal agents of canker diseases on a wide range of host plants worldwide . The taxonomy and phylogeny of Diatrypaceae need to be resolved, as many genera are polyphyletic. Hence, fresh collections and sequences are required to define genera and establish their phylogenetic placement within the family. Diatrypella was introduced by Cesati and De Notaris (1863) with D. verruciformis (Ehrh.) Nitschke as the type. The genus is characterised by conical to truncate, cushion-like or discoid stromata usually delimited by a black zone in host tissues, umbilicate or sulcate ostiolar necks, cylindrical, polysporous, long-stalked asci and allantoid, hyaline or yellowish ascospores in their sexual morph and a libertella-like coelomycetes asexual morph (Kirk et al. 2008. Both Cryptovalsa and Diatrypella have polysporous asci and cannot easily be distinguished, based only on morphological comparisons (Acero et al. 2004, Vasilyeva andStephenson 2005). Therefore, molecular data are essential for defining genera in Diatrypaceae (Mehrabi et al. 2015). There are 65 names of Diatrypella in Species Fungorum (2020) (http://www.indexfungorum.org/names/names.asp), but only 15 have molecular data in GenBank (Hyde et al. 2020).
In this study, we introduce a new genus, Paraeutypella, which shows eutypella-like morphology, but is distinct phylogenetically. Paraeutypella comprises three species viz. Paraeutypella guizhouensis sp. nov. and P. citricola (basionym: Eutypella citricola) and P. vitis (basionym: Sphaeria vitis). Diatrypella longiasca sp. nov. is also introduced, which forms a distinct clade in Diatrypella sensu stricto. Species novelties are confirmed by morphological comparisons along with micro-photographs and the phylogeny of combined ITS and β-tubulin sequence data.

Sample collection and morphological observations
Dead twigs of Acer palmatum and undetermined plants were collected from China (Guiyang, Guizhou Province) during September to October 2019. Samples were observed with a stereomicroscope (SZX16, Olympus). Hand sections of the ascomata were mounted in distilled water and the following characters were measured: diameter and height of ascomata, width of the peridium, diameter and height of ostiolar necks, length and width of asci and ascospores. Melzer's Reagent was used for testing the ascal apical ring reaction. Images were captured with a Canon EOS70D digital camera fitted to a compound microscope. Measurements were made with the Tarosoft (R) Image Frame Work programme and images used for figures processed with Adobe Photoshop CS6 software (Adobe Systems, USA). Single spore isolation was performed according to  and germinating spores were transferred to potato dextrose agar (PDA-Shanghai Bio-way Technology Co. Ltd.). The pure cultures were incubated at 18-20ºC for four weeks. The type specimens were deposited in the Cryptogamic Herbarium, Kunming Institute of Botany, Academia Sinica (HKAS), Chinese Academy of Science, Kunming and Chinese Academy of Science Herbarium (HMAS), Beijing, China. Ex-type cultures were deposited in the Kunming Institute of Botany Culture Collection (KUMCC). Facesoffungi and Index Fungorum numbers are provided as mentioned in Jayasiri et al. (2015) and Index Fungorum (http://www.indexfungorum.org) respectively.

DNA extraction, PCR amplifications and sequencing
Fungal isolates were grown on PDA for 3-4 weeks at 25°C and total genomic DNA was extracted from 50 to 100 mg of axenic mycelium scraped from the edges of the growing cultures (Wu et al. 2001). EZgne fungal gDNA extraction kit (BIOMIGA, Hangzhou City, TM Zhejiang Province, China) was used to extract DNA by following the manufacturer's protocol. DNA extracts were stored at -4°C for use in regular work and duplicated at -20°C for long term storage.
DNA sequence data were obtained from the internal transcribed spacer (ITS) and partial βtubulin gene. ITS and β-tubulin were amplified by using the primers ITS5/ITS4 (White et al. 1990) and T1/T22 (O'Donnell and Cigelnik 1997), respectively. Polymerase chain reaction (PCR) was carried out in a volume of 25 μl, which contained 9.5 μl of ddH O, 12.5 μl of 2× PCR Master Mix (2× Bench Top Taq Master Mix, BIOMIGA, China), 1 μl of DNA template and 1 μl of forward and reverse primers (10 μM each) in each reaction. The PCR thermal cycle programme for all gene amplifications was as follows: initialisation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 50s, elongation at 72°C for 90s and final extension at 72°C for 10 min. Purification and sequencing of PCR products were done by Sangon Biotech, Shanghai, China.

Sequence alignment
The sequence data generated in this study were analysed with closely-related taxa retrieved from GenBank (Table 1), based on BLASTn searches (https:// www.ncbi.nlm.nih.gov) and recently published data (Mehrabi et al. 2019, Dayarathne et al. 2020b). ITS and β-tubulin were used for the analyses according to the previous studies listed above. Sequences (ITS and β-tubulin) were aligned using MAFFT v. 6.864b (Katoh et al. 2019) and manually improved when necessary in BioEdit v. 7.0 (Hall 1999). The single gene alignments were used to perform model test in MrModeltest 2.3 to estimate the best-fit evolutionary model under the Akaike Information Criterion (AIC) (Nylander 2004) and resulted in a GTR+I+G substitution model for each. Ambiguously aligned areas of each gene region were excluded and gaps were treated as missing data. Missing characters were assessed to be unordered and equally weighted.

Species
Strain no. GenBank Accession no.

Phylogenetic Analyses
Maximum Likelihood (ML) analysis was performed using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using the GTR+I+G model of evolution. Bootstrap supports were obtained by running 1,000 pseudoreplicates. Bayesian analysis was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) to evaluate Bayesian posterior probabilities (BYPP) (Rannala andYang 1996, Zhaxybayeva andGogarten 2002) by Markov Chain Monte Carlo sampling (BMCMC). GTR+I+G was used as the substitution model. Six simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 200 generation. The distribution of log-likelihood scores was examined to determine the stationary phase for each search and to decide if extra runs were required to achieve convergence, using the programme Tracer 1.5. The first 10% of generated trees were discarded and remaining 90% of trees were used to calculate posterior probabilities of the majority rule consensus tree. All trees were visualised in  Culture characteristics -Colonies on PDA reaching 21 mm diam. after 2 weeks at 20-25 C, medium dense, circular to slightly irregular, slightly raised, cottony surface; colony from above: at first white, becoming buff; from below: yellowish white at margin, yellow to brown at centre; mycelium yellowish.

Etymology
The specific epithet longiasca refers to the long asci.

Etymology
With reference to the morphological resemblance to Eutypella.

Notes
Paraeutypella is introduced to accommodate three species viz. P. guizhouensis sp. nov., as well as P. citricola and P. vitis, two species previously placed in Eutypella sensu lato. Paraeutypella is typified by P. guizhouensis, which was collected from undetermined dead twigs. Paraeutypella can be distinguished from Eutypella species by stromata with perithecia in groups of 4-25 arranged in a valsoid configuration, 3-6 sulcate, long ostiolar necks, while stromata of Eutypella comprise groups of 20-70 perithecia having comparatively shorter ostiolar necks with sulcate or smooth ostiolar necks. Strains of both genera appear in distinct clades in a phylogeny based on ITS and Beta tubulin data (Fig. 1)
Culture characteristics -Colonies on PDA, reaching 21 mm diam. after 2 weeks at 20-25 C, medium dense, circular to slightly irregular, slightly raised, cottony surface; colony from above: at first white, becoming buff; from below: yellowish-white at margin, yellow to brown at centre; mycelium yellowish.

Etymology
The specific epithet guizhouensis refers to the locality in which the fungus was collected.
Culture characteristics -Colonies on PDA, reaching 21 mm diam. after 2 weeks at 20-25 C, medium dense, circular to slightly irregular, slightly raised, cottony surface; colony from above: at first white, becoming buff; from below: yellowish-white at margin, yellow to brown at centre; mycelium yellowish.

Phylogenetic analyses
The combined ITS and β-tubulin matrix comprises 79 sequences that represents the genera in Diatrypaceae including the outgroup taxa. The best scoring RAxML tree is shown (Fig. 1) (Fig. 1). KUMCC 20-0016 and KUMCC 20-0017 formed a separate clade basal to E. vitis with high statistical support (94% ML) (Fig. 1). These species form a separate clade from the Eutypella clade. A novel genus is needed to accommodate these species, hence we introduce Paraeutypella.

Discussion
This study introduces a new genus, Paraeutypella and accepts 22 genera in Diatypaceae. According to the previous analyses of combined ITS and β-tubulin sequence data, the genus Eutypella has been often identified as polyphyletic in Diatrypaceae (Trouillas et al. 2011, Mehrabi et al. 2016, Mehrabi et al. 2019, Dayarathne et al. 2016, Dayarathne et al. 2020a, Dayarathne et al. 2020b) and determined in our study as well (Fig. 1) Eutypella citricola groups separately from Eutypella sensu stricto with Eutypella vitis and our newly-generated strains. These new strains are introduced as a new genus, Paraeutypella with three species viz. P. citricola, P. guizhouensis and P. vitis. We studied the morphological characteristics of the species belonging to this clade and found considerable morphological differences from Eutypella sensu stricto. The differences include stromata with 4-25 groups of perithecia in a valsoid configuration, 3-6 sulcate, long ostiolar necks; thus, we consider them to belong in a distinct genus from the Eutypella and hence, we introduce the novel Paraeutypella.
There does not appear to be any host-specificity since members of Diatypaceae are found on a wide range of hosts in various habitats. Diatypaceae species frequently have been identified as saprobes on the decaying wood of angiosperms. Few endophytes, such as Diatrypella frostii Peck and Peroneutypa scoparia (Schwein.) Carmarán & A.I. Romero, have been reported (de Errasti et al. 2010, Vieira et al. 2011, Grassi et al. 2014. Therefore, the family may have the potential for switching nutritional modes during the degradation of plant material (de Errasti et al. 2010, Grassi et al. 2014 (Fig. 1). This may be due to lack of single-copy nuclear genes like β-tubulin or misidentified species.