New contributions to Diatrypaceae from karst areas in China

Abstract In this study, fungal specimens of the family Diatrypaceae were collected from karst areas in Guizhou, Hainan and Yunnan Provinces, China. Morpho-molecular analyses confirmed that these new collections comprise a new genus Pseudodiatrype, three new species (Diatrypelancangensis, Diatrypellapseudooregonensis and Eutypacerasi), a new combination (Diatrypellaoregonensis), two new records (Allodiatrypethailandica and Diatrypellavulgaris) from China and two other known species (Neoeutypellabaoshanensis and Paraeutypellacitricola). The new taxa are introduced, based on multi-gene phylogenetic analyses (ITS, β-tubulin), as well as morphological analyses. The new genus Pseudodiatrype is characterised by its wart-like stromata with 5–20 ascomata immersed in one stroma and the endostroma composed of thin black outer and inner layers of large white cells with thin, powdery, yellowish cells. These characteristics separate this genus from two similar genera Allodiatrype and Diatrype. Based on morphological as well as phylogenetic analyses, Diatrypelancangensis is introduced as a new species of Diatrype. The stromata of Diatrypelancangensis are similar to those of D.subundulata and D.undulate, but the ascospores are larger. Based on phylogenetic analyses, Diatrypeoregonensis is transferred to the genus Diatrypella as Diatrypellaoregonensis while Diatrypellapseudooregonensis is introduced as a new species of Diatrypella with 8 spores in an ascus. In addition, multi-gene phylogenetic analyses show that Eutypacerasi is closely related to E.lata, but the ascomata and asci of Eutypacerasi are smaller. The polyphyletic nature of some genera of Diatrypaceae has led to confusion in the classification of the family, thus we discuss whether the number of ascospores per asci can still be used as a basis for classification.

Members of Diatrypaceae occur on a wide range of hosts in terrestrial and marine environments worldwide, some of which are important plant pathogens (Moyo et al. 2018a;Mehrabi et al. 2019;Dayarathne et al. 2020;Konta et al. 2020 Tul. And Eutypella (Nitschke) Sacc., are responsible for canker diseases in grapevine (Trouillas et al. 2011;Gao et al. 2013;Moyo et al. 2018b). Besides cankers of grapevine, some species have been reported as the causal pathogentic agents of fruit trees and woody plants in Europe and the USA (Trouillas et al. 2011;Gao et al. 2013).
Thirteen species of Cryptosphaeria and Diatrype were introduced by Vasiljeva and Ma (2014) from north-eastern China, which includes two new species and four new records. China has the largest range of karst distribution in the world. The landform of karst can be found in almost all Provinces of China, with the most extensive distribution in Guizhou and Yunnan Provinces (Miao et al. 2007). Karst virgin forest is a relatively stable ecosystem with rich biological resources, highly primitive and maintaining stable biological diversity (Dong et al. 2002). The special karst and ecological environment is home to a rich diversity of diatrypaceous fungi.
In this study, we revisit species of Diatrypaceae collected from karst areas in Guizhou, Hainan and Yunnan Provinces of China. Based on morpho-molecular analyses, one new genus and three new species are introduced; in addition, a new combination and two new records from China are reported. Descriptions and illustrations of new taxa and new records are provided.

Fungi collection, isolation and identification
Samples of decaying wood were collected from October 2019 to November 2020 in forests and nature reserves of Guizhou, Hainan and Yunnan Provinces in China. The specimens were observed with a stereomicroscope while microscopic images of the samples were taken using a Nikon ECLIPSE Ni compound microscope, with a Canon EOS 700D digital camera. Measurements were taken with Tarosoft (R) Image Frame Work (v.0.9.7). More than 30 asci and ascospores were measured for each specimen examined. Photoplates were arranged and improved by using Adobe Photoshop CS6 software. Isolations of fungi were made by single spore isolation (Chomnunti et al. 2014) and germinated spores were transferred to potato dextrose agar (PDA) medium for purification. The specimens were deposited at the Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (KUN-HKAS) and Herbarium of Guizhou Medical University (GMB). Strains of the new genus and new species are maintained in the Guizhou Medical University Collection Centre (GMBC).

DNA extraction, Polymerase Chain Reaction (PCR) and phylogenetic analyses
Genomic DNA was extracted from fungal mycelium following the manufacturer's protocol of the BIOMIGA Fungal gDNA isolation Kit (BIOMIGA, Hangzhou City, Zhejiang Province, China). Extracts of DNA were stored at -20 °C.
PCR profiles for the ITS and LSU are as follows: initially at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 94 °C for 1 minute, annealing at 52 °C for 1 minute, elongation at 72 °C for 1.5 minutes and a final extension at 72 °C for 10 minutes. PCR profile for the RPB2 is as follows: initially at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 95 °C for 1 minute, annealing at 54 °C for 2 minutes,

Phylogenetic analyses
Phylogenetic analyses were performed by searching homologous sequence data of the family Diatrypaceae in the GenBank database, selected from NCBI and recently published papers (Mehrabi et al. 2019;Dayarathne et al. 2020;Konta et al. 2020;Dissanayake et al. 2021;Zhu et al. 2021). After the preliminary identification results of the sequences, multiple sequence alignments (ITS and β-tubulin) were aligned using Bi-oEdit v. 7.0 (Hall 1999

Phylogenetic analyses
Based on RAxML and BYPP analyses, phylogenetic analyses were similar in overall tree topologies and did not differ significantly. The dataset consists of 105 taxa for representative strains of species in Diatrypaceae, including outgroup taxa with 1071 characters, including gaps (ITS: 1-486, β-tubulin: 486-1071). The RAxML analyses resulted in a best scoring likelihood tree selected with a final ML optimisation likelihood value of -15731.506304, which is shown in Fig. 1. The phylogenetic tree, based on combining ITS and β-tubulin sequence data, is also shown in Fig. 1 and contains 17 clades within Diatrypaceae. Below, we list the placements of new taxa:  Clade 1: Diatrypella pseudooregonensis and Diatrypella oregonensis clustered with the species of Diatrypella in Clade 1 with high bootstrap support, Diatrypella pseudooregonensis is introduced as an 8-spored new species of Diatrypella and Diatrype oregonensis is renamed as Diatrypella oregonensis. Clade 4: Pseudodiatrype formed a separate branch in a clade (Clade 4) basal to the genus Allodiatrype. Clade 7: Diatrype lancangensis clusters with the species of Diatrypella and Diatrype in an unresolved clade. However, Diatrype and Diatrypella have previously shown confused classification which is difficult to distinguish, based on phylogenetic aspects alone. Therefore, we introduce Diatrype lancangensis as a new species of Diatrype, based on phylogenetic analyses and morphological differences (Table 2). Clade 8: Eutypa cerasi forms a distinct lineage which is sister to Eutypa lata (EP18, RGA01) ( Fig. 1).
Notes. The genus Diatrype was introduced by Fries (1849). The genus is characterised by stromata widely effuse or verrucose, flat or slightly convex, with discoid or sulcate ostioles at the surface, 8-spored and long-stalked asci and hyaline or brownish, allantoid ascospores. In this study, we introduce a new species of Diatrype from China.
Culture characteristics. Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became luteous, dense but, thinning towards edge, margin rough, white from above, reverse white at margin, pale yellow to luteous at centre, no pigmentation produced on PDA medium, no conidia observed on PDA or on OA media. Note. Our new strain, GMBC0045 falls into the unresolved clade (Clade 7) which comprises five Diatrypella and one Diatrype species (Fig. 1), this clade is consistent with the study of Konta et al. (2020). The taxonomic confusion of Diatrypaceae has led to difficulties in separating the genera. We consider that the new species belongs to the genus Diatrype, based on the stromata features mentioned above which closely resemble descriptions of Diatrype subundulata Lar. N. Vassiljeva & Hai X. Ma and Diatrype undu- lata (Pers.) Fr. (Vasilyeva et al. 2014). However, the ascospores of these species are larger than the ascospores of D. subundulata and D. undulata (Table 2). Phylogenetic analyses also showed that D. lancangensis falls on a separate branch that clustered with species of Diatrypella and Diatrype (Fig. 1). Hence, by combining morphological characteristics and phylogenetic analyses, it seems appropriate to categorise this species as Diatrype.
In the phylogenetic analyses, it can be seen that Clade 7 can be defined as a new genus, but it is difficult to find the common morphological similarities among these species. More specimens and sequence or chemical composition analysis are needed in the future to determine whether Clade 7 can be a new genus. The characteristics of the stromata of Diatrypella spp. in clade 7 are solitary and scattered, which is distinctly different from widely effuse, flat and slightly convex stromata of Diatrype lancangensis and Diatrype palmicola Hyde et al. 2020b;Zhu et al. 2021). And in the recent study, Zhu et al. (2021) proposed that the species of Diatrypella in Clade 7 were isolated from Betula spp., it may have host specificity. Because of the above two reasons, we think it is better to classify our strains into Diatrype. Description. Saprobic on decaying branches of an unidentified plant. Sexual morph: Stromata scattered or aggregated on host, wart-like, pustulate, visible as black, rounded to irregular in shape on host surface, erumpent through host bark, 5-20 ascomata immersed in one stroma. Endostroma consists of outer layer of black, small, dense, thin parenchymal cells and inner layer of white, large, loose parenchymal cells, thin, pale yellow, powdery near margin of the black cells. Ostiole opening through host bark and appearing as black spots, separately, papillate or apapillate, central. Perithecium immersed in stroma, globose to subglobose, glabrous, with cylindrical neck, brevicollous or longicollous. Peridium is composed of an outer layer of dark brown to black, thin-walled cells, arranged in textura angularis, the inner layer of hyaline thinwalled cells of textura angularis. Asci 8-spored, unitunicate, clavate, long-stalked, apically rounded, apical rings inamyloid. Ascospores irregularly arranged, allantoid, slightly or moderately curved, smooth, subhyaline, aseptate, usually with two oil droplets. Asexual morph: undetermined.
Note. The genus Pseudodiatrype is introduced to accommodate the new collection made from Hainan Province of China and typified by Pseudodiatrype hainanensis. Pseudodiatrype is monotypic and, morphologically, resembles Diatrype and Allodiatrype Konta & K.D. Hyde. However, Pseudodiatrype can be distinguished from Diatrype by its 5-20 ascomata immersed in a stroma, while the stroma of species of Diatrype is distributed over large areas, sometimes covering the surface of the host (Vasilyeva and Ma 2014;Konta et al. 2020). Pseudodiatrype differs from Alloiatrype by having its 5-20 ascomata immersed in a stroma, whereas the stroma of Allodiatrype has only 1-10 ascomata. Moreover, the endostroma of Allodiatrype is composed of dark brown outer layer cells and yellow inner layer cells (Konta et al. 2020), which are different from the endostroma of Pseudodiatrype having black outer and inner cells surrounded by powdery, pale yellow cells. In addition, the sizes of stroma and ascospores are different from species of Diatrype and Allodiatrype (Table 2). In the phylogenetic analyses, species of Pseudodiatrype appeared in a separate branch which is distinct from other genera within Diatrypaceae (Fig. 1) Fig. 3 Holotype. GMB0054.
Culture characteristics. Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became pale brown, dense, but thinning towards the edge, margin rough, white from above, white at margin and light brown at centre from Note. Morphologically, Diatrype has 8 ascospores in a single ascus, while Diatrypella has more than eight ascospores in each ascus . However, previous research (e.g. Acero et al. 2004 andTrouillas et al. 2011) suggested that both Diatrypella and Diatrype are polyphyletic within the family. In the phylogenetic analyses, Diatrypella pseudooregonensis grouped closely to the D. verruciformis and thus, we consider this new species to belong in the genus Diatrypella, because it is doubtful whether the number of ascospores per asci is useful as a basis for generic classification. 1.3 mm, n = 30) pustulate, visible as black, rounded to irregular in shape on host surface, semi-immersed, erumpent through host bark, with 2-8 ascomata immersed in one stroma. Endostroma consists of outer dark brown, small, dense, thin parenchymal cells and an inner layer of white, large, loose parenchymal cells. Ostiole opening separately, papillate or apapillate, central 710.7-787.2 μm high, 270.2-422 μm diam. (av. = 742 × 363 μm, n = 10). Perithecium immersed in stroma, round to oblong, with cylindrical neck, brevicollous or longicollous. Peridium composed of outer layer of dark brown to black, thin-walled cells, arranged in textura angularis, inner layer of hyaline thin-walled cells of textura angularis. n = 30), polysporous, clavate, long-stalked, apically rounded. Ascospores 8-11 × 1-2 μm (av. = 8.9 × 1.7 μm, n = 30), overlapping, crowded, allantoid, slightly or moderately curved, smooth, subhyaline, yellowish in mass, aseptate, usually with two oil droplets. Asexual morph: undetermined.
Culture characteristics. Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became pale yellow, irregular in shape, medium dense, flat or effuse, slightly raised, with edge fimbriate, fluffy to fairly fluffy, white Additional sequences. GMB0050 (LSU: MW797052). Note. The ITS sequence data were subjected to BLAST in NCBI and the results showed that it is 100% similar to Allodiatrype thailandica. Additionally, based on morphological and phylogenetic analyses, this strain was identified as the A. thailandica. The stromata are similar, but the ascospores of GMB0050 are longer and wider than the ascospores of strain MFLUCC 15-3662 (3.8-6.9 × 1-1.4 μm) isolated from the holotype specimen, but it is similar to the strain MFLU 17-0735 (6.5-10.7 × 1.6-2.7 μm) ). Here, we use the ITS sequence similarity between the new collection and the type strain of Allodiatrype thailandica as basis for identification. A. thailandica has been reported in Thailand in 2016 as Diatrype thailandica and recognised as A. thailandica by Konta et al. (2020). This is the first report of Allodiatrype thailandica from China. Note. The morphological characteristics of this specimen are consistent with those of N. baoshanensis a species described by Phookamsak et al. (2019). Based on phylogenetic and morphological analyses, we consider that this specimen is Neoeutypella baoshanensis. Neoeutypella baoshanensis was described as the type species of Neoeutypella on dead wood of Pinus armandii Franch. from Yunnan Province in China . This is the first record of N. baoshanensis from Guizhou Province, China.
Culture characteristics. Ascospores germinating on PDA within 24 hours. Colonies on PDA, white when young, became pale yellow, irregular in shape, medium dense, flat or effuse, white from above, reverse white at margin, pale yellow at centre, no pigmentation produced on PDA medium, no conidia observed on PDA or on OA media. Notes. Eutypa lata is an important pathogen that has a wide range of hosts. However, the classification of E. lata is confusing because there are many variants in previous studies; now all are classified as E. lata (Index Fungorum 2020). Morphologically, the new collection GMB0048 has similar stromata with Eutypa lata, but the ascomata of the new collection are smaller than the ascomata (400 μm diam.) of the original description of E. lata (Tulasne & Tulasne, 1863). The ascomata and asci of the new collection are smaller than the ascomata (400-600 μm diam.) and asci (110-180 × 5-7 μm) of the description of E. lata (Rappaz 1987). Additionally, in the phylogenetic analyses, E. cerasi is located on a branch that forms a sister clade with EP18 and RGA01 and CBS 290.87 basal to E. cerasi. Therefore, combining phylogenetic and morphological analyses, we introduce Eutypa cerasi as a new species of Eutypa. Notes. Paraeutypella was introduced by Dissanayake et al. (2021) to accommodate Paraeutypella guizhouensis and the genus currently comprises three species. The genus is characterised by poorly developed stromata erumpent through the bark, grouped and irregularly shaped, sometimes confluent, dark brown to black, spindle-shaped, 8-spored asci and allantoid, overlapping, subhyaline ascospores (Trouillas et al. 2011;de Almeida et al. 2016;Dissanayake et al. 2021). In this study, we illustrate Paraeutypella citricola collected from Guizhou Province in China. Notes. The ITS sequence data were compared by using NCBI and the result showed that it is 100% similar to the ex-type strain (HVVIT07) of P. citricola. The morphological features of the new collection are consistent with those described by Dissanayake et al. (2021). This collection is identified as a P. citricolca, based on morphological and molecular data.

Discussion
In this study, one new genus, three new species, two new records from China, a novel combination and two known species were reported from karst areas of China. We used molecular data to delimit the species of Diatrypaceae. The new genus Pseudodiatrype is morphologically similar to Allodiatrype and Diatrype, but distinct in the size of stromata, number of ascomata and colour of endostroma; it also formed a distinct branch in the phylogenetic analyses (Fig. 1). Diatrype oregonensis was transferred to Diatrypella oregonensis based on the phylogenetic analyses. Based on phylogenetic analyses, Diatrypella pseudooregonensis was introduced as an 8-spored species of Diatrypella. Our phylogenetic analyses, based on ITS and β-tubulin, agree with the previous studies (Acero et al. 2004;Trouillas et al. 2011;Mehrabi et al. 2015Mehrabi et al. , 2016de Almeida et al. 2016;Shang et al. 2017;Dissanayake et al. 2021;Zhu et al. 2021). However, several genera are not monophyletic;for example, Cryptosphaeria, Diatrype, Diatrypella, and Eutypa. The identification of species of Diatrypaceae has been a problem due to the polyphyletic generic concepts based on the features of the stromata in early research (Fries 1823). Recently, new approaches have been proposed for classifying Diatrypaceae. Acero et al. (2004) proposed to classify them by ITS sequence-based phylogenetic analyses, while Carmarán et al. (2006) suggested that the identification should be based on the morphology of the asci. However, due to the lack of type specimens, the lack of β-tubulin sequence and polyphyletic origins have resulted in molecular data that correlate poorly with morphological criteria used to delineate genera and species within the Diatrypaceae (Acero et al. 2004). Moreover, Acero et al. (2004) has mentioned that Diatrypella quercina should be placed in the genus Diatrype despite its polysporous asci since the molecular data placed Diatrypella quercina in the branch of the genus Diatrype.
Diatrype and Diatrypella have morphologically similar verruculose stromata and allantoid ascospores and the polysporous or 8-spored ascus serve as a basis for distinguishing the two genera. However, in phylogenetic analyses, species of these two genera overlap. In this study, we used the phylogenetic analyses as the main basis for classification following Vasilyeva and Stephenson (2005) and Liu et al. (2015). Clade 1 contains Diatrypella verruciformis which is the type species of Diatrypella, of which Diatrypella pseudooregonensis, Diatrypella oregonensis have 8-spored, and other species in clade 1 have polyspored ascus. Clade 12 contains the Diatrype type species Diatrype disciformis, of which Diatrype iranensis and Diatrype macrospora have polyspored ascus, and other species in clade 12 have 8-spored ascus. Hence, we concluded that the number of ascospores in each ascus cannot be used as a criterion for distinguishing Diatrypella from Diatrype.
The phylogenetic tree shows that the classification of Diatrypaceae is confusing. Members of Diatrypella (D. favacea, D. hubeiensis, D. pulvinata and D. yunnanensis) cluster with Diatrype palmicola and Diatrype lancangensis.Maybe this clade should be identified as a new genus. We will discuss its classification status after more strains, more gene sequences and new taxonomic features are collected. Some species of Diatrypella (D. iranensis and D. macrospora) which have polysporous ascus are placed between species of Diatrype, and they are transferred to Diatrype iranensis and Diatrype macrospora by Zhu et al. (Zhu et al. 2021). Diatrype enteroxantha is often derived from the sister clade of Allodiatrype rather than the Diatrype clade. Additionally, Eutypa microasca (BAFC51550) clusters with Peroneutypa species (Clade 17). The above-mentioned confusion also showed in the original publication and other recent studies (Grassi et al. 2014;Mehrabi et al. 2016;Shang et al. 2018;Hyde et al. 2019;Phookamsak et al. 2019;Konta et al. 2020). Therefore, addressing the taxonomic confusion of this family requires a re-examination of older taxa, based on morphological studies, epitypification and multi-gene phylogenetic analyses (Ariyawansa et al. 2014).