Taxonomy and Phylogeny of Novel and Extant Taxa in Pleosporales Associated with Mangifera indica from Yunnan, China (Series I)

Pleosporales is the largest fungal order with a worldwide distribution in terrestrial and aquatic environments. During investigations of saprobic fungi associated with mango (Mangifera indica) in Baoshan and Honghe, Yunnan, China, fungal taxa belonging to pleosporales were collected. Morphological examinations and phylogenetic analyses of ITS, LSU, SSU, rpb2 and tef1-α loci were used to identify the fungal taxa. A new genus, Mangifericomes; four new species, namely Mangifericomes hongheensis, Neomassaria hongheensis, Paramonodictys hongheensis, and Paramonodictys yunnanensis; and six new host and country records, namely Byssosphaeria siamensis, Crassiparies quadrisporus, Paradictyoarthrinium aquatica, Phaeoseptum mali, Torula fici, and Vaginatispora amygdali, are introduced. Photoplates, full descriptions, and phylogenetic trees to show the placement of new and known taxa are provided.


Introduction
Mango (Mangifera) belongs to the genus Mangifera and the family Anacardiaceae. Mangifera contains 69 species, while the most cultivated fruit tree species is Mangifera indica, which has over 1000 varieties worldwide, but only a few varieties are cultivated and traded on a large scale [1,2]. Mangifera indica is one of the five most economically significant fruit crops worldwide, while it is the second most planted fruit after banana throughout the tropics and subtropics [3,4]. DeCandolle (1884) [5] estimated that mangoes' cultivation history can trace back 4000 years in India and Southeast Asia, as the fossil evidences discovered show Mangifera indica first appeared during the quaternary period. However, the origin of Mangifera indica has been debated for years, and some believe mango evolved from several related species in and around Malay Archipelago and China, which may be camera (Canon EOS 600D, Canon Inc., Tokyo, Japan) mounted on a compound microscope (Nikon ECLIPSE Ni, Nikon., Tokyo, Japan). Measurements of microstructures were obtained by the Tarosoft (R) Image Frame Work program, while further processing was done in Adobe Photoshop CS3 Extended v. 10.0 (Adobe ® , San Jose, CA, USA).
Single spore isolation was carried out following the methods outlined in Senanayake et al. [37] by using potato dextrose agar (PDA) and incubating at 27 • C for 12-48 h. Germinated conidia or ascospores were observed with a stereo microscope, transferred to new PDA plates, and incubated at room temperature for 1 week. Culture characteristics were observed after 2 weeks. Specimens were deposited in the herbarium of the Kunming Institute of Botany Academia Sinica (HKAS), while living cultures are maintained at the Kunming Institute of Botany Culture Collection (KUMCC). Index Fungorum numbers were registered as described in Index Fungorum [38].

DNA Extraction, PCR Amplification and Sequencing
Genomic DNA were extracted from fungal mycelium by using Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux ® , Beijing, China), following the manufacturer's instructions. The extracted DNA was stored at 4 • C for the Polymerase Chain Reaction (PCR), while a part of the DNA was maintained at −20 • C for long-term storage. The PCR mixture contains 8.5 µL of double-distilled water (ddH 2 O), 12.5 µL of 2×Power Taq PCR MasterMix (mixture of EasyTaqTM DNA Polymerase, dNTPs, and optimized buffer, Beijing Bio Teke Corporation (Bio Teke), China), 1 µL of each forward and reverse primers (10 pmol), and 2 µL of DNA. The internal transcribed spacer (ITS) region was amplified with the primers ITS4 and ITS5, the 18s small subunit (SSU) region amplified by primers NS1 and NS4 [39], the nuclear ribosomal 28s large subunit (LSU) region amplified by the primers LROR and LR5 [40], the partial RNA polymerase II subunit (rpb2) region with primers fRPB2-5F and fRPB2-7cR [41], and the partial translation elongation factor 1-alpha (tef1-α) gene with primers EF1-983F and 2218R [42]. The conditions for PCR of ITS, SSU, LSU, and tef1-α genes constituted an initial denaturation step of 2 min at 95 • C, followed by 35 cycles of 30 s at 95 • C, 50 s at 55 • C, 1 min at 72 • C, and a final denaturation step of 10 min at 72 • C. For the rpb2 (fRPB2-5F and fRPB2-7cR) gene, the initial denaturation occurred at 95 • C for 3 min; denaturation at 95 • C for 45 s, annealing at 57 • C for 50 s, and extending 90 s at 72 • C for 35 cycles; and extending at 72 • C for 10 min. PCR products were sent to Beijing Bio Teke Corporation for purification and sequencing. To ensure the accuracy of the sequencing, the above methods were repeated to obtain extra sequences for new fungal colonies.

Phylogenetic Analyses
Sequence data both reverse and forward generated in this study were assembled using the Geneious (Restricted) 9.1.2 (https://www.geneious.com, accessed on 25 January 2022) and subjected to BLASTn searches in the nucleotide database of GenBank (http: //blast.ncbi.nlm.nih.gov/, accessed on 25 January 2022) to determine their most probable closely related taxa. Single-gene sequence alignments were made with the server version of MAFFT (www.ebi.ac.uk/Tools/mafft, accessed on 25 January 2022) [43] and edited manually in BioEdit 7.2.3 [44]. The uninformative gaps and ambiguous regions were removed by trimAL v1.2 (http://trimal.cgenomics.org, accessed on 25 January 2022) and combined multi-genes sequencing manually in BioEdit. The fasta files were transferred to PHYLIP (for ML) and NEXUS (for BI) format in Alignment Transformation Environment (ALTER) online program [45]. The maximum likelihood analysis (ML) was generated on the CIPRES Science Gateway v.3.3 (http://www.phylo.org/portal 2, accessed on 25 January 2022 [46]) selecting RAxML-HPC2 on XSEDE (8.2.12) [47], with GTRGAMMA substitution model with 1000 bootstrap iterations. The Bayesian analysis performed by using MrBayes v.3.2.2 [48]. The best models of evolution were estimated by using MrModeltest v. 2.3 [49] and PAUP v. 4.0b10 [50]. Bayesian analyses of six simultaneous Markov chains were run for 1,000,000 to 50,000,000 generations (depending on individual fungal groups) and trees were sampled at one tree every 100th or 1000th generation. Phylogenetic trees were visualized using FigTree v1.4.0 [51], and the trees were edited by Microsoft PowerPoint and inserted reliable bootstrap support values from ML and BI.
Culture characteristics: Ascospore germinated within 18 h, moderately growing on PDA, reaching 1-20 mm diameter after two weeks at 27 • C, grayish brown to white at the margin, circular, effuse to low convex, indistinctly striate, entire edge to above; white at the margin, reddish brown to pale brown to reverse, without pigments produced from PDA.  [56], collected and isolated from the endocarp of Amygdalus persica in Japan. Our isolate V. amygdali KUMCC 21-0334 was morphologically similar to member of Vaginatispora and Leptoparies. However, based on phylogenetic analyses of combined SSU, LSU, rpb2 and ITS sequence data, our isolate clusters together with V. amygdali (KT 2248, holotype) with high bootstrap support ( Figure 1). The BLASTn results of LSU, ITS, and SSU of our strain gives 99% similarity with V. amygdal (KT 2248). Therefore, based on morphology and phylogeny, we introduce and describe our strain as V. amygdali from China on a dead branch of Mangifera indica. This is a new host and a new country record (Table 1).
Notes: The new genus Mangifericomes was established as a monotypic genus in Pleosporales genera incertae sedis, which was isolated from Mangifera indica in China.
Mangifericomes hongheensis as the type species is characterized by ellipsoid and muriform, pale brown to brown ascospores, wrapped in a gelatinous sheath, while the other genera, namely. Murispora, Phaeoseptum, Halojuella, Julella, and Pleospora, also have muriform, yellow-brown to dark-brown ascospores, with or without a sheath [76][77][78][79]. However, M. hongheensis differs in having 7-11-transversely septate and 5-8 longitudinal septa in each spore, oblong, more obtuse, and flatter at both ends, and the pseudoparaphyses are sparse, short-branched, and thick-walled. Furthermore, M. hongheensis clustered in an individual group ( Figure 5) and separated well with other genera, which has muriform ascospores in phylogenetic trees.
Culture characteristics: Ascospores germinated within 20 h on water agar, with colonies on PDA reaching 7-12-mm diameter after 1 week on the PDA medium, circular, convex, raised, erose or dentate, moderately dense, above cottony with abundant white, slight gray to white from margin to center; reverse: visible as white loop at the margin, brown to dark in the center, sunken, without pigments produced in PDA.   , and our isolates show completely different ascospores compared with Brunneoclavispora bambusae MFLUCC 11-0177 (Didymosphaeriaceae) even though they are relatively closely related in phylogeny [80]. Our new species is very similar to Halojulella avicenniae (Halojulellaceae, Pleosporales) in immersed ascomata, muriform ascospores, and gelatinous sheath [77], but Halojulella avicenniae differs in having dark brown, thick-walled peridium; consisting of textura angularis to globosa cells; and having an ascospore often more obtuse and flatter at both ends (6-7 transverselly, 2-3 longitudinal vs. 7-11 transverselly, and 5-8 longitudinal). Therefore, we introduce Notes: Initially, Neomassaria fabacearum was introduced as the type in a monotypic novel genus Neomassaria (Massariaceae) [82], However, Ariyawansa et al. [30] collected a neomassaria-like species (Neomassaria formosana) from stem of Rhododendron sp. in Taiwan Province of China. The maximum likelihood and Bayesian phylogenetic analyses formed a separate sister group with Neomassaria fabacearum and was well-separated with Massaria species (Massariaceae); hence, a new family, Neomassariaceae, was established with a single genus, Neomassaria, and two species (Neomassaria fabacearum and N. formosana). Sexual morph of the genus Neomassaria is characterized by having immersed, subglobose to globose ascomata, central ostiole, peridium comprising cells of textura angularis, pseudoparaphyses, 8-spored, bitunicate oblong to cylindrical, pedicellate asci, containing ellipsoid to fusiform, 1-septate, hyaline ascospores, and with or without a gelatinous sheath [30,82], while asexual morph has not yet been obtained.
Culture characteristics: Ascospore germinated within 20 h on water agar, growing on PDA reaching around 10-mm diameter after 2 weeks incubation at 27 • C above: circular, flat to effuse, rough on surface, wrinkled, dark-brown to black well-defined at margin, dense; reverse: black, without pigments produced in PDA. Notes: Ariyawansa et al. [30] and Hongsanan et al. [81] were referred for the phylogeny, and LSU, SSU, tef1-α, rpb2 and ITS gene were used for the phylogenetic analyses in Pleosporales. From the phylogenetic trees, we found our isolates (KUMCC 21-0340, KUMCC 21-0344) well separated and closely related with other two species in Neomassariaceae, namely Neomassaria formosana (NTUCC 17-007 and NTUCC 17-009) and N. fabacearum (MFLU 16-1875), with high statistical support values (ML: 100%, BI: 1, Figure 5). However, these three strains have few differences in morphology. Neomassaria fabacearum is characterized by having immersed ascomata, ostiole central, fusiform, ellipsoid to broadly fusiform ascospore surrounded by a gelatinous sheath, comprising two equal cells, narrow in both ends, and initially placed under family Massariaceae [82] Neomassaria formosana was established by Ariyawansa et al. [30] and described with immersed ascomata, erumpent, compressed neck, produce fusoid to ellipsoid, oblong or slight flexuous ascospores (20-30 × 3-7 µm), visibly distinct oil droplets in each ascospore but without sheath [30], while our strain has semi-immersed ascomata, widens at the base, without erumpent neck, ascospore (14-17 × 4-8 µm) fusoid, fine at the bottom when it is immature, becomes ovoid later, broadens at upper region, rounded at both ends, distinctly constricted at the septum, without a sheath. In addition, based on BLASTn search, the closest matches of LSU show Neomassaria formosana strain NTUCC 17-009 and formosana strains just overlapped 89% in tef1-α region. We compared the differences of base pairs of rpb2 from N. formosana and our strains, and the similarity was less than 90%, and therefore, based on morphological examinations and phylogenetic analyses, we introduce our strains as a new species N. hongheensis.  Notes: Paramonodictys was first introduced by Hyde et al. [83] with P. solitarius as the type species, which is known to be saprobic on decaying wood in terrestrial habitats; later, on another collection, it was reported from freshwater by Dong et al. [84] This genus is morphologically characterized as having superficial black colonies, monoblastic conidiogenous cells, pyriform or clavate, brown to olivaceous brown dictyosporous, subglobose to globose conidia. Paramonodictys presently has been investigated only as an asexual morph, and it has a complex placement with Monodictys sp. (KH331 and MAFF 243825) in phylogenetic analyses (Figure 8) [84].
Culture characteristics: Conidia germinated within 18-20 h on PDA. Colonies rapidgrowing on PDA reached about 15-mm diameter, after 1 week at room temperature in natural light, the upper surface of colonies appear circular, umbonate, slightly radial striations, circinate, fluffy, entire edge, initially grayish to pale brown, finally exhibiting dark brown when it matures after 1-2 months. Reverse dark brown, reddish-brown near the margin, sunken at the center, without pigments produced in PDA. Notes: In our study, Paramonodictys hongheensis KUMCC 21-0343 has a high similarity with Monodictys melonopa (PAN 32767) in morphology, which was introduced by Prasher and Verma [85], conidia multicellular, muriform, subglobose to oval, pale-brown in the lower parts, and the conidia of our isolate was smaller than conidia of M. melonopa (19-26 × 19-22 µm vs. 26-46 × 19-28 µm); unfortunately, we lack genes of M. melonopa to compare it with our isolate. Based on our phylogeny, our new isolates formed a separate branch basal to Monodictys sp., Paramonodicty solitarius, and P. yunnanensis with high bootstrap support in ML and BI (Figure 8). The morphological characteristics of our new isolate and other Paramonodictys spp. are very similar [83,84]. In addition, comparison of the nucleotides across the tef1-α gene region showed a quite few numbers of base pairs were different compared with Monodictys sp. KH331 (difference: 911/948 bp, 3.9%, 0 gaps) and Paramonodictys solitarius MFLUCC 17-2353 (890/921 bp, 3.4%, 0 gaps). Therefore, based on conidial morphology comparison and phylogenetic analyses, Paramonodictys hongheensis is established as a new species on Mangifera indica from China. Notes: The morphological characteristics of Paramonodictys yunnanensis sp. nov. fits well with the generic type of Paramonodictys. Our new species is similar to P. solitaries in having brown to olivaceous-brown and obovoid to oblong conidia; however, the conidiogenous cells of P. yunnanensis are shorter than P. solitarius, while colonies on PDA have distinct differences with P. solitarius MFLUCC 17-2353 (brown, erose margin vs. olivaceous brown, entire edge) [84]. In the NCBI BLASTn of ITS sequence, our strain P. yunnanensis (KUMCC 21-0337) highly overlapped with P. solitarius (MFLUCC 17-2353 and GZCC 20-0007) at 98.97% and 98.74% similarity, while LSU (874 bp) BLASTn showed high similarity with Monodictys sp. (KH 331 and MAFF 243825) at 99.77% and 99.08%, the SSU (1028 bp). The rpb2 BLASTn result was quite close to Paramonodictys solitarius GZCC:20-0007 (99%), while the tef1-α showed only 86.5% (837/935 bp, 3 gaps) similarity to P. solitarius GZCC 20-0007 and Monodictys sp. MAFF 243825. Thus, P. yunnanensis is described as a new species in Monodictys and Paramonodictys complex clade. Moreover, the multi-gene (SSU, LSU, ITS, and tef1-α) phylogeny generated herein reveal P. yunnanensis at basal clade of P. solitarius with well-separated branch with high bootstrap support value in ML and BI (Figure 8). Therefore, our isolate can be described as a new species on Mangifera indica from China.

Notes:
The monotypic genus Paradictyoarthrinium was established by Matsushima [63] with P. diffractum as the type species, which was collected from dead twigs in a stream in South Africa. This genus contains only P. aquatica, P. diffractum, P. hydei and P. tectonicola, in Index Fungorum [38], while those hydrophilous or terricolous taxa were collected from China and Thailand [86,87]. The genus is characterized by superficial, powdery colonies, conidiophores macronematous, unevenly dictyoseptate, muriform, conidiogenous cells blastic, integrated, terminal, determinate, subglobose to irregular, brown to black conidia congregate on the top, sometimes developing in branched chains, with 1-2 short chains [64,86]. Sexual morphology has not been documented in this genus. The phylogeny of Paradictyoarthrinium and closely related genera is shown in Figure 11. Notes: The comparison of morphological characteristics of colonies and conidiogenous cells show that our isolate is highly consistent with Paradictyoarthrinium diffractum in morphologically. However, our strain has slightly olivaceous to brown conidia, while the previous description recorded brown to dark brown conidia [13,63,64]. However, based on LSU, ITS, and rpb2, BLASTn results show our isolate has 100% similarity with P. diffractum (MFLUCC13-0466, BCC 8704, MFLUCC 12-0557). Our phylogenetic tree topology of the ML and BI analyses were similar to Liu et al. [86], while our isolate clusters together with P. diffractum (MFLUCC12-0557, MFLUCC 13-0466) with high bootstrap support ( Figure 11). In this study, Paradictyoarthrinium diffractum (KUMCC 21-0336) represents a new host record and country record for this fungus (Table 1).  Notes: Phaeoseptum was first introduced by Zhang et al. [78] with the type species P. aquaticum, which was found on submerged branch of Robinia pseudoacacia in France [78]. This genus was initially placed in family Halotthiaceae but later was transferred to Phaeoseptaceae [88]. There are six records in Index Fungorum [38]. This genus is characterized by immersed, subglobose, papillate, ostiolate ascomata, trabeculate pseudoparaphyses, mostly 8-spored asci (excluding P. hydei with 32 ascosporesin asci), cylindrical-clavate asci with a small ocular chamber or apical ring when it is immature, fusiform, light brown to brown ascospores, dictyosporous with thickened transversal septa, range of 1-3 seriate [60,77,87]. In this genus, only the sexual morph has been reported from China, France, India, and Thailand, while habitats are mostly freshwater, estuarine, or various terres-trial plants [65,78,[88][89][90]. The phylogeny of Phaeoseptum and closely related genera is shown in Figure 13.

Discussion
Mango plantations in Yunnan Province, China, represent one of the largest plantation groups in China, contributing to the annual total economic output with CNY 1.8 billion. The area of land under mango cultivation was 74,100 hm 2 in 2018, and the annual production reaches 0.47 megatons [96]. Baoshan and Honghe are two large mango planting areas in Yunnan, mainly attributable to the suitable temperature and rainfall. Flowering occurs from January to March, and the fruiting period is April to July. The most widely cultivated mango varieties in these areas are Keitt, Guifei, Sannian, Nang Klangwan, and JinHwang [7,[97][98][99]. Honghe, located in the dry and hot river valley (north tropical climate regions), has an average annual temperature of 24.9 • C, annual sunshine time of 2300 h, and average annual precipitation of 800-1000 mm, while Baoshan, located in west of Yunnan (south subtropical climate regions), reports an average annual air temperature of 21.5 • C, annual sunshine time of 2300 h, and average annual precipitation of 750-900 mm [99][100][101][102].
Mango, an important and economically useful across the world, is largely cultivated in subtropical to tropical regions, while mango-associated fungi, especially pathogens, are always discovered [103][104][105][106][107]. Although more than 2000 fungal records on mango have been documented, only a few saprobic fungi species associated with mango have been introduced; among the fungi already reported from mango, less fungal species have been reported from the order Pleosporales [12]. In this study, we introduce saprobic fungi in Pleosporales that were collected from Yunnan Province, China. Mangifericomes is introduced here as a new fungal genus associated with mango. In addition, further research is needed to confirm whether fungi in mango are "host-specific" or if they can be "host generalists" that jump within different hosts.