Insight into the Systematics of Microfungi Colonizing Dead Woody Twigs of Dodonaea viscosa in Honghe (China)

Members of Dodonaea are broadly distributed across subtropical and tropical areas of southwest and southern China. This host provides multiple substrates that can be richly colonized by numerous undescribed fungal species. There is a severe lack of microfungal studies on Dodonaea in China, and consequently, the diversity, phylogeny and taxonomy of these microorganisms are all largely unknown. This paper presents two new genera and four new species in three orders of Dothideomycetes gathered from dead twigs of Dodonaea viscosa in Honghe, China. All new collections were made within a selected area in Honghe from a single Dodonaea sp. This suggests high fungal diversity in the region and the existence of numerous species awaiting discovery. Multiple gene sequences (non-translated loci and protein-coding regions) were analysed with maximum likelihood and Bayesian analyses. Results from the phylogenetic analyses supported placing Haniomyces dodonaeae gen. et sp. in the Teratosphaeriaceae family. Analysis of Rhytidhysteron sequences resulted in Rhytidhysteron hongheense sp. nov., while analysed Lophiostomataceae sequences revealed Lophiomurispora hongheensis gen. et sp. nov. Finally, phylogeny based on a combined dataset of pyrenochaeta-like sequences demonstrates strong statistical support for placing Quixadomyces hongheensis sp. nov. in Parapyrenochaetaceae. Morphological and updated phylogenetic circumscriptions of the new discoveries are also discussed.


Introduction
Fungi are cosmopolitan, featuring a broad geographic distribution and high level of diversity compared to plants and other organisms [1]. 140,000 fungal species have been listed in Kirk [2], and one recent overview of global fungi and fungus-like taxa by Wijayawardene et al. [3] listed approximately 100,000 known taxa. However, both numbers represent less than 5% of global fungal estimates [4,5]. There is a need to bridge the gap between our understanding of these missing fungi and their diversity. Numerous diverse habitats and substrates remain unexplored. It has also been observed that several countries and regions are bountiful repositories of many missing fungi, such as northern Thailand [6]. Despite this, fungi in Asia are relatively understudied [5]. Even though the Greater Mekong Subregion (GMS) hosts a high level of biodiversity and forms an integral part of the Indo-Burma Biodiversity Hotspot, fungi from this region largely remain a mystery. Yunnan Province, China, as part of the GMS, is home to an extremely wide variety of ecosystems. Mycologists working in Yunnan have recently focused their attention on abundant "lessresearched habitats" for fungal occurrences, including caves, forests, grasslands, lakes, karst landscapes and mountains; accordingly, there is a rich body of literature documenting novel discoveries across the region [7][8][9][10][11][12][13][14][15][16][17][18][19].  [41,52] a act: actin; btub: β-tubulin; cal: calmodulin; ITS: part of rDNA 18S (3' end), the first internal transcribed spacer (ITS1), the 5.8S rRNA gene, the second ITS region (ITS2), and part of the 28S rRNA (5' end); LSU: large subunit (28S); rpb2: RNA polymerase II second largest subunit; SSU: small subunit rDNA (18S); tef 1: translation elongation factor 1-alpha gene. b fRPB2-5f and fRPB2-414R were used only for Teratosphaeriaceae analysis. c All the PCR thermal cycles include initiation step of 95 • C: 5 min, and final elongation step of 72 • C: 10 min and final hold at 4 • C.

Phylogenetic Analyses
Single-locus data sets were scanned for topological incongruences between loci for members of the analyses. Conflict-free alignments were concatenated into a multi-locus alignment that underwent maximum-likelihood (ML) and Bayesian (BI) phylogenetic analyses. Evolutionary models for BI and ML were selected independently for every locus using MrModeltest v. 2.3 [60] under the Akaike Information Criterion (AIC) implemented in PAUP v. 4.0b10.
The CIPRES Science Gateway platform [61] was used to perform RAxML and Bayesian analyses. ML analyses were made with RAxML-HPC2 on XSEDE v. 8.2.10 [62] employing the GTR+GAMMA swap model with 1000 bootstrap repetitions.
MrBayes analyses were performed setting GTR+I+GAMMA for 2-5 million generations, sampling every 100 generations and ending the run automatically when standard deviation of split frequencies dropped below 0.01 with a burnin fraction of 0.25. ML bootstrap values equal or greater than 60% and Bayesian posterior probabilities (BYPPs) greater than 0.95 were placed above each node of every tree.

Global Checklist of Fungi on Dodonaea Viscosa
Information for the global checklist ( Table 2) was retrieved from the Agriculture Research Service Database generated by the United States Department of Agriculture (USDA) [64], related books and research papers. This checklist includes fungal species associated with Dodonaea viscosa and the countries from which they were recorded.

Phylogenetic Analyses
Four phylogenetic analyses were performed using the acquired sequences from Gen-Bank (Table 3). The first is a phylogenetic overview of the genera treated in Teratosphaeriaceae (Figure 1), while the remaining three alignments represent the species in Rhytidhysteron (Figure 2), an overview of the phylogeny of the genera treated in Lophiostomataceae ( Figure 3) and Parapyrenochaeta, and allied genera in Pleosporineae ( Figure 4). Other details related to ML and BI analyses from different datasets are presented in Table 4. The acquired phylogenetic results are discussed where applicable in the notes below.
Neovaginatispora clematidis Penidiellopsis radicularis Quixadomyces    GenBank accession numbers with * are resulting from EF1-728F and EF-2 primers and -means missing data or not used in the phylogenetic analyses. The newly generated sequences are indicated in bold.

Taxonomy of Fungi Colonising Dodonaea Viscosa Twigs
In the current study, two new genera and four novel species were found. These taxa are subsequently described below.
Class It is saprobic on dead twigs and branches in terrestrial habitats. Sexual morph: the ascomata is a scattered, immersed to semi-immersed, subglobose to conical or shaped irregularly, glabrous, brown to dark brown ostiolate. The ostiole is a short papillate, black, smooth periphysate. The peridium comprises cells of textura angularis. The hamathecium comprises numerous, filamentous, branched, septate, pseudoparaphyses. The asci are eightspored, bitunicate, fissitunicate, clavate, with a pedicel, apically rounded with or without an ocular chamber. The ascospores overlap the biseriate, are ellipsoidal to sub-fusiform, hyaline, one-septate, with small to large guttules in each cell, with the ends remaining rounded, surrounded by a distinct mucilaginous sheath. Asexual morph: Coelomycetous. The conidiomata are sporodochial on PDA, globose, solitary or aggregated, semi-immersed, black, exuding yellow conidial masses. Conidiophores and conidiogenous cells were not observed in vitro. The conidia are solitary, aseptate, globose to ellipsoid, with the hyaline becoming medium to golden brown, and finely verruculose.
Type species: Haniomyces dodonaeae Haniomyces dodonaeae Wanas. and Mortimer sp. nov. (Figure 5) semi-immersed to erumpent, subglobose to conical or shaped irregularly, flattened base, glabrous, brown to dark brown ostiolate, fused with host tissues. The ostiole is a short papillate, black and smooth, with hyaline periphyses (15-25 µm long, 1.5-2 µm wide). The peridium 5-10 µm wide at the base, 10-20 µm wide at sides, comprising 2-4 layers, outer layer pigmented, comprising reddish brown to dark brown, with thin-walled cells of textura angularis, and an inner layer composed of hyaline, loosen, cells of textura angularis. Culture characteristics: the colonies on PDA reached a 3 cm diameter after 2 weeks at 20 • C. They were circular has a serrate margin, whitish at the beginning, becoming brown at the centre and brownish green towards the margin after 4 weeks. They were slightly raised, and reverse blackish brown. The hyphae septate were branched, hyaline, thin, and smooth-walled.
Known distribution: Yunnan, China, on Dodonaea viscosa. × 840 µm, n = 10), arising singly or in small groups, sessile, and slightly erumpent from the substrate. The receptacle is cupulate, black, flat or slightly concave, with a slightly dentate margin. The excipulum are 70-100 µm wide, with the ectal excipulum narrow layered, deep, and thick-walled, with black cells of textura globulosa to textura angularis; the medullary excipulum is composed of narrow, long, thin-walled, hyaline to brown cells of textura angularis. The hamathecium are 2.5-4 µm wide, numerous, propoloid, pseudoparaphyses, exceeding asci in length, apically swollen, branched and reddish-orange pigmented. The branched apices form a layer on hymenium to develop pseudo-epithecium. The asci are 140-180 × 12-16 µm (M = 163.3 × 13.8 µm, n = 20), eight-spored, long cylindrical, short pedicellate, and is rounded at apex. The ascospores 20-33 × 9-13 µm (M = 28.2 × 11.2 µm, n = 30), overlap the uniseriate, are hyaline to light brown, one-septate, with wrinkled walls when young, becoming dark brown at maturity. They are ellipsoid with conical ends, regularly three-septate, and rarely muriform with one longitudinal septum, smooth walled, guttulate. Asexual morph: Undetermined. Culture characteristics: Colonies on PDA reached a 4 cm diameter after 2 weeks at 20 • C. The colony was dense, circular, slightly raised, and the surface was smooth, with an undulated edge, with floccose which were greenish grey at the centre and brown towards margin from the top and reverse dark brown. The hyphae septate were branched, hyaline, thin, and smooth-walled.
Known distribution: Yunnan, China, on Dodonaea. The generic epithet stems from the combined two words "lophio" and "murispora", referring to muriform ascospores in Lophiostomataceae.
It is saprobic on woody substrates in terrestrial habitats. Sexual morph: The ascomata is a solitary or gregarious, semi-immersed, erumpent through the host surface, coriaceous to carbonaceous, dark brown to black, globose to subglobose or conical ostiolate. The ostiole is a slit-like, central papillate, with or without a crest, opening by an apical, lysigenous pore or dehiscence, comprising hyaline periphyses or hyaline to lightly pigmented, pseudoparenchymatous cells. The peridium is broad at the apex and thinner at the base, comprising two strata with several layers of brown or lightly pigmented to hyaline cells of textura angularis to textura prismatica, fusing and indistinguishable from the host tissues. The hamathecium comprises many branched, septate, cellular pseudoparaphyses, located between and above the asci, embedded in a gelatinous matrix. The asci are eight-spored, bitunicate, fissitunicate, cylindric-clavate, pedicellate, and apically rounded, with an ocular chamber. The ascospores are uni-to bi-seriate, partially overlapping, and are hyaline when immature, becoming brown to dark brown when mature. They are ellipsoidal to fusiform, muriform, two-to-eight-transversely septate, with one-to-two-longitudinal septa, constricted at the central septum, with or without a mucilaginous sheath. Asexual morph: Coelomycetous. The conidiomata is pycnidial, phoma-like, solitary, gregarious, dark brown to black, immersed or slightly erumpent, coriaceous to carbonaceous, papillate or apapillate. The conidiomata wall is multi-layered, with three to four outer layers of brown-walled pseudoparenchymatous cells, with the inner most layer being thin and hyaline. The conidiophores are long, septate, and sparsely branched, which are formed from the inner most layer of the pycnidium wall. The conidiogenous cells are phialidic, cylindrical, hyaline, flexuous and smooth, with a short collarette. The conidia are hyaline, aseptate, straight to curved, ellipsoidal with rounded ends, thin-walled, smooth, and numerous.

MycoBank: MB 837998
Etymology: The specific epithet is derived from Honghe County, the region of Yunnan Province in which this species was gathered.
Culture characteristics: the colonies on PDA reached a 4 cm diameter after 2 weeks at 20 • C. They were circular, had a serrate margin, and were whitish at the beginning, becoming greenish-brown 4 weeks later. They were slightly raised, and reverse dark brown. The hyphae septate were branched, hyaline, thin, and smooth-walled.
Known Culture characteristics: The colonies on PDA reached a 4 cm diameter after 2 weeks at 20 • C. They were circular, had a serrate margin, and were greenish brown after 4 weeks. They were slightly raised, and reverse dark brown. The hyphae septate were branched, hyaline, thin, and smooth-walled.

Discussion
Teratosphaeriaceae was introduced by Crous et al. [187]. Given that it is composed of 61 genera, it is regarded as one of the largest families in Dothideomycetes [188]. Members of this family are adapted to a broad range of life modes and can be saprobic, plant and human pathogenic, rock-inhabiting and endophytic; accordingly, they are widely distributed across varied terrain [49,136,139,188,189]. We have included representative sequence data of all available genera listed in Hongsanan et al. [188] for the phylogenetic analyses (except Davisoniella, Pachysacca and Placocrea, which lack DNA-based sequence data). Among them, Aulographina was grouped in Venturiales, and Leptomelanconium was related to Helotiales in the initial analysis. Therefore, they were excluded from the final analysis ( Figure 1). In addition, representative taxa for Piedraia were included in the final dataset that were phylogenetically closely related to Teratosphaeriaceae. However, this genus is still considered a member in Piedraiaceae. The phylogeny generated herein (Figure 1) is congruent with those of other published studies to resolve intergeneric relationships in Teratosphaeriaceae [49,188]. In the combined LSU, ITS, rpb2, act, cal and tef 1 data analysis, 58 clades are recognized from the ingroup taxa. Two strains from our new collections constitute a distinct monophyletic lineage (subclade 17, Figure 1) within the genera in Teratosphaeriaceae, which we introduce as a new genus.
The phylogeny (Figure 1) reveals a close relationship between two strains of the newly collected fungus (Haniomyces dodonaeae) to Camarosporula persooniae, Lapidomyces hispanicus, Neophaeothecoidea proteae, Teratosphaeriaceae sp. (CCFEE 5569), Xenoconiothyrium catenata and Xenophacidiella pseudocatenata, with 87% ML and 1.00 BYPP support values. Among them, only Camarosporula persooniae is reported from the sexual morph, and despite the high degree of phylogenetic similarity, these two species are morphologically dissimilar [136]. Neophaeothecoidea is more closely related to Haniomyces in the phylogenetic results, but this relationship lacks statistical support. In addition, Neophaeothecoidea is reported as a hyphomycete [188], whereas Haniomyces produces a coelomycetous asexual morph.
Out of the 61 genera listed in Teratosphaeriaceae, only 24 genera are described with sexual morphs. We suggest that the sexual morphs of these genera require further examination with increased collections to verify the accurate treatment of and relationships to remaining species. During asexual stages of Teratosphaeriaceae, most members are pathogenic, whereas they are non-pathogenic during sexual stages. This is an important distinction for identifying opportunistic pathogens, as members of this family can easily spread diseases between locations. The new taxon, Haniomyces dodonaeae, fits morphologically well into Teratosphaeriaceae by its periphysate ostiole and hyaline ascospores with a single septum in each. However, the dimensions of the asci and ascospores are significantly larger than the existing sexual reports of this family. The golden brown, ellipsoidal conidia of Haniomyces dodonaeae are similar to those in Neophaeothecoidea and Readeriella. Phylogenetically, Haniomyces dodonaeae has a close proximity to Neophaeothecoidea proteae. This relationship, however, is not strongly supported in the ML and BI analyses (Figure 1). Neophaeothecoidea proteae was originally isolated as a coelomycete (Phaeothecoidea proteae) based on its yeast-like growth in culture [190]; however, it is currently accounted for in a hyphomycetous genus. Comparison of the 805 base pairs (bp) across the LSU gene region of Haniomyces dodonaeae shows 17 bp (2.1%) differences exist in comparison with Neophaeothecoidea proteae. Similarly, comparison of the 356 bp of the rpb2 gene region shows 56 bp (15.73%) differences in comparison with Neophaeothecoidea proteae.
Rhytidhysteron was introduced by Spegazzini [191] to account for R. brasiliense and R. viride collected from southern Brazil in 1877 and 1880, respectively. Spegazzini [56] did not designate any type; therefore, Clements and Shear [192] designated R. brasiliense as the type species. Subsequently, few species were introduced to this genus based on morphological evidence [193][194][195][196]. In recent studies, more species have been introduced based on both morphology and DNA-based sequence data [55,56,172,177,178,183]. Presently, there are 23 species mentioned in Species Fungorum [197], including saprobic to weakly pathogenic taxa that grow on woody plants in terrestrial habitats [56,181]. Species of Rhytidhysteron are typically involved in wood degradation and occur primarily on the woody parts of a broad range of hosts [64,188].
We introduce a new species into Rhytidhysteron from a dead twig of Dodonaea sp. in Honghe, China, and its relationships with other species are presented based on multigene phylogenetic analyses (Figure 2). Our analysed molecular data generated phylogenies consistent with those of Mapook et al. [55] and Hyde et al. [56]. The novel species, Rhytidhysteron hongheense, is phylogenetically closely related to R. camporesii (KUN-HKAS 104277) and Rhytidhysteron chromolaenae (MFLUCC 17-1516), and these three constitute a strongly supported monophyletic clade. The ascospore and asci characteristics between the three species are similar, but the colour of hysterothecia in R. chromolaenae (green) is different from the other two (black). The pseudo-epithecium of R. camporesii is brown to purple, whereas it is reddish orange in R. hongheense. The significance of these morphological characteristics in species delineation should be further investigated in terms of phylogenetic signals. A pairwise comparison of 521 ITS (+5.8S) sequence data showed 31 (5.95%) bp differences between R. hongheense and R. camporesii as well 28 (5.37%) bp differences between R. hongheense and R. chromolaenae. Currently, there are only two Rhytidhysteron species, viz. Rhytidhysteron magnoliae and Rhytidhysteron thailandicum, reported from China [56,198], making this report the third of its kind from China and first from Honghe Prefecture.
Lophiostomataceae species are usually characterized by a slot-like ostiole on the top of the flattened neck, occurring mainly on twigs, stems or the bark of different woody and herbaceous plants in terrestrial, freshwater and marine environments as saprobes [129,140,188]. Thambugala et al. [129] undertook a comprehensive study of this family and accepted 16 genera. Subsequently, 12 new genera have been introduced by recent publications, and currently the family comprises 28 accepted genera [188]. The most recent multi-locus phylogenetic backbone tree to the family is presented in this study, including a novel genus (Lophiomurispora) found in Honghe County, Yunnan Province, China.
Lophiomurispora morphologically resembles Coelodictyosporium, Platystomum and Sigarispora with its crest-like ostiole and brown, multi-septate ascospores. However, these genera are revealed as phylogenetically distant in multi-gene phylogenetic analysis (Figure 3). Lophiomurispora has a close phylogenetic relationship to Desertiserpentica (Figure 3). However, Desertiserpentica is only known from its hyphomycetous asexual morph [54], whereas Lophiomurispora differs from Desertiserpentica by its coelomycetous asexual morph. Five strains of Lophiomurispora clustered in Lophiostomataceae as a strongly supported monophyletic clade ( Figure 3) in both ML and BI of a concatenated SSU, LSU, ITS, tef 1 and rpb2 dataset. All specimens were collected from dead twigs of Dodonaea viscosa at the Centre for Mountain Futures (CMF) in Honghe. There was no significant difference between morphological characteristics and DNA-based sequence comparisons between these collections. Therefore, we introduce them as different collections of Lophiomurispora hongheensis.
Crous et al. [131] introduced Quixadomyces for a fungus that was collected from Brazil on decaying bark. However, they did not observe the development of any internal structures. This fungus slightly resembles species in Pleosporales with its setose pycnidia [131,188]. In a multi-gene (concatenated LSU, SSU, ITS, rpb2, tef 1 and btub) phylogenetic analysis, the ex-type strain of Quixadomyces cearensis (HUEFS 238438) clustered with two of our new strains as a monophyletic clade with poor bootstrap support ( Figure 4). We introduce this isolate as a novel species belonging to this genus, Q. hongheensis. Based on the features of conidiogenous cells and conidia of Quixadomyces hongheensis, no substantial morphological differences exist to warrant two generic ranks. Therefore, this genus could potentially be reclassified as a synonym of Parapyrenochaeta in future studies. Because we did not perform extensive taxonomic reassessment using multiple fresh collections (especially sexual morphs of both genera), we will not attempt to synonymize any extant taxa.
Owing to lack of details on the internal structures of Quixadomyces cearensis, it is difficult to compare morphological characteristics such as conidiogenous cells and conidia between the new collection and this species. Lacking sufficient morphological evidence to perform accurate comparisons, we analysed nucleotide differences between these two strains. Comparing the 544 ITS (+5.8S) nucleotides of the two strains (HUEFS 238438 and KUMCC 20 0215) revealed 32 (5.88%) nucleotide differences. Therefore, it would seem prudent to treat our isolate as a new species in Quixadomyces as Q. hongheensis.
Nearly a century's worth of taxonomic investigation into Dodonaea viscosa has yielded only 58 fungal records [ Table 2]. These are mainly reported as saprobes or pathogens, but very few of these taxa are confirmed by both morphological and phylogenetic evidence. Many of these published records lack illustrations, descriptions or DNA sequence data, resulting in unclear taxonomic relationships. Even though Dodonaea viscosa is widely distributed across southwest and southern China, e.g., Fujian, Guangdong, Guangxi, Hainan, Sichuan and Yunnan [199], there is only one report for the fungus Pseudocercospora mitteriana on this host from China [124]. Previous taxonomic studies have suggested that increased collections might lead to the discovery of many new fungal species, and we, too, believe that Dodonaea is likely teeming with fungal diversity. More Dodonaea collections across different geographic regions are urgently needed, along with accompanying work in culture isolation, morphological description, DNA sequence analyses, phylogenetic relationship investigation, and accurate identification and classification. This study provides a case study for Dodonaea viscosa as a worthwhile host for the further study of microfungal associations and hints that it may potentially host numerous unknown fungal species.

Data Availability Statement:
The datasets generated for this study can be found in the NCBI GenBank, MycoBank and TreeBASE.