Taxonomic Novelties of Woody Litter Fungi (Didymosphaeriaceae, Pleosporales) from the Greater Mekong Subregion

Simple Summary The Greater Mekong Subregion (GMS) has a diverse geographic landscape, and due to its varied environmental conditions, it harbors numerous florae, fauna, and microorganisms. Thus, the biodiversity in this region is exceptionally high. Over recent decades, the number of studies on microfungal diversity in the GMS increased rapidly. However, in the GMS the fungi of terrestrial habitats such as woody litter is still poorly researched. This paper introduces one monotypic genus, five novel species, and two new host records in Didymosphaeriaceae-inhabiting woody plant litter from the GMS and provides morpho-molecular justifications. Abstract The Greater Mekong Subregion (GMS) is known as a diverse geographic landscape and one of the richest biodiversity hotspots in the world with a high fungal diversity. Collections were carried out in terrestrial habitats to determine the diversity of woody litter fungi in the GMS, with an emphasis on northern Thailand and the Yunnan Province of China. Morphological characteristics and multigene phylogenetic analyses of combined SSU, LSU, ITS, and tef1-α supported the placement of the new isolates in the family Didymosphaeriaceae. The phylogenetic affinities of our isolates are illustrated through maximum likelihood and Bayesian inference analyses. Seven species of woody litter fungi were identified, comprising a new monotypic genus, Septofusispora; five novel species (Chromolaenicola sapindi, Dictyoarthrinium thailandicum, Karstenula lancangensis, Septofusispora thailandica, and Spegazzinia jinghaensis); and new host records of two species (Austropleospora archidendri, and Montagnula donacina). Furthermore, this study provides a synopsis of the Montagnula aff. donacina species based on their morphological characteristics, which can be useful in the species-level identifications in this genus.


Introduction
The Greater Mekong Subregion (GMS) is a global biodiversity hotspot with a 2.5 million km 2 land area [1], including Cambodia, Lao PDR, Myanmar, Thailand, the People's Republic of China, and Vietnam. Due to its varied environmental conditions, the GMS harbors abundant biodiversity [2,3]. Numerous studies have shown that China (Yunnan Province) and Thailand have the potential to support a high diversity of macroand micro-fungi, many yet to be discovered [3][4][5][6][7][8][9]. For instance, many saprobic taxa have The newly generated sequences are indicated in bold. T refers to ex-type strains and NA refers to "no data in GenBank".

Phylogeny
The combined dataset of SSU, LSU, ITS, and tef 1-α comprised 109 strains of Didymosphaeriaceae and 2 strains of Periconia didymospora (MFLU 15-0057 and MFLU 15-0058), the latter 2 strains from Periconiaceae as the outgroup taxa ( Table 1) Didymosphaeriaceae comprises 33 genera, but molecular data are available only for 28 of them. Thus, sequence data representing 28 genera were used in the phylogenetic analyses. Phylogenetic trees resulting from ML and BI ( Figure 1) analyses have similar overall topologies compared to the trees illustrated in Dissanayake et al. [30], Jayasiri et al. [33], and Samarakoon et al. [34]. These results show that the KUMCC 21-0647 and KUMCC 21-0652 isolates formed a monophyletic clade independent from all others (Alloconiothyrium, Kalmusia, and Xenocamarosporium) (  (Figure 1), but they appear in a different branch, perhaps due to the lack of homologous SSU and tef 1-α sequences from the type collection.
Culture characteristics: The colonies on PDA, reaching 15-20 mm diam. at 14 days at room temperature (25-30 °C ), superficial, entire margin, umbonate at center, rough surface, with dense mycelia, velvety, raised, gray at the center, white at the edge; reverse atrovirens, darkening towards center and white at the edge.
Notes: Austropleospora archidendri was introduced by Ariyawansa et al. [92] as a new combination of Paraconiothyrium archidendri based on the combined phylogeny of LSU, SSU, β-tubulin, and ITS sequence data. In the present study, the multi-gene phylogenetic analyses indicated that our new strain, KUMCC 21-0680, formed a sister clade with A. archidendri (MFLUCC 17-2429) with 100% ML bootstrap and 0.95 BYPP support (Figure 1). Since the type species lacks SSU and tef 1-α sequences, the nucleotide base pair comparisons of LSU and ITS demonstrated that our new strains are identical to the type of the Austropleospora archidendri strain and other species (Table 1). Our strain, KUMCC 21-0680, is similar to A. archidendri (CBS 168.77, MFLUCC 17-2429) in having doliiform conidiogenous cells and sub-globose-to-ovate, brown, aseptate conidia [28,33]. Austropleospora archidendri was reported as a pathogen on A. bigeminum leaves in Thailand and a saprobe on the pods of a Leucaena sp. in Myanmar [28,33]. Therefore, we report our strain KUMCC 21-0680 as a new record of A. archidendri on woody litter of Euphoria longana in Thailand.     Notes: Dictyoarthrinium thailandicum is introduced as a new species based on its distinct morphology and the phylogeny of the combined SSU, LSU, ITS, tef 1-α dataset. This species is phylogenetically distinct from other Dictyoarthrinium species and formed a clade sister to D. musae with 76% ML bootstrap and 1.00 BYPP support (Figure 1). This species is similar to D. musae in having black colonies, cup-shaped conidiophore mother cells, and cylindrical conidiogenous cells. However, the size of the conidiophores and conidia of D. thailandicum (180 × 4.5 µm, 10 × 9.7 µm) is comparatively larger than those of D. musae (81.5 × 1.6 µm, 8.7 × 7.9 µm) [34].
Notes: Wanasinghe et al. [42] synonymized Munkovalsaria donacina and M. appendiculata under Montagnula based on the phylogenetic analyses of the combined LSU, SSU, and ITS sequence data. Generally, M. donacina is characterized by immersed-to-erumpent, single, or gregarious ascomata with a single ostiole, bitunicate, clavate or cylindrical asci with a pedicel and an ocular chamber, ellipsoid, unicellular, 1-septate ascospores strongly constricted at the septum with the upper cell wider and the lower cell rounded [80,100]. Spegazzinia was introduced by Saccardo [101] with S. ornata as the type species. Hyde et al. [95] accommodated Spegazzinia in Sordariomycetes (Apiosporaceae), and based on morphological and molecular evidence, Tanaka et al. [73] transferred Spegazzinia to Didymosphaeriaceae in Dothideomycetes. This was supported by Jayasiri et al. [33], Samarakoon et al. [52], and Thambugala et al. [41]. Currently, 14 taxa are listed in Species Fungorum [45]. Spegazzinia is a widely distributed genus with species reported as saprobes on decaying leaves, wood, fruit, and bambusae from Australia, Brazil, China, Cuba, Ghana, and Thailand [38,41,52,[102][103][104][105], and endophytes from lichen and leaves in Brazil and India [87,106]. The Spegazzinia species have also been reported from the soil in Congo and estuarine sediment [94,107]. Morphologically, most species of Spegazzinia have two types of conidia in the same mycelium: α conidia are composed of 4-8 subglobose, very dark cells with very long spines, while β conida are subspherical or broadly ellipsoid in general, flattened in one plane, cruciately septate or muriform, almost always pale brown and smooth [38]. This paper introduces two new isolates of the Spegazzinia species observed from decaying wood in terrestrial habitats in China and Thailand.
Out of 33 genera, Kalmusia, Montagnula, Paraphaeosphaeria, Paraconiothyrium, Phaeodothis, Pseudocamarosporium, Pseudopithomyces, and Spegazzinia are well studied compared to other genera in Didymosphaeriaceae, but many species are likely awaiting discovery [77]. Barria, Cylindroaseptospora, Kalmusibambusa, Lineostroma, Neptunomyces, Vicosamyces, and Xenocamarosporium are still monotypic [45], and new species discovery is expected [77]. In this study, we added taxonomic novelties from the GMS to better understand the morphological and phylogenetic relationships of Didymosphaeriaceae. Septofusispora, typified by S. thailandica, is introduced to accommodate terrestrial dothideomycetes species with a characteristic morphology compared to the extant genera (Alloconiothyrium, Kalmusibambusa, Kalmusia, and Xenocamarosporium) in Didymosphaeriaceae. This genus is characterized by its clavate asci, fusiform, guttulate ascospores with 4-5 transverse septa, whereas Kalmusia has ovoid-to-clavate, 3-septate ascospores (sometimes muriform), with a mucilaginous sheath. Kalmusibambusa differs from Septofusispora by having ellipsoidal-to-fusiform, 3-septate ascospores with a wide mucilaginous sheath [41,81,90,91]. Alloconiothyrium and Xenocamarosporium are known only from their asexual morphs [28,63]; therefore, they cannot be morphologically compared with the teleomorph of Septofusispora. The phylogenetic analyses showed that Septofusispora is distinctly separated from its closely related taxa in this family. Therefore, based on morphological characters and the SSU, LSU, ITS and tef1-α sequence data, we recognize Septofusispora as a new genus in the family Didymosphaeriaceae.
Alloconiothyrium was introduced by Verkley and coauthors [28] with A. aptrootii as the type species, which is characterized by having pycnidial or eustromatic conidiomata, holoblastic, annellidic conidiogenous cells, olivaceous-brown and irregularly outlined conidia with a rough surface [28]. However, Ariyawansa and coauthors [63] introduced Alloconiothyrium camelliae as a new species with uni-loculate, globose-to-subglobose conidiomata, ampulliform-to-doliiform or cylindrical conidiogenous cells and smooth-walled conidia. Furthermore, the multi-gene phylogenies of Ariyawansa and coauthors [63] and our multi-gene phylogenies show that Alloconiothyrium aptrootii is well-separated from A. camelliae. Therefore, we suggest that A. camelliae is a monotypic genus of Didymosphaeriaceae. Further studies are needed for a better understanding of the morphological and phylogenetic relationships of Alloconiothyrium.
Karstenula is an ambiguous genus that exhibits morphological similarities with different families [29,31]. Usually, the sexual morph of Karstenula was thought to be characterized by having cylindrical or clavate asci, and brown ascospores with transverse septa and sparse longitudinal septate as dominant characters [31]. For instance, Karstenula adenocarpi has oblong ascospores with three transverse septa and 1-several longitudinal septa [110]; Karstenula calligoni has clavate asci, and fusoid ascospores with 5-7 transverse septa and a longitudinal septum [111]; Karstenula guttulata has cylindrical asci, and ellipsoid, oblongto-oval ascospores with 4-6 transverse septa and 1-2 longitudinal septa [112]; Karstenula rhodostoma has cylindrical asci, and ellipsoid ascospores with three transverse septa and a vertical septum in one or two central cells [31]. As mentioned by Constantinescu [98], the anamorph of Karstenula rhodostoma is identical to the coelomycete Microdiplodia frangulae, characterized by ampulliform-to-doliiform or cylindric-to-ampulliform conidiogenous cells, and cylindrical, yellow-to-golden-brown conidia with one septum. Karstenula lancangensis is similar to the asexual morph of Karstenula rhodostoma in having cylindric, 1-septate, brown conidia. However, in our phylogenetic analysis, Karstenula lancangensis forms a well-supported clade sister to K. rhodostoma in Didymosphaeriaceae (Figure 1). Our study provides a reference for further understanding the asexual morphology of Karstenula.
Montagnula donacina is a prevalent species distributed almost all over the world. It has been isolated from 38 plant species within 24 families [27,80], from which 20 hosts have been from India. However, M. donacina has been rarely reported in the GMS, with only two hosts (Althaea rosea and Trachycarpus fortunei) recorded from China and one host (Nephelium litchi) from Myanmar [100,113,114]. The present study reports two M. donacina collections from the hosts E. acuminata and Betula sp. in China and Thailand (the first report of this species in this country). The morphological comparisons between species of Montagnula, putatively related to M. donacina ( Table 2), showed that the ascospores of M. graminicola are light brown, verruculose, and have a mucilaginous sheath without guttules. In contrast, all the other species in this clade have no significant apomorphic morphological traits, except for slight differences in the size of ascomata, asci, or ascospores (Table 2), which can be due to ecological factors [115]. In addition, as mentioned in the notes of M. donacina, these species are not significantly distinct in phylogeny. Therefore, based on the current morphological data and phylogenetic analyses, we suggest that M. chromolaenicola, M. puerensis, M. saikhuensis, and M. thailandica can be considered conspecific with M. donacina. However, even though M. donacina is widely reported from different hosts, molecular data from only two collections are available in GenBank. Therefore, more extensive studies are needed, applying a combination of different species delimitation criteria to more sequence data obtained from additional samples [116,117] in order to resolve and define the species boundaries in the Montagnula donacina complex. Finally, Montagnula jonesii was introduced by Tennakoon et al. [49] based on morphology coupled with the analysis of the combined LSU, SSU, ITS, and tef 1-α sequence data. Despite the fact that our phylogenetic tree based on multigene data showed that Montagnula jonesii is not monophyletic with the taxa in Montagnula s. str., single-gene analyses (not shown) showed that LSU, SSU, and tef 1-α data support that M. jonesii belongs in Montagnula, and only ITS suggests a different placement. Therefore, we believe that the published ITS sequence of M. jonesii may be incorrect and needs to be conducted again.

Data Availability Statement:
The datasets generated for this study can be found in the GenBank, NCBI and the accession numbers are given in Table 1. Newly introduced fungal names were registered at the Index Fungorum and the identification numbers are shown in their respective entries.