Taxonomy and Phylogenetic Appraisal of Dothideomycetous Fungi Associated with Magnolia, Lilium longiflorum and Hedychium coronarium

This paper highlights the taxonomy of some interesting saprobic microfungi associated with dead plant materials of Hedychium coronarium, Lilium longiflorum, and Magnolia species. The taxa reported in this study belong to the orders Pleosporales and Kirschsteiniotheliales (Dothideomycetes). These taxa were identified based on multi-locus phylogeny of nuclear ribosomal DNA (rDNA) (LSU, SSU, and ITS) and protein-coding genes (tef1-α and rpb2), together with comprehensive morphological characterization. Two novel saprobic species, Leptoparies magnoliae sp. nov. and Neobambusicola magnoliae sp. nov., are introduced from Magnolia species in Thailand. Another new species, Asymmetrispora zingiberacearum sp. nov., is also described from dead stems of H. coronarium, which is the first asexual morph species of the genus Asymmetrispora. In addition, Ramusculicola thailandica and Kirschsteiniothelia thailandica are reported as new host records from dead twigs of Magnolia species. Sphaerellopsis paraphysata is reported as a new host record from L. longiflorum. Newly described taxa are compared with other similar species and detailed descriptions, micrographs, and phylogenetic trees to show the positions are provided.


Introduction
The exploration of the tropics to discover and describe fungal life has recently gained immense scientific interest. In this contribution, we investigate novel and existing fungal species and their host plant associations. Fungi play fundamental ecological roles as decomposers, mutualists, and pathogens [1]. They also help carbon cycling and biogeochemical processes and mediate mineral nutrition of plants in forest ecosystems [2][3][4]. Fungi have an important contribution to ecosystems, as higher saprobic fungal diversity intensifies the decomposition process and higher mycorrhizal diversity enhances plant diversity, ecosystem functioning, and nutrient assimilation [5]. Mycologists have proposed different estimations for existing and described fungal species numbers. Hawksworth and Lucking [6] stated that the estimated fungal species ranged from 2.2 to 3.8 million fungal species, and only 120,000 (8%) have been described. This implies that a large number of fungi still remain to be discovered. The possible reasons for the observed discrepancy can be due to fungi being poorly studied in many countries, regions, and host species [7].
Tropical rainforests harbor the highest fungal diversity [3]. Fungi present in tropical regions and their vegetations are not well studied; thereby, fungal novelties might

Samples Collection, Morphological Studies, and Isolation
Dead twigs attached to Magnolia spp. in Chiang Mai Province, Thailand, were collected in this study. Dead stems of H. coronarium and leaf litter from L. longiflorum in Taiwan Province of China were also collected in this study. These plant specimens were transferred to the laboratory and examined with a JNOEC JSZ4 stereomicroscope. Morphological characteristics were observed using an OLYMPUS SZ61 compound microscope. Photographs of morphological characters were captured with a Canon EOS 600D digital camera mounted on a Nikon ECLIPSE 80i compound microscope. The Tarosoft (R) image framework v. 0.9.0.7 was used to measure all microscopic measurements. The Adobe Photoshop CS3 Extended version was used to process photographs further. Pure fungal cultures were isolated following the protocol described in Senanayake et al. [20]. Germinating ascospores and conidia were transferred aseptically to potato dextrose agar (PDA) for further cultural and molecular analyses. Culture characteristics of pure fungal cultures, such as growth rate and colony characteristics, were observed and recorded at room temperature (25 • C).
The collections made in the current study were deposited at the Mae Fah Luang University Herbarium (Herb. MFLU), Chiang Rai Province, Thailand, the herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, Yunnan Province, China, and National Chiayi University Herbarium (NCYU). The living fungal cultures recovered in this study were deposited at Mae Fah Luang University Culture Collection (MFLUCC) and National Chiayi University Culture Collection (NCYUCC). Faces of Fungi numbers and Index Fungorum numbers were registered as described in Jayasiri et al. [21] and Index Fungorum [22], respectively.

DNA Extraction and PCR Amplification
Genomic DNA was extracted using one-week-old fungal cultures on PDA [23]. The mycelia were scraped off from pure cultures, and genomic DNA was extracted using Biospin fungus genomic DNA kit (BioFlux ® , Hangzhou, China) according to the manufacturer's instructions. In addition, genomic DNA was extracted from the fruiting bodies on the natural substrate of Leptoparies magnoliae (MFLU 18-1291), Neobambusicola magnoliae (HKAS 107122), and Ramusculicola thailandica (HKAS 107136) using a DNA extraction kit (BioFlux ® , Hangzhou, China) according to the manufacturer's instructions. The DNA products were kept at 4 • C for DNA amplification and maintained at −20 • C for long-term storage.
Selected genes, such as the partial gene regions of Internal Transcribed Spacers (ITS) and 28 S ribosomal RNA (LSU), 18 S ribosomal RNA (SSU), and Translation Elongation Factor 1-alpha (tef1-α), were amplified using appropriate primers via polymerase chain reaction (PCR). The LSU region was amplified with primer pair LR0R and LR5 [24]. The SSU region was amplified with primer pair NS1 and NS4, and the ITS region was amplified with primer pair ITS5 and ITS4 [25]. The part of the tef1-α region was amplified with primer pair EF1-983F and EF1-2218R [26]. The total volume of the final PCR mixture was 25 µL, which was composed of 1 µL of DNA template, 1 µL of each forward and reverse primer, 12.5 µL of 2×Easy Taq PCR SuperMix (a mixture of EasyTaq TM DNA Polymerase, dNTPs, and optimized buffer, Beijing TransGen Biotech Co., Ltd., Beijing, China), and 9.5 µL of ddH 2 O. PCR amplification of LSU, SSU, ITS, and tef1-α included an initial denaturing step of 94 • C for 3 min., followed by 40 amplification cycles of 94 • C for 45 s, 55 • C for 50 s, and 72 • C for 1 min. and a final extension step of 72 • C for 10 min.
PCR purification and sequencing of amplified PCR products were conducted at Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China.
Sequences of the individual genes were aligned with MAFFT v. 7 online version [27] with default settings. The alignments were manually improved where necessary and to exclude incomplete portions at the ends of the sequences before the analyses using BioEdit v. 7.0.5.2 [28]. The newly generated sequences in this study were deposited in GenBank, and accession numbers were mentioned in relevant entries. Details of the sequences used for phylogenetic analysis are provided in supplementary documents (Tables S1-S4).

Figure 1.
Phylogram generated from maximum likelihood analysis is based on combined LSU, SSU, and ITS sequence data. Related sequences of Leptosphaeriaceae were obtained from Doilom et al. [33]. ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are indicated above the branches. The tree was rooted to Didymella exigua (CBS 183.55). The newly generated sequences are indicated in red. Type and ex-type strains are in bold.
Sulcatisporaceae LSU, ITS, SSU, and tef1-α Phylogeny The combined dataset of LSU, ITS, SSU, and tef1-α comprised 18 strains, representing Bambusicolaceae D.Q. Dai and K.D. Hyde, Sulcatisporaceae, and Latoruaceae Crous with Didymosphaeria rubi-ulmifolii Ariyaw., Camporesi, and K.D. Hyde (MFLUCC 14-0024) as the outgroup taxon. The topology of the resulting phylogram of maximum likelihood analysis is largely similar to Bayesian analysis. The combined gene analyses comprised 3500 characters after alignment (1000 characters for LSU, 1000 characters for SSU, 550 characters for ITS, and 950 characters for tef1-α). The best RAxML tree with a final Figure 1. Phylogram generated from maximum likelihood analysis is based on combined LSU, SSU, and ITS sequence data. Related sequences of Leptosphaeriaceae were obtained from Doilom et al. [33]. ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are indicated above the branches. The tree was rooted to Didymella exigua (CBS 183.55). The newly generated sequences are indicated in red. Type and ex-type strains are in bold.

Figure 2.
Phylogram generated from maximum likelihood analysis is based on combined LSU, SSU, ITS, tef1-α, and rpb2 sequence data. Related sequences of Leptoparies and closely related genera in Lophiostomataceae were obtained from Andreasen et al. [34]. ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are indicated above the branches. The tree was rooted to Teichospora trabicola (C134). The newly generated sequences are indicated in red. Type and ex-type strains are in bold.
Teichosporaceae LSU, ITS, SSU, tef1-α, and rpb2 Phylogeny The combined dataset of LSU, ITS, SSU, tef1-α, and rpb2 comprised 76 strains, representing Teichosporaceae with Didymella exigua (CBS 183.55) as the outgroup taxon. The topology of the resulting phylogram of maximum likelihood analysis is largely similar to Bayesian analysis. The combined gene analyses comprised 4200 characters after alignment (900 characters for LSU, 1000 characters for SSU, 500 characters for ITS, 900 characters for tef1-α, and 900 characters for rpb2). The best RAxML tree with a final likelihood value of −24,166.180022 is presented. The matrix had 1659 distinct alignment patterns, with 43.60% undetermined characters or gaps. Estimated base frequencies were as follows: A = Figure 2. Phylogram generated from maximum likelihood analysis is based on combined LSU, SSU, ITS, tef1-α, and rpb2 sequence data. Related sequences of Leptoparies and closely related genera in Lophiostomataceae were obtained from Andreasen et al. [34]. ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are indicated above the branches. The tree was rooted to Teichospora trabicola (C134). The newly generated sequences are indicated in red. Type and ex-type strains are in bold.
Teichosporaceae LSU, ITS, SSU, tef1-α, and rpb2 Phylogeny The combined dataset of LSU, ITS, SSU, tef1-α, and rpb2 comprised 76 strains, representing Teichosporaceae with Didymella exigua (CBS 183.55) as the outgroup taxon. The topology of the resulting phylogram of maximum likelihood analysis is largely similar to Bayesian analysis. The combined gene analyses comprised 4200 characters after alignment (900 characters for LSU, 1000 characters for SSU, 500 characters for ITS, 900 characters for tef1-α, and 900 characters for rpb2). The best RAxML tree with a final likelihood value of −24,166.180022 is presented. The matrix had 1659 distinct alignment patterns, with 43.60% undetermined characters or gaps. Estimated base frequencies were as follows:  Phylogram generated from maximum likelihood analysis is based on combined LSU, SSU, ITS, and tef1-α sequence data. Related sequences of Sulcatisporaceae were obtained from Phukhamsakda et al. [35,36]. ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are indicated above the branches. The tree was rooted to Didymosphaeria rubi-ulmifolii (MFLUCC 14-0024). The newly generated sequences are indicated in red. Type and ex-type strains are in bold.  Phylogram generated from maximum likelihood analysis is based on combined LSU, SSU, ITS, and tef1-α sequence data. Related sequences of Sulcatisporaceae were obtained from Phukhamsakda et al. [35,36]. ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are indicated above the branches. The tree was rooted to Didymosphaeria rubi-ulmifolii (MFLUCC 14-0024). The newly generated sequences are indicated in red. Type and ex-type strains are in bold.   Phylogram generated from maximum likelihood analysis is based on combined LSU, ITS, SSU, tef1-α, and rpb2 sequence data. Related sequences of Teichosporaceae were obtained from Tennakoon et al. [37]. ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are indicated above the branches. The tree was rooted to Hermatomyces tectonae (MFLUCC 14-1140) and H. thailandica (MFLUCC 14-1143). The newly generated sequences are indicated in red. Type and ex-type strains are in bold.

Figure 5.
Phylogram generated from maximum likelihood analysis is based on combined LSU, SSU, and ITS sequence data. Related sequences of Kirschsteiniothelia species were obtained from Sun et al. [38]. ML bootstrap values equal to or greater than 75% and Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are indicated above the branches. The tree was rooted to Pseudorobillarda eucalypti (MFLUCC 12-0422) and P. phragmitis (CBS 398.61). The newly generated sequences are indicated in red. Type and ex-type strains are in bold.
Culture characteristics: Colonies on PDA, 15-20 mm diam. after three weeks at 25 • C; colonies from above: medium dense, irregular, slightly raised, surface smooth with undulate edge, with smooth aspects, yellowish brown at the margin, dark grey to dark brown in the center; reverse: yellowish brown at the margin, dark brown to black in the centre, mycelium dark grey to brown.
Known hosts and distribution: on rusts on Pennisetum sp. in Brazil, Ravenelia macowania in South Africa [45], on Liriope spicata in China [47], on dead leaves of Lilium longiflorum in Taiwan    tally well with the type in having semi-immersed to superficial, globose to subglobose, multiloculate conidiomata and fusiform to ellipsoidal, one-septate, hyaline conidia with mucilaginous appendages at both ends [45]. However, our collection slightly differs from the type of S. paraphysata in having smaller conidiomata (70-120 µm vs. 450 µm) and lacking paraphyses [45]. Multi-gene phylogeny (LSU, SSU, and ITS) also indicates that our collection clusters with other S. paraphysata isolates in a well-supported clade (100% ML, 1.00 BYPP, Figure 1). Thus, we identify the new isolate as S. paraphysata and considered it as a new host record from L. longiflorum in Taiwan [49]. The type genus is Lophiostoma, and the type species is L. macrostomum [49]. Members of this family are generally distributed in temperate regions. Species of this family are saprobes or necrotrophs that grow on herbaceous and woody plants from terrestrial and aquatic habitats including freshwater and marine environments [50,51]. Members of this family can be identified by their coriaceous to carbonaceous ascomata with the slit-like ostiole [42,50,51].
Neobambusicola magnoliae N.I. de Silva and S. Lumyong, sp. nov. Index Fungorum number: IF559915, Faces of fungi number: FoF12710, Figure 8. Etymology: Name reflects the host genus Magnolia, from which the new species was isolated.
Asymmetrispora Thambugala & K.D. Hyde Type: Asymmetrispora tennesseensis (Mugambi, A.N. Mill. and Huhndorf) Thambug. and K.D. Hyde, Fungal Diversity: 50, (2015) Notes: Asymmetrispora was introduced by Thambugala et al. [51] to accommodate two species, namely A. tennesseensis (type) and A. mariae, which were previously known as Misturatosphaeria tennesseensis and M. mariae, respectively. Subsequently, Jaklitsch et al. [57] synonymized this genus under Teichospora based on the broad genus concept. However, Tennakoon et al. [37] provided a comprehensive taxonomic revision for the family and reestablished Asymmetrispora based on its distinct morphological variations and phylogenetic evidence. Currently, only two sexual morph species are accepted in this genus [48]. In this study, we introduce the first asexual morph species of the genus Asymmetrispora, namely Notes: According to the multi-gene phylogenetic analyses (LSU, SSU, ITS, tef1-α, and rpb2), our strains (MFLU 19-2813 and NCYU 19-0115) grouped within Asymmetrispora isolates (A. tennesseensis and A. mariae) in a strongly supported clade (96% ML, 1.00 BYPP, Figure 4). Both A. tennesseensis and A. mariae species have been recorded as their sexual morphs. Thus, we could not compare the morphological differences between our new collection and A. tennesseensis and A. mariae. Therefore, we compared the ITS (+5.8 S) and tef1-α gene regions' base pair differences. There are 10 base pair differences (1.96%) across 510 nucleotides across the ITS (+5.8 S) gene region and 26 base pair differences (3.59%) across 724 nucleotides across the tef1-α gene region between our collection (MFLU 19-2813) and A. mariae (CBS 140732). In addition, there are 29 base pair differences (3.86%) across 724 nucleotides across the tef1-α gene region between our collection (MFLU 19-2813) and A. tennesseensis (ANM 911). Therefore, we introduce our collection as a new species, A. zingiberacearum, from dead stems of H. coronarium (Zingiberaceae). It will be interesting to add fresh collections to expand this genus and to resolve the sexual and asexual connection of Asymmetrispora species. Notes: Thambugala et al. [51] introduced Ramusculicola to accommodate the type R. thailandica. Species of Ramusculicola are saprobic on dead twigs [37]. The sexual morph is characterized by immersed ascomata, eight-spored, bitunicate, fissitunicate, cylindrical asci, hyaline, fusiform to cylindrical, usually 1-3-septate ascospores with thin mucilaginous sheath [51]. Two species were recorded for Ramusculicola in Index Fungorum [22].  Etymology: The species name reflects the host family Zingiberaceae, from which the holotype was collected.
Culture characteristics: Colonies on PDA, 15-20 mm diam. after three weeks at 25 • C; colonies from above: circular, medium dense, slightly raised, surface smooth with entire edge, velvety appearance with smooth aspects, white to cream at the margin, grey in the center; reverse: yellowish brown at the margin, light brown in the center, mycelium white to whitish cream or grey.
Known hosts and distribution: from Ficus macrocarpa in Thailand [38] and Magnolia sp. in Thailand (this study). Material

Discussion
Studying the fungi in the tropics, particularly in Thailand and Taiwan Province of China, will provide important information towards establishing the numbers of fungi. The current study reveals three new species viz. Asymmetrispora zingiberacearum in Taiwan Province of China, Leptoparies magnoliae and Neobambusicola magnoliae in Thailand, and three new host records viz Kirschsteiniothelia thailandica and Ramusculicola thailandica in Thailand and Sphaerellopsis paraphysata in Taiwan Province of China. Considering the fact that tropical fungi are poorly documented and only a small percentage of global fungi have been discovered, which is between 2.6 and 4.5% of the 2.2-3.8 million estimated species, it is expected to reveal a large number of undiscovered taxa in the tropics. [7]. Thailand is geographically located in the core of the Greater Mekong Subregion with tropical seasonal forests and various types of floristic compositions [61,62]. Similarly, Taiwan Province of China has tropical to subtropical climatic zones that are attributed to the luxuriant vegetation, resulting in tremendous biodiversity [37]. These tropical regions have terrestrial ecosystems that have a great influence on regional and global energy and water cycling because these are located in regions of high solar radiation and evaporation [61]. Previous investigations have identified several novel fungal species and reported new host and geographical records in Thailand. For example, Doilom et al. [63] studied both asymptomatic stems and dead wood and symptomatic branches, stems, and leaves of Tectona grandis (Lamiaceae) in Thailand. Their investigation revealed 14 species and 14 new host and geographical records. In another study, Tibpromma et al. [64] identified seven new species and nine known species of endophytes associated with leaves of Pandanaceae collected from southern Thailand. Mapook et al. [65] introduced 12 new genera, 47 new species, and 12 new host records of microfungi associated with the invasive weed Chromolaena odorata collected in northern Thailand. In addition to these studies, Tennakoon et al. [66] explored microfungi associated with the leaf litter of different plants in Taiwan Province of China and revealed two new families, three new genera, 41 new species, and 54 new host records. These intensive studies generated important findings to support the fact that the tropics harbor enormous diversity of plant-associated microfungi and also provide a host-fungus database for future studies and increase knowledge of fungal diversity, as well as new fungal discovery.
Dothideomycetes are the largest and most ecologically diverse class of Ascomycota. Dothideomyceteous members exhibit as endophytes, pathogens, saprobes, or epiphytes on various hosts, in terrestrial as well as aquatic habitats [18,65]. We investigated saprobic Dothideomycetes on dead twigs and leaves from three different plant species, namely Hedychium coronarium and Lilium longiflorum and Magnolia species. In this study, we introduced A. zingiberacearum as new species on dead stems of H. coronarium (Zingiberaceae). Thambugala et al. [51] introduced the genus Asymmetrispora and incorporated two species, A. tennesseensis and A. mariae, that were previously known as Misturatosphaeria tennesseensis and M. mariae, respectively. Two species were accepted in Asymmetrispora in Species Fungorum [48]. Asymmetrispora tennesseensis was isolated from woody branches in the USA [67], while A. mariae was isolated from the bark and wood of Robinia pseudoacacia in Europe (Austria, France, and Germany) [57]. The novel species of Asymmetrispora introduced in this study, A. zingiberacearum, is also a saprobic species that was isolated from dead stems of H. coronarium in Taiwan Province of China. This indicates that Asymmetrispora are found in different geographical locations such as the USA, Europe (Austria, France, and Germany), and Taiwan Province of China. Another interesting fact is that both A. tennesseensis and A. mariae were described using their sexual morph characteristics. Asymmetrispora zingiberacearum is the first asexual morph (coelomycetous) that is recorded for this genus. The second host plant species investigated in the current study is L. longiflorum, a bulbous plant species of the Liliaceae, endemic to the Ryukyu archipelago and Taiwan [68]. Lilium longiflorum is regarded as an important species in world horticulture [68]. The current study reports Sphaerellopsis paraphysata from dead leaves of L. longiflorum (Liliaceae) in Taiwan Province of China for the first time. One of the findings here is that our collection slightly differs from the type of S. paraphysata in having smaller conidiomata and lacking paraphyses ( Figure 6). This new information can be used to amend the morphology of S. paraphysata, which is useful for fungal identification.
Magnolia species are widely distributed in temperate and tropical Southeast and East Asia [69]. These plant species are important as ornamental plants due to their attractive flowers and foliage and are used as timber and medicine by local communities [69,70]. However, Magnolia species face habitat destruction, and 48% of all Magnolia species are considered endangered [71]. Hence, it is important to develop ex situ conservation for these endangered, endemic, and economically valuable plant species [71]. Microfungi on Magnolia species have been studied by various scientists around the world. According to Farr and Rossman [72], 1100 fungal taxa have been recorded from Magnolia species worldwide; however, 52 taxa have been reported in Thailand. In this study, we introduced two novel species, Leptoparies magnoliae (Lophiostomataceae) and Neobambusicola magnoliae (Sulcatisporaceae) from Magnolia species in Thailand. Both Leptoparies and Neobambusicola genera consist of only one known species to date [22]. Leptoparies was introduced by Hashimoto et al. [52] with the type species Leptoparies palmarum. Neobambusicola was introduced by Crous et al. [55] with the type species N. strelitziae. Further, Kirschsteiniothelia thailandica and Ramusculicola thailandica were reported herein as new host records from Magnolia species in Thailand. These novel findings demonstrate that Magnolia species are ideal candidates for studying microfungi, as they provide a suitable host environment for diverse microfungal occurrences. In addition, the current study added three new saprobic species as A. zingiberacearum, L. magnoliae, and N. magnoliae to the genera, Asymmetrispora, Leptoparies and Neobambusicola, respectively. Therefore, it will be interesting to explore and study fresh collections to uncover the hidden taxonomic diversity from the species-poor genera such as Asymmetrispora, Leptoparies, and Neobambusicola. Thus, these additional representative species will help to populate and better understanding of the genus.
Modern molecular techniques have exploded the ability to recognize fungal diversity and understand diverse fungal communities. In this study, the phylogeny of Kirschsteiniothelia was constructed using combined LSU, SSU, and ITS sequence data as provided in Boonmee et al. [59], Bao et al. [73], and Sun et al. [38]. Molecular phylogenetic studies were carried out mostly including LSU, SSU, mtSSU, and ITS, as well as the protein genes, such as rpb1, rpb2, tef1-α, β-tubulin, and ACT for Pleosporales taxa [15]. The use of a single molecular marker has not been successful in resolving numerous relationships. However, concatenated genes with additional protein-coding markers such as tef1-α and rpb2 provide more precise phylogenetic affiliations of the members in Dothideomycetes [53,74]. Phylogenetic analyses of Leptosphaeriaceae were constructed using combined LSU, SSU, and ITS sequence data following the studies of Doilom et al. [33], Tennakoon et al. [75], and Wanasinghe et al. [76]. Since many strains of Leptosphaeriaceae lack tef1-α sequence data and other protein-coding markers, the phylogenetic studies were restricted to combined LSU, SSU, and ITS sequence data in accordance with Doilom et al. [33]. Generic delimitation in Teichosporaceae has been debatable in previous studies during the last decade because of insufficient taxon sampling and genetic marker coverage [37]. This species-rich family was erected by Barr [56] with eight genera, viz. Bertiella, Byssothecium, Chaetomastia, Immotthia, Loculohypoxylon, Moristroma, Sinodidymella and the genus type Teichospora based on morphological characteristics. The latest treatment of Tennakoon et al. [37] took advantage of both molecular and morphological approaches to delimitate species in Teichosporaceae. Their phylogenetic analyses based on a multigene-matrix of five genetic markers (ITS, LSU, SSU, tef1-α, and rpb2) and increased taxon sampling shed new light on the relationships of different genera within Teichosporaceae. However, the majority of strains lacked rpb2 sequence data. It is, therefore, necessary to collect fresh samples and obtain molecular data including proteincoding markers such as tef1-α and rpb2 for existing and novel species to investigate their phylogenetic relationships in order to achieve better identification and classification. Some of the more advanced molecular studies have examined a great amount of molecular data and identified fungal diversity at the generic level. Nevertheless, this results in the majority of uninformative data. Therefore, further studies should incorporate not only phylogenetic studies and morphological comparisons but also host associations and geographical information of microfungi.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof8101094/s1, Table S1: GenBank and culture collection accession numbers of species included in this phylogenetic study (Sphaerellopsis tree). The newly generated sequences are shown in red.; Table S2: GenBank and culture collection accession numbers of species included in this phylogenetic study (Leptoparies tree). The newly generated sequences are shown in red.; Table S3: GenBank and culture collection accession numbers of species included in this phylogenetic study (Neobambusicola tree). The newly generated sequences are shown in red.; Table S4: GenBank and culture collection accession numbers of species included in this phylogenetic study (Teichosporaceae tree). The newly generated sequences are shown in red.; Table S5: GenBank and culture collection accession numbers of species included in this phylogenetic study (Kirschsteiniothelia tree). The newly generated sequences are shown in red. Data Availability Statement: All sequences generated in this study were submitted to GenBank (https://www.ncbi.nlm.nih.gov, accessed on 1 October 2022).